The Role of Lipid Fluidity in the Function of the Thylakoid

Home , Chloroplast membrane, Membrane fluidity

)

The Role of Lipid Fluidity in the

Function of the Thylakoid Membrane

by Robert Curtis Ford

Thesis submitted for the degree of Doctor of Philosophy in the

University of London, and for the Diploma of Membership of the

Imperial College of Science and Technology.

1 ABSTRACT

A fluorescence method for estimating the fluidity of the thyla-

koid membrane was developed and compared with other more establis-

hed techniques using ESR spin-label probes. The fluorescence method

was found to have several advantages over the ESR spin-labelling

technique in that only small quantities of sample were required

(equivalent to 20 micrograms Chi.) and information on both the

rate of motion as well as the static order of the lipid acyl chains

could be obtained.

Under physiological conditions a highly fluid lipid environment

was shown to exist in the thylakoid membrane for the probe DPH with

a viscosity of 0.3 poise for wobbling motions within a restricted

cone of half-angle 50°. Treatments causing a reduction in lipid

fluidity were found to inhibit photosynthetic electron flow between

Photosystem 2 and Photosystem 1 which are organized into separate

regions of the thylakoid membrane. Reduction in the fluidity of the

lipid matrix was also found to restrict salt-induced changes in

chlorophyll fluorescence which have been associated with lateral

protein movement.

The lipid matrix in stromal lamellae fractions was found to be

much less ordered (order parameter, 0.38) than the granal membrane

fractions (order parameter, 0.61). The differences observed were

interpreted in terms of the high protein:lipid ratios in the granal

fractions (1.9:1) compared to the stromal lamellae (1.2:1), and the importance of the protein:lipid ratio in the ordering of the thylakoid lipid matrix is discussed in detail.

2 Acknowledgements

I would like to thank everyone who has given help and encouragement to me during the last three years, and in particular I would like to thank Prof. James Barber, ray supervisor, whose enthusiasm for the study of photosynthesis has proved highly infectious. Much of this work would not have been possible without the helpful and patient instruction of Dr. David Chapman who has introduced me to the techniques of lipid extraction and analysis. I would also like to acknowledge the help of Dr. Raymond Cox at Odense University in

Denmark who provided facilities and expertise for the measurement of the ESR spectra of spin-labels.

Many others have contributed to this work by their constructive criticism and discussion of ideas and experiments, and in particular I would like to thank Drs. Fred Chow, Yasusi Yamamoto, Paul

Millner, Lars Olsen, Alison Telfer, Barry Rubin and Nigel Packham.

I also acknowledge the invaluable technical support of Kathy Wilson and John De-Felice.

Special thanks in particular to Alice Leeming for her help and encouragement over the last few years, and for her diligent pruf reeding.

Finally I would like to thank the Science Research Council and

Standard Telecommunications Laboratories Ltd. for financial support for this work.

3 Contents

List of Tables 7

List of Figures 8

Symbols and Abbreviations 12

1 Introduction 14

1.1 General Introduction 14

1.2 The Two Light Reactions 16

1.3 Light-Harvesting Mechanisms: The Photosystems 18

1.4 Structure of the Chloroplast 20

1.4.1 The Thylakoid Membrane 20

1.4.2 The Envelope Membrane 22

1.5 Organization of the Electron Transport Components 23

1.5.1 Inter-System Electron Transfer Components 27

1.5.1.1 Plas toquinone 27

1.5.1.2 Primary Acceptors From PS 2 28

1.5.1.3 The Cytochromes 30

1.5.1.4 Plastocyanin 32

1.5.2 Photosystem 1 Reaction Centre: P700 33

1.5.3 Reactions on the Reducing Side of PS1 34

1.5.4 Photosystem 2 Reaction Centre: P680 37

1.5.5 Reactions on the Oxidizing Side of PS2 37

1.6 The Production of ATP 40

1.7 Lipid Composition and Structure of the Thylakoid Membrane 43

1.7.1 The Use of EPR spin-labels 49

1.8 The Effect of Temperature 51

1.9 Other Experiments on Thylakoid Membrane Fluidity 52

1.10 Summary 57

4 2 Materials and Methods 59

2.1 Plant Material 59

2.2 Chloroplast Preparations 59

2.2.1 Preparation of Class 1 Intact Chloroplasts 60

2.2.2 Preparation of Class 2 Broken Chloroplasts 61

2.2.3 Determination of Chlorophyll 61

2.3 Preparation of Thylakoid Membrane Fractions 62

2.3.1 Incubation Media 62

2.3.2 Fractionation Procedure 63

2.4 Preparation of Cholesterol-Treated Thylakoid Membranes 64

2.4.1 Determination of Cholesterol 66

2.5 Isolation and Analysis of Chloroplast Lipids 67

2.5.1 Fatty-Acid Analysis 67

2.5.2 Isolation of Chloroplast Lipids 69

2.6 Preparation of Lipid Vesicles 70

2.6.1 Chloroplast Lipids 70

2.6.2 Other Lipids 70

2.6.3 Lipid Enrichment Experiments 71

2.7 Protein Analysis 71

2.8 Electron Transport Measurements 72

2.8.1 Steady-State Electron Transport 72

2.8.2 Flash-Induced Electron Transport 73

2.9 Chlorophyll Fluorescence Measurements 76

2.9.1 Salt-Induced Chlorophyll Fluorescence Changes 76

2.9.2 Modulated Chlorophyll Fluorescence 76

2.10 ESR Measurements: Spin Labels 78

2.10.1 Measurement of First-Derivative ESR Spectra 78

2.10.2 Calculation of Rotational Correlation Times 79

5 2.10.3 Calculation of Order Parameters 80

2.11 Fluorescence Polarization Measurements 85

2.11.1 Preparation of DPH-Labelled Membranes 85

2.11.2 Steady-State Fluorescence Polarization Measurements 86

2.12 Time-Resolved Fluorescence Measurements 88

3 Results 91

3.1 Lipid Fluidity Measurements 91

3.1.1 A Fluorescent Probe: DPH 91

3.1.1.1 Fluorescence Polarization Measurements with DPH 109

3.1.1.2 Steady-State Fluorescence Polarization Measurements 110

3.1.1.3 Time-Resolved Fluorescence Depolarization Measurements 113

3.1.2 ESR Spin-Label Probes 120

3.2 Lipid Fluidity and Function 130

3.2.1 Effect of Temperature 131

3.2.2 Effect of Cholesterol 133

3.2.3 Effect of Ageing on Lipid Fluidity 151

3.3 Lateral Heterogeneity in the Thylakoid Membrane 155

3.3.1 Characterization of the Thylakoid Membrane Fractions 156

3.3.2 Fluidity Measurements of Stromal and Granal Membranes 157

3.3.3 Composition of the Stromal and Granal Fractions 166

3.3.4 Lipid Fluidity and the Temperature Sensitivity of Photo- system2 170

4 Discussion 175

4.1 Comparisons With Other Biological Membranes 175

4.2 Distribution of the Probes 185

4.3 Possible Links Between Lipid Fluidity and Lateral Diffusion 187

4.4 The Effect of Lipid Fluidity on Electron Flow 191

6 4.5 Stromal and Granal Membrane Fractions 197

4.6 Future Studies 204

5 References 208

Appendix 1 235

Appendix 2 240

List of Tables

1. Effect of temperature on photosynthetic processes in higher

plants. 53

2. Effect of temperature on photosynthetic processes in algae. 55

3. Fluorescence lifetimes of DPH in thylakoid systems. 101

4. Motional parameters of DPH in thylakoid membrane systems. 119

5. Spin-label to chlorophyll ratios and order parameters in

thylakoid membranes. 130

6. Order parameters in soya lipid/cholesterol vesicles. 142

7. Order parameters in cholesterol-treated thylakoid membranes. 143

8. PS 1 and PS 2 activities in pea thylakoid fractions. 156

9. DPH fluoresence polarization in pea thylakoid fractions. 159

10. Motional parameters of DPH in pea thylakoid fractions. 160

11. Levels of protein, acyl lipid and chlorophyll in pea thylakoid

fractions. 168

12. Proteintlipid ratios in pea thylakoid membrane fractions. 168

13. Fatty-acid composition of pea thylakoid membrane fractions. 169

14. Comparison of the steady-state fluorescence polarization values of DPH in various biological membranes. 176

15. Comparison of the (time-resolved) motional properties of DPH in various biological membranes. 177

7 List of Figures

1. Diagrammatic representation of the light reactions of photosyn-

thesis. 24

2. Two-dimensional representations of a lipid bilayer in the gel and liquid-crystalline states. 45

3. Structures of the major thylakoid acyl lipids. 48

4. Flow diagram of the fractionation procedures adopted to isolate granal and stromal membrane fragments. 65

5. Block diagram of the single-beam flash spectrophotometer. 74

6. Block diagram of the apparatus used to measure modulated chlorophyll fluorescence from intact leaves. 77

7. Diagrammatic representations of first-derivative ESR spectra of fast and slow moving spin-labels. 84

8. Corrected excitation and emission spectra of DPH in various environments. 93

9. Corrected excitation and emission spectra of DPH in thylakoid membranes and soya-phospholipid liposomes. 94

10. Chlorophyll fluorescence excitation and emission spectra in the presence and absence of DPH. 96

11. Effect of increasing DPH levels on chlorophyll fluorescence.98

12. Fluorescence decay of DPH in thylakoid membranes. 102

13. Fluorescence decays of DPH in stromal and granal membrane fractions. 103

14. Lifetimes of DPH fluorescence in lipid-enriched thylakoid membranes. 106

15. Time-course of entry of DPH into the thylakoid membrane. 108

8 16. Fluorescence polarization values of DPH during the time-course

of entry into the thylakoid membrane. Ill

17. Wavelength-dependence of the steady-state fluorescence

polarization values of DPH. 112

18. Wobbling-in-cone model for the motion of DPH within a lipid

bilayer (a), and the actual anisotropy decay for the probe in

thylakoid membrane (b). 117

19. Total and difference fluorescence decays of DPH in the

thylakoid membrane. 121

20. Total and difference fluorescence decays of DPH in DGDG

vesicles. 122

21. Structures of the spin-labels used in ESR measurements of

thylakoid membrane fluidity. 124

22. Theory of ESR spectra and spin-label motion. 126

23. Removal of the ESR spin-label signal in the thylakoid membrane

in the absence of DCMU. 128

24. Steady-state DPH polarization values of DPH in thylakoid mem-

branes and DPPC vesicles at various temperatures. 132

25. Difference absorption spectra of cytochrome f^ and b^^ at 20°C

in thylakoid membranes. 134

26. Arrhenius plots of cytochromes b^g and f_ reduction kinetics under conditions of cyclic elctron flow. 135

27. Fluorescence polarization values of DPH in soya-lipid/cholesterol vesicles. 137

28. Incorporation of cholesteryl hemisuccinate into thylakoids 138

29. Incorporation of cholesterol into thylakoids. 139

30. Effect of cholesteryl hemisuccinate on the steady-state DPH fluorescence polarization from thylakoid membranes. 141 31. First-derivative ESR spectra of 5-doxyl stearate in cholesteryl hemisuccinate-treated membranes. 144

32. The effect of cholesterol on the steady-state rates of linear

electron flow. 145

33. Flash-induced cytochrome f_ absorption changes under linear electron flow and in the presence of DCMU. 148

34. Effect of cholesteryl hemisuccinate on the reduction kinetics of cytochrome f_ in the presence of different electron acceptors.148

35. Effect of cholesteryl hemisuccinate at various incubation levels on cytochrome f_ reduction kinetics in the presence of methyl viologen. 150

36. Effect of cholesteryl hemisuccinate on cytochromes b^gg JL reduction and oxidation kinetics under psuedo-cyclic electron flow.

152

37. Effect of ageing on DPH fluorescence polarization values and the salt-induced chlorophyll fluorescence increase in thylakoid membranes. 154

38. Polarization values of DPH fluorescence in stromal and granal membrane fragments. 158

39. Effect of temperature on the steady-state polarization of DPH fluorescence in stromal and granal membrane fragments. 161

40. First-derivative ESR spectra of 5-doxyl decane in stromal and granal membrane fragments. 164

41. First-derivative ESR spectra of 12-doxyl stearate in stromal and granal membrane fragments. 164

42. First-derivative ESR spectra of 5-doxyl stearate in stromal and granal membrane fragments. 164

43. Rotational correlation times of 5-doxyl decane and 12-doxyl 10 stearate in stromal and granal membrane fragments. 166

44. Arrhenius plot of the temperature-dependence of the rate of rotation of 5-doxyl decane in stromal and granal membrane fragments. 167

45. Chlorophyll fluorescence increase in heated leaves in State 1 or

State 2. 173

46. Chlorophyll fluorescence increase in heated thylakoids in the stacked or unstacked condition. 173

47. Block diagram of the nanosecond fluorescence spectrophotometer.

239

11 Symbols and Abbreviations

The following symbols and abbreviations have been used in the

text:

ANS 1-anilinonaphthalene-8-sulphonate

b6 cytochrome b^g

Chi. Chlorophyll

Choi. Cholesterol or cholesteryl hemisuccinate

(specified in the figure legends).

DAD 2,3,5,6tetramethyl p-phenylenediamine (diaminodurene)

DCIP 2,6-Dichlorophenolindophenol

DCMU 3-(3',4'-dichlorophenyl)-1,1-dimethylurea

DGDG Digalactosyl diacylglycerol

DPH 1,6-diphenyl-l,3,5-hexatriene

DPPC Dipalmitoyl phosphatidylcholine

EDTA Ethylenediaminetetra-actetate

ESR Electron Spin Resonance f cytochrome f

3- FeCN6 Ferricyanide complex

Fe.S Iron-sulphurce ntre

FWHM Full Width at Half-Maximum (intensity)

HEPES N-2-hydroxyethylpiperazone-N-2-ethanesulphonic

acid HPLC High-performance liquid chromatography MES 2-(N-Morpholino ) ethane sulphonic acid MGDG monogalactosyl diacylglycerol MV Methylviologen NADP Nicotinamide adenine dinucleotide phosphate P Polarization (of fluorescence) 12 PMS Phenazine methosulphate

PS Photosystem (1 or 2)

PQ,PQH2,PQH- Plastoquinone, Plastoquinol, Plas tohydrosemiquinone

PVP Polyvinyl pyrrolidone

P700 Reaction centre of Photosystem 1

P680 Reaction centre of Photosystem 2 r Anisotropy (of fluorescence)

THF Tetrahydrofuran

TLC Thin-layer liquid chromatography

Tricine N-tris(Hydroxymethyl) methylglycine

Tris Tris (hydroxymethyl) methylamine

13 Preface

As our knowledge of the processes involved in photosynthetic

energy and electron transfer increases, an awareness of the

dependence of these events on the physical state of the membrane in

which they take place is emerging. This is particularly true of

oxygen-evolving organisms because the transfer of electrons^ from + water to NADP requires the co-operative interaction of at least

three membrane protein complexes. Not only will the efficiency of

the process be dependent on the redox gradient, but also on the

spatial relationship between the components which must be optimised

to allow electron transfer to occur and for the two photosystems to

obtain a balanced supply of absorbed quanta under differing light-

ing conditions.

The work presented in this thesis has therefore been directed

towards a clearer understanding of the energy and electron transfer

processes in terms of the viscosity of the lipid matrix in which

the functional components are embedded. To this end, the aim which has been broadly outlined above has been approached with three main experimental objectives. a) The development of techniques for the measurement of lipid viscosity and ordering in the thylakoid membrane. b) The manipulation of the physical properties of the lipid matrix and subsequently the monitoring of electron transport and light energy distribution changes in the modified thylakoids. c) A characterization of the lipid and protein composition of two different regions of the thylakoid membrane (the stromal and granal lamellae) which contain separate photosystems, and which show large differences in the physical state of the lipid matrix.

The extent to which these experimental objectives have been achieved and the relevance of the results to the understanding of photosynthetic processes will be discussed later, however at this point it may be useful to summarize the general conclusions that have been reached from these studies.

The fluidity of the lipid matrix of the thylakoid membrane has been measured for the first time by the technique of time-resolved fluorescence polarization, and it has been possible to draw detailed comparisons between the thylakoid system and other biological membranes studied by this technique. In all respects the thylakoid lipid matrix appears to be a highly fluid system where the rapid, motion of membrane components would be possible.

Treatments which reduce the dynamic properties of the system cause an inhibition of electron transport and restrict the cation- induced energy transfer changes associated with the lateral redis- tribution of pigment-protein complexes.

Within the membrane the close interaction of lipid and protein gives rise to an increased ordering of the lipid chains in regions where higher levels of protein exist. 1. INTRODUCTION

1.1 General Introduction

"Die Pflanzen nehmen eine Kraft, das Licht, auf, und bringen eine

Kraft hervor: die chemische Differenz."

Julius Robert Mayer 1845 in NThe Organic Motion in its Relation to

Metabolism'.

This quotation from the work of Robert Mayer, describes the process which is under study in this thesis, that is the production of chemical potential from the light energy absorbed by green plants.

The history of the partial elucidation of the mechanisms taking part in this process began in the seventeenth century with the observations of van Helmont and Stephen Hales on the growth of plants, but it was not until this century that the isolation and characterization of some of the components and reactions involved was possible. From the experiments of Joseph Priestly, Jan Ingen-

Housz, Jean Senebier and Nicholas de Saussure a general equation for the light-driven process of photosynthesis was understood by the beginning of the twentieth century:

lai ts Light + C02 + H20 P ; 02 + Organic Material

The progress made in this century towards a more complete understanding of photosynthetic mechanisms has been, to a much greater degree, the product of work from a large number of scientists.

However some important discoveries particularly stand out as lan- dmarks in the development of our present day knowledge.

In 1938 Robert Hill first showed that an illuminated, cell-free

14 preparation (of chloroplasts) could produce oxygen and reduce other

compounds, in particular ferric salts. The preparation could not

reduce carbon dioxide however (Hill 1938). During the next few

years many other xHill oxidants' were found, and in 1951 Vishniac

and Ochoa showed that the pyridine nucleotide, nicotinamide adenine

dinucleotide phosphate ( NADP+) could also be utilized as a Hill

oxidant by chloroplasts.

Three years later Arnon et al discovered the process of photo-

synthetic phosphorylation; they observed that the illumination of

isolated chloroplasts resulted in the formation of high energy

phosphate bonds in the production of adenosine triphosphate (ATP)

from adenosine diphosphate (ADP) and orthophosphate (Pi) (Arnon et

al 1954). Later the same workers found that the phosphorylation of

ADP in chloroplasts was coupled with NADP reduction, and that

under different conditions, chloroplasts were able to produce ATP

at high rates without the accumulation of NADPH or the evolution of

oxygen; a process which has been termed cyclic phosphorylation.

Two processes were identified by 1958, cyclic and non-cyclic

photophosphorylation, and the two products of the %light reactions'

of photosynthesis; ATP and NADPH were recognized (Arnon et al

1958). The two products provide the reducing equivalents (in the

form of NADPH), and additional energy (in the form of ATP), to drive the reduction of carbon dioxide in the so-called Mark reactions'of photosynthesis. The term Mark reactions' was coined because these reactions can occur under certain conditions without the need of light provided that NADPH and ATP are present (Werden et al 1975). The sequence of reactions involved in the utilization of NADPH and ATP in the reduction of carbon dioxide was first

15 mapped out by Bassham and Calvin (1957) who used radioactively

labelled carbon dioxide to trace the fate of carbon through the

various intermediate sugars.

This thesis will be concerned primarily with the xlight react-

ions' of photosynthesis, that is, the events leading up to the

production of ATP and NADPH. The following pages will consist of a

resumfe of the present-day understanding of the sequence of electron

transfer processes which comprise the light reactions of photosyn-

thesis.

1.2 The Two Light Reactions

Much of the current understanding of the light reactions of photosynthesis has developed from the concept of the co-operation of two light-driven reactions in the electron transport chain. This idea was first put forward by Hill and Bendall (1960) who used it to account for the participation of cytochromes in the light reactions. They argued that if only one light reaction was necessary to reduce NADP+ at a redox potential of -0.3 V, and to accept electrons from water at a potential of +0.8 V, then any intermediates in this scheme would necessarily have redox potentials outside that range for the energetically favourable flow of electrons. They pointed out that the redox potentials of the recently discovered chloroplast cytochromes, cytochrome b^gg ( 0.0 V ), and cytochrome f_ ( +0.4 V ) would not fit into a scheme involving only one photosystem.

In 1961 Duysens and co-workers also presented a scheme based on two light reactions, and named them Photosystem 1 and Photosystem

2. They based their conclusions on observations of cytochrome _f

16 absorption changes in the red alga, Porphyridium cruentum in the

presence of red (680nm) and green (560nra) exciting light (Duysens

et al 1961). This work established the idea of the xpush-pull' nature of the two photosysterns, that is, by using far red light,

which is only absorbed by the pigments associated with photosystem

1 they were able to observe the oxidation of the cytochrome. This oxidation was reversed when excitation light of a wavelength absorbed by both photosystems was used. Similar results have also been obtained for other intermediate electron transport components such as plastoquinone (Amesz 1964), plastocyanin (Malkin and Bearden

1973) and XQ' the primary electron acceptor of Photosystem 2 the redox state of which can control the level of chlorophyll fluorescence (Duysens and Sweers 1963).

These discoveries could be interpreted in terms of earlier observations of Emerson and Lewis (1943), who studied the wavelength dependence of the quantum efficiency of photosynthesis

(mainly in algal systems). They found that the action spectrum of photosynthesis decreased very sharply at wavelengths above 680nm, much more sharply than the decrease in the absorption of the pigments which continued above 700nm. Emerson called this effect the xred drop', which is sometimes known as the xfirst Emerson effect'.

Emerson also noticed that the red drop could be shifted to much longer wavelengths by the addition of supplementary light of shorter wavelength (Emerson et al 1957), and the rate of photosynthesis was higher in the presence of both types of light than the sum of the rates when the two light sources were supplied separately. This effect is known as ^enhancement' or the 'second Emerson effect'.

17 Thus a drop in the quantum yield of photosynthesis is seen when

more of the incident light is absorbed by one photosystem, which

then becomes limited by the other photosystem, resulting in the

wastage of part of the light energy absorbed.

As will be described later, higher plants and algae have a

complex control mechanism to optimise the distribution of light

energy between the two photosystems. If artificial electron donors

are supplied to Photosystem 1, then no red drop effect is observed

as expected (McSwain and Arnon 1968, Avron and Ben-Hayyiml969).

1.3 Light-Harvesting Mechanisms: The Photosystems

The present day concept of the photosystems is as a complete

physical structure of defined conformation and size. The light-

harvesting pigments are not randomly distributed in the vicinity of

the reaction centres, but are part of highly organized three-

dimensional arrays of molecules encapsulated in large protein com-

plexes which can be isolated in a reasonably intact form (Thornber

1975, Thornber and Barber 1979, Markwell et al 1981).

The pigment molecule chlorophyll a_ is present in all oxygen-evol

ving species together with other associated pigments; chlorophyll_b

(in higher plants and green algae), chlorophyll £ (in brown algae),

and the phycobilins (in red algae and cyanobacteria). Carote .noids

are also present in most oxygen-evolving species, but these pig-

ments may have a secondary role in the protection of the photosyn-

thetic machinery against attack by free-radicals formed at high

illumination intensities.

Emerson and Arnold (1932) estimated that approximately 2,500 molecules of chlorophyll co-operate in the production of one molecule of oxygen. It is now widely accepted that at least eight

18 quanta of light are required to evolve one oxygen molecule (Emerson

1958), suggesting a value of about 300 chlorophyll molecules per

reaction centre. Emerson concluded that the majority of the pig-

ments were merely acting to trasfer excitation energy to the reac-

tion centre.

Further evidence for the %light harvesting' role of the access-

ory pigments was provided by the work of Duysens on the action

spectrum of chlorophyll fluorescence in the red-alga Porphyridium

cruentum. He found that chlorophyll fluorescence was greater with

excitation light absorbed mainly by the phycobilins rather than

with excitation light absorbed mainly by chlorophyll a_ (Duysens

1951). Duysens found that the efficiency of transfer of light

energy from the phycobilins to the reaction centre was extremely high, of the order of 100% efficient.

The physical properties of the photosynthetic pigments in vivo are greatly different from the properties of the isolated species, and this is a reflection of the unusual and varied environments in which the light-harvesting pigments are situated. Chlorophyll ji as it occurs in the chloroplast is not homogeneous; various differences in the protein environment (the degree of aggregation, or the polarity for example) can cause a shift and broadening in the spectrum so that several different forms can be identifiedj chlorophyll 670, 680, 690, 700. The longest wavelength form is almost entirely in the protein complex associated with photosystem 1, whereas the pigments such as chlorophyll _b (in higher plants) and phycobilins (in red and blue-green algae) appear to be associated with the pigment-protein complex comprising photosystem 2 (Thomas

* -often termed the fcyanobacteria1. 19 1962, Govindjee and Govindjee 1975).

1.4 Structure of the Chloroplast

Antony van Leeuwenhoek was probably the first person to notice

the presence of green vesicles within plant tissues, which he

described in two letters to the Royal Society in 1676 and 1678. In

the first letter he described the spiral chloroplast of Spirogyra,

and noted that it was not uniformly green, but that granules or

vesicles were embedded in it. The second letter described the

xgreen globules' present in grass leaves. These were remarkable

observations with the primitive microscopes in use at the time.

With the development of light microscopy and the improvement of

staining techniques in the 19th and 20th centuries it was possible

to discern some of the fine structure of the chloroplast. The

occurence of grana were noted, and it was realized that grana were

much more distinctly seen in shaded leaves rather than those gro-

wing in full sunlight (Weier 1938). The advent of electron micros-

copy allowed a much fuller picture of the structure of the chlorop-

last to be visualised.

Early electron microscope studies revealed that the chloroplast

was surrounded by a double outer membrane called the envelope.

Within the envelope there was a second membrane system of lamellar

sacs, termed the xthylakoid' membrane (Menke 1962) .

1.4.1 The Thylakoid Membrane

The thylakoid membrane encloses a space, and thus the internal

chloroplast medium is separated into two compartments, the outer medium or stroma and the intra-thylakoid space. This property of

the thylakoid membrane is vital for the production of a chemical * from the Greek, 0vAaKO£lSr)O (sac-like) 20 and electrical gradient between the two compartments of the chloro-

plast which is then used to drive the synthesis of ATP as will be

described later. The grana observed in the light microscope were

identified as being composed of appressed regions of the thylakoid

membrane stacked upon each other in the form of discs up to 1

micrometer in diameter ( see Coombs and Greenwood 1976). The granal

stacks are interconnected by non-appressed membrane regions termed

the stromal lamellae.

Freeze-fracture electron microscopy has revealed that the thyl-

akoid membrane contains large particles of diameter from 8 to 18

nanometers. The particles are thought to be pigment-protein com-

plexes, and the size and distribution of the particles is different

in the stromal and granal membrane regions (Goodenough and Staehe-

lin 1971). The large particles seen on the EFg (ex oplasmic face,

stacked) face of the granal regions are thought to be photosystem 2

units associated with light-harvesting chlorophyll protein partic-

les which are seen on the protoplasmic face, PFg (Staehelin 1975,

Simpson 1979). The smaller and more widely spaced particles seen on

the unstacked e xoplasmic (EFU) face are generally associated with

Photosystem 1 pigment-protein complexes.

The stromal lamellae and granal stacks can be separated by mechanical and detergent fractionation procedures (Boardman 1970).

It has been shown that vesicles prepared from the stromal lamellae by these fractionation techniques contain very few Photosystem 2 pigment-protein complexes whilst the granal membrane fractions are enriched in Photosystem 2 complexes. (Anderson and Boardman 1966).

More recently phase partition techniques using high polymer concen-

21 trations have been used to purify a Photosystem 2 fraction

prepared by mechanical (Yeda pressure cell) fractionation (Ander-

sson and Akerlund 1978). This Photosystem 2 fraction contains

virtually no Photosystem 1 complexes, and the vesicles formed

appear to have an inverted orientation, that is the vesicles are

xinside —out'. This has led to the theory that the xinside-out'

vesicles are formed from the appressed regions of the thylakoid

membrane stacks (Andersson and Anderson 1980). Recent models of the

thylakoid membrane have put the Photosystem 1 complexes in the

stromal lamellae and the Photosystem 2 complexes in the appressed

regions, with inter-mixing in the end-membranes of the stacks

(Andersson 1978, Barber 1980, Anderson 1980). The concept of the

physical separation of the Photosystem 1 and Photosystem 2 units has added an extra insight in the current model of the structure of

the thylakoid membrane, and has raised new problems in the understanding of the mechanism of electron flow in photosynthesis.

1.4.2 The Envelope Membrane

Early preparations of chloroplasts from higher plants lacked the external envelope membranes through harsh extraction procedures. It was realized that the presence of the outer envelope membranes was required for active preparations of chloroplasts which could fix carbon dioxide at rates comparable to those of intact leaves or plants. Chloroplasts isolated in this condition were first obtained by Jensen and Bassham (1966) .'Broken' chloroplasts lacking the envelope lose the soluble co-factors and enzymes necessary for the fixation of carbon dioxide, which can be reconstituted by the external addition of the necessary co-factors.

22 The envelope membranes do not participate in the light or dark

reactions of photosynthesis and contain no chlorophyll, rather they

appear to have a regulatory role in controlling the exchange of

metabolites between the chloroplast stroma and the cytosol. The

outer membrane is permeable to a number of low molecular weight

substances and the inner membrane acts as an osmotic barrier bet-

ween the internal chloroplast medium (stroma) and the cytoplasm

(Heldt et al 1974, Heldt 1976). Thus the envelope acts as a select-

ively permeable barrier to the concentration-gradient controlled

diffusion of cations and metabolites such as some sugars and carb-

oxlic acids. The envelope is impermeable to ATP and NADPH, and

therefore the export of energy and reducing equivalents out of the

chloroplast is facilitated by various shuttles involving dicarboxy-

lic acids and phosphorylated sugars (Heber 1974). Carbon dioxide

seems to be freely permeable, probably crossing the membrane as the gas (Gimmler et al 1974).

1.5 Organization of the Electron Transport Components

Electron transfer between water and NADP+ is often described as being mediated by a chain consisting of one PS 2 reaction centre, one PS 1 reaction centre and one complement of inter-system components (see Figure 1). This picture of an isolated photosynthetic unit (or ^quantasome') is probably misleading since much evidence has been accumulated which suggests that electrons pass through various pools of carriers, allowing the interaction of many PS 2 units with many PS 1 reaction centres. The two reaction centres do not occur in strict 1:1 proportions (Melis and Brown 1980) depending on the growth conditions and in some cases no PS 2 activity is Photosystem 1

P700 -1-0 Photosystem 2

P680* / e - X e Pheo- A B (Fe.S -0-5 phytin 2c Ferredoxin NADPH ^ (i Q A 0-0-- V cyt b563 PQ " PQH - ^ 7 2e+ 2 2e •-o Rieske 2H" < volts) Fe.S Oxygen cyt-f J. pC P7W + 05-- Evolving e Complex H 0 ^ o 2 2 Cytochrome b/f 4e; complex S-States + hV + 10- :.P680 ^hV

+ 1-5—

FIGURE 1 Diagrammati^representation of the light reactions of photo-

synthesis, incorporating two light-driven reactions in Photosystem 2 and

Photosystem 1. The major protein complexes involved are drawn as blocks.

The process is depicted against a redox scale so that the 'downhill1

flow of electrons is energetically favourable.

24 present (e.g. in the bundle sheath cell chloroplasts of C^ plants,

Woo et al 1970). Also some of the electtbn transfer components such as cytochrome f_ are present in varying proportions to the reaction centres, depending on the growth conditions (Boardman et al 1975).

At least two of the chain components have "distributive" functions

(Wood and Bendall 1976), that is they exist as pools which inter- connect different chains and have no fixed relationship with any one photocentre. These are the pools of plastoquinone and plastocyanin molecules. It is this Mistributive' function that will be closely examined later in this thesis with reference to the effect of membrane fluidity on electron transfer in the thylakoid membranes.

A third distributive mediator could be soluble ferredoxin which is localized on the outside of the thylakoid sacs which is thought to link the the iron-sulphur centres of PS 1 with the ferredoxin-

NADP reductase and possibly cytochrome b^^^ in cyclic electron flow around PS 1 (Bohme and Cramer 1972, Hauska et al 1974). Arnon has postulated that an additional role for ferredoxin may exist as an electron acceptor from PS 2 with a distributive role between different PS 2 units (Arnon et al 1981).

In summary, the idea of a complete photosynthetic unit with one complement of electron transport components and the two photosystems would now seem to be highly unlikely. Moreover, recent work from the laboratories of Andersson and Barber has led to the concept that the two photosystems are segregated into different do- mains of the thylakoid membrane, and that relatively large distances separate the two light reactions. The advantages gained by

25 higher plants by the lateral separation of the two photosystems are not clear at the present time (see Barber 1980 for a review). One possible explanation stems from the fact that the segregation of the two photosystems is more pronounced in plants grown under low light intensities and in shaded leaves where the majority of the light energy will be absorbed by Photosystem 1. The light energy available to Photosystem 2 under these conditions can be used more efficiently if the Photosystem 2 complexes are close together since they can act as a xpool' for the distribution of light energy from one Photosystem 2 unit to another. Moreover the lateral separation of Photosystem 1 and 2 units reduces the %spill-over' of excitation energy from Photosystem 2 to Photosystem 1. Thus the presence of grana stacks and the separation of the two light-driven processes of the electron transport chain may represent a control of the distribution of light energy between the photosystems.

The lateral segregation of the photosystems seems to require that at least one mobile electron carrier exists to connect the two light reactions. Possible candidates for this role will be discussed in the following section on inter-system electron transport components.

26 1.5.1 Inter-System Electron Transfer Components

1.5.1.1 Plastoquinone

The most abundant redox mediator in chloroplasts is plastoquin-

one, with a molar ratio of 1:7 to chlorophyll; about 80% of this

pool is plastoquinone A, and 20% plastoquinone C. These highly

hydrophobic compounds were first extracted from plant material by

Crane and Lester (1959) using petroleum ether. They found that the

compound, which they called Q254 (absorption max. 255nm), was pre-

sent in larger quantities than Coenzyme Q^q in leaf tissues. Using

differential centrifugation it was possible to conclude that plast-

oquinone was present in chloroplasts, whilst Coenzyme Q^q was

present in mitochondria.

Subsequent studies of plastoquinone in chloroplasts showed that

the compound was reduced when extracted after illumination, al-

though the data was very variable. Less equivocal evidence for the redox role of plastoquinone was obtained by Bishop (1959) and

Heninger and Crane (1967) who found that the photosynthetic capacity of lyophilized chloroplasts which had been extracted with petroleum ether (which removes plastoquinones and carote noids) could be restored by the re-addition of the plastoquinone extract. The conclusions from these studies were also supported by the measurement of ultra-violet absorption changes in intact membrane systems in Witt's laboratory (Klingenberg et al 1962), and by Amesz (1964).

Plastoquinone is thought to have two roles: as a two electron carrier, and also as a two proton carrier. The electrons are passed along to subsequent acceptors in the chain, but the protons, bound to plastoquinone on the outside of the thylakoid sac, are released

27 on the inside (where the plastoquinol is oxidized) to contribute to

a high-energy proton-motive force. Thus the polar head-groups of

the plastoquinone molecules must cross the thylakoid membrane in

some fashion, a function which probably requires mobility and a

fluid environment for the molecule.

1.5.1.2 Primary Electron Acceptors from PS 2_

The position of plastoquinone as an electron acceptor after PS 2

is now well established, although it does not accept electrons

directly from PS 2. The existence of a primary electron acceptor,

xQ'-the quencher of chlorophyll fluorescence was first deduced by

Duysens and Amesz (1962) in their experiments on the effect of far-

red light on chlorophyll fluorescence from PS 2. The identity of

XQ' is still unknown, however it may be a quinone molecule, tightly

bound to protein as a semiquinone anion (Van Gorkom 1974). The

redox state of XQ' controls the chlorophyll fluorescence from PS

2, that is, if XQ' is largely reduced then the electron xtrap' of

PS 2 is closed and because charge separation by the reaction centre

chlorophyll cannot be stabilised the energy is wasted as fluoresce-

nce. The redox potential of XQ' was first determined by Kok using

chlorophyll fluorescence measurements and by measuring the quantum

yield of photoreduction of Hill oxidants having different redox

potentials in a Scenedesmus mutant lacking PS 1 (Kok and Chenaie

1966, Kok and Datko 1965). The results in both cases agreed at a

potential of +180 mV.

Another component associated with the electron accepting side of

PS 2 has been identified by Knaff and Arnon (1969). This species, known as C550, shows a light-induced oxidation followed by an absorption change at 550nm in the presence of ferricyanide (cyto-

28 chromes b^gg and f are already oxidised). The reaction is independ-

ent of temperature, occurring at ?7K , and is stimulated by PS 2

light suggesting that a primary process around PS 2 is involved.

The existence of an earlier electron acceptor before Q and C550

has been postulated, and this species may be a pheophytin molecule

close to P680 which has been detected from EPR and ENDOR spectros-

copy (see later Fujita et al 1978). Both XQ' and C550 are

thought to be bound by proteins close to the PS 2 reaction centre

in a large protein complex.

Electron flow out of PS 2 proceeds via a secondary electron

acceptor before reaching the plastoquinone pool, this acceptor,

originally known as XB' or %R' acts as a two-electron and proton

accumulator. The reducing equivalents are then passed on to the

plastoquinone pool. XB' was discovered by Bernadette Bouges-Bouquet

(1973) who observed oscillations with a periodicity of two in the

number of electrons transferred from PS 2 to the plastoquinone pool

after a series of short saturating flashes of light. Velthuys and

Amesz (1974) independently identified the same component which they

called XR'. Their experiments involved the measurement of the

increase in chlorophyll fluorescence on the addition of dithionite

or DCMU (to reduce XQ') following a series of short light flashes.

The percentage increase in chlorophyll fluorescence was thus a measure of the redox state of XQ', which was found to vary with a periodicity of two. The scheme they devised to explain the results is shown below:

29 FLASH No 0 1 2 3

Redox State of Q RED OX RED OX

Reaction Q .R Q .R Q .R Scheme U U _ Q.R- Q.R Q.R*2Z-

PQ PQ2'

More recent modifications of this scheme have suggested that two

plastoquinone molecules are involved in the transport of electrons

away from PS 2, Qa and Qb (Velthuys 1981). In this scheme Q is

represented by Qa, and is able to pass one electron at a time to Q^

which is a two-electron and proton carrier. Q^ is only bound in the

semi-reduced form, and after the addition of two electrons and

protons is released into the plastoquinone pool.

1.5.1.3 The Cytochromes

Electrons from the plastoquinone pool are passed on to cytochrome components of the electron transport chain. This process is probably mediated by a protein complex often termed the complex' which contains an iron-sulphur centre, named the Rieske centre which was first discovered in chloroplasts by its electron paramagnetic resonance signal at g=1.90 (Malkin and Aparicio 1975).

The mid-point redox potential of the Reiske centre was also determined and found to be about +290 mV (pH independent). Cytochrome components are also present in this complex which has not been fully characterized yet, although it appears that cytochrome f_ and cytochrome b^g are tightly bound to this complex. Cytochrome bc^

30 (low potential form) has been identified in impure preparations of

the b^/f complex (Nelson and Neumann 1972), although it is not

present in more purified particles (Hurt and Hauska, 1981).

The sequence of electron transfer between the cytochromes has

been the subject of much debate and controversy since they were

first identified in plant material. Cytochrome f_ was purified from

higher plant chloroplasts by Davenport and Hill (1952); they ident-

ified it as a xc'-type cytochrome, that is, the haem group is attached to the protein by stable (covalent) linkages. The cytochrome also had a high redox potential (+365mV), similar to previously isolated c-type cytochromes. Cytochrome £_ is thought to donate electrons to plastocyanin, the immediate electron donor to

PS 1, although there is still some controversy as to whether cytochrome f_ can also donate electrons directly to PS 1 (Malkin et al

1973, Trebst 1974).

The roles of cytochrome b^g and the two forms of cytochrome

^559 (kigh and low potential types) are much less clearly understood than the role of cytochromef^ although all of these cytochromes show light-induced redox changes.

Cytochrome b^g was discovered in chloroplasts by Davenport

(1952), who noticed that a sharp band in the absorption spectrum of barley chloroplasts was produced at 563nm when a trace of reducing agent (sodium hyposulphite) was added. The addition of a small amount of ferricyanide caused the removal of the absorption band as the cytochrome b^g was completely oxidised. Hill and Bendall

(1965) found that the redox potential of cytochrome b^g was about

0 mV, placing it before cytocH5me f in a linear electron transport

31 scheme between the two photosysterns. Today, cytochrome b^^ is

generally associated with cyclic electron flow around PS 1, poss-

ibly being involved in a %Q'-cycle type scheme with plastoquinone

(Simonis and Urbach 1973, Arnon and Chain 1979, Olsen et al 1980b).

The cytochrome b^g was first identified in spinach chloroplasts by Cramer and Butler (1967) who looked at light-induced absorption changes. They called the cytochrome ^^q', and suggested a role for it near PS 2. In fact two forms of cytochrome b^^^ exist in the chloroplast, the low—potential form (E° of about 0 mV), and the high-potential form with a very high redox potential for a b-type cytochrome (+370 mV). The high-potential form is converted into the low-potential cytochrome b,^ by heating, detergent extraction and other extraction procedures (de la Rosa et al 1981). These observations account for the wide disagreement in the early reports of the redox potential of cytochrome b^g (Bendall 1968, Hind and

Nakatani 1970, Fan and Cramer 1970, Knaff and Arnon 1971). It is thought that the high-potential form of cytochrome b^g has a role in electron transport around PS 2; the cytochrome is often present in preparations enriched in PS 2 reaction centres.

1.5.1.4 Plastocyanin

Plastocyanin is a blue coloured copper-containing protein which can be readily isolated from chloroplasts after sonication or detergent treatment. Photosystem 1 preparations obtained by detergent or sonication treatment have reduced NADP+-reducing activity unless plastocyanin is added back. The redox potential of plastocyanin is measured by EPR spectroscopy since the visible absorption spectrum is too broad; Malkin et al (1973) have published a redox potential of +340 mV for plastocyanin. Evidence for its close 32 association with PS1 comes from the similarities between the red-

uction kinetics of PS 1 (P700+) and the oxidation kinetics of

plastocyanin (Trebst 1974, Olsen et al 1980a).

Plastocyanin is a soluble protein, located on the inside surface

of the thylakoid membrane sacs, it is feasible that it could be a mobile electron carrier between the two photosystems, although its

reaction kinetics should not be affected by membrane fluidity, but possibly by other factors such as the viscosity and surface charge density of the region close to the PS 1 reaction centre (Olsen and

Cox 1982).

Indirect evidence for the possible role of plastocyanin as a mobile electron carrier in photosynthesis has come from studies on the distribution of cytochromes b^g and _f in inside-out vesicles and in stromal lamellae vesicles (Cox and Andersson 1981, Nakatani et al 1982). It has been shown that the cytochromes b^g and _f are approximately evenly distributed between stromal and granal regions of the thylakoid membrane, which possibly suggests that a subsequent electron acceptor (plastocyanin) is responsible for the distribution of electrons from the b^/f complexes in the appressed regions of the grana to the PS 1 units in the stromal lamellae and end-membranes. More recently, however, highly purified inside-out vesicles have been prepared by phase-partition methods which contain very low levels of the cytochromes b^g and f_ (Henry and

Moller 1981).

1.5.2 Photosystem J_ Reaction Centre: P700

Bessel Kok (1956) was the first to observe a light-induced

33 absorption change around 700nm in chloroplast preparations. The

signal was further characterized by the same worker, who was able

to isolate a detergent-solubilised complex containing the chloro-

phyll a_ pigment form absorbing at 700nm, which he called xP700'(Kok

1961). In this work he found that P700 had a redox potential of

+410 mV. Later investigations of the redox potential of P700+ have

shown that a slightly higher potential of about +490 mV is observed

with milder extraction procedures (Setif and Mathis 1980).

The identity of the P700 species has been investigated in the

laboratory of Katz using electron-spin resonance (ESR) and electron

-nuclear double resonance (ENDOR) spectroscopy. They compared

'Signal 1'- the photo-electron spin-resonance signal associated

with the primary charge separation in PS 1 (Beinert et al 1962)

with ESR signals from various chlorophyll species which they had

synthesised (Norris et al 1971, Norris et al 1974). They concluded

that Signal 1 was consistent with an unpaired spin derealization over two chlorophyll molecules, suggesting that P700 is due to a chlorophyll dimer.

1.5.3 Reactions on the Reducing Side of PS 1

Most of the evidence for electron acceptors on the reducing side of PS 1 has been obtained from electron paramagnetic resonance (EPR) spectroscopy of intact thylakoid membranes and PS 1 detergent particles. Malkin and Bearden (1971) identified a group of light- induced EPR signals with g-values of 2.05, 1.94 and 1.86 which were characteristic of a reduced iron-sulphur protein. These signals are very similar to the signals of ferredoxin (although the ferredoxin had previously been washed out of the chloroplast preparation);

34 therefore it was concluded that the signal arose from tightly-bound

xferredoxin' close to the PS 1 reaction centre. The observations of

Malkin and Bearden were confirmed by other workers (Evans et al

1972), and it was shown that the %bound ferredoxin' was similar to

the iron-sulphur protein showing light-induced absorption changes

at 430nm (P430) which was first observed in PS 1 particles (Hiyama

and Ke 1971). The bound ferredoxin seems to have the properties

required of an electron acceptor from PS 1; the redox potential of

the species was shown to be about -530 mV (Lozier and Butler 1974).

The EPR signals of the iron-sulphur centre is only seen at very low

temperatures (less than 20 K) since recombination with P700 is

very rapid at higher temperatures, nevertheless, a correlation

between the decay kinetics of P700+ and the EPR signal has been

made (Ke et al 1974).

The iron-sulphur centre EPR signal is today allocated two diff-

erent identities, centre A and centre B, with slightly different

redox potentials (-550 mV for A,-590 mV for B), however it is not

clear whether the two centres act in series or in parallel (Heath-

cote and Evans 1981). The centres A and B are not reduced at more

oxidizing potentials than -500 mV, and at about -560 mV the EPR

signal with g values at 2.05, 1.94 and 1.86 occur, corresponding to

the reduction of centre A. At lower potentials the reduction of centre B occurs, with the appearance of EPR signals at g = 1.92 and

1.89 (Malkin 1982).

A more direct electron acceptor from P700 called xX' was detected with lower redox potential than centres A and B estimated to be about -730 mV by Evans et al 1975. The component xX' may also be an iron-sulphur centre similar to centres A and B (Golbeck et al 1978), however the reduction of X by P700 at liquid helium temperatures is reversible, which is not the case for centres A and B. The characteristic EPR signals of X are at g-values 2.08, 1.88 and

1.78.

Evidence for an intermediate electron acceptor, A^, between X and P700 has come from the detailed analysis of the kinetics of charge separation in Photosystem 1 (Sauer et al 1978). EPR and optical analysis of A^ has suggested that it is a monomer chlorophyll molecule or possibly a pheophytin molecule (Baltimore and

Malkin 1980, Fujita et al 1978, Fajer et al 1980).The redox potential of this species can only be roughly estimated since chemical reducing agents with a sufficiently low potential are not available; Fujita et al (1978) have assigned a value of -880 mV to the

A^ species.

All of the above membrane-bound electron transfer processes occuring on the reducing side of PS 1 which have been described proceed at very low temperatures suggesting that primary events are involved.

A summary of the known electron transfer events on the reducing side of Photosystem 1 is presented below:

1 * P700 P700+

Aj —• X —• centre B

-880 mV -730 mV\ -590 mV

centre A ^ ferredoxin » NADP

-550 mV -420 mV -320 mV

36 Electron transport proceeds from the primary acceptors via the

soluble proteins ferredoxin and ferredoxin-NADP-reductase located

on the outside of the thylakoid membrane before reaching the final

electron acceptor, NADP+. The reducing equivalents are then used in

CO2 fixation and other reductive pathways or are re-cycled in

cyclic electron transport for the production of ATP.

1.5.4 Photosystem 2 and Reaction Centre: P680

Knowledge concerning the reaction centre of photosystem 2 is

poor in comparison to the degree of understanding about photosystem

1, however some advances towards the characterization of the react-

ion centre has been possible since the flash-induced signal at 680

nm was identified in Witt's laboratory in 1967 (Doring et al 1967,

Doring et al 1969). The direct detection of the photosystem 2

reaction centre by EPR spectroscopy has been achiieved in chloro-

plast preparations lacking PS 1 activity. This has been achieved in

two ways: using mutant species which lack PS 1 (Nugent et al 1980),

and by the use of oxidizing agents which will completely oxidize PS

1 but not PS 2 (Bearden and Malkin 1973).

1.5.5 Reactions on the Oxidizing Side of PS 2

Signal 2 is the broad EPR signal centred around a g-value at

2.0043 with a width of 20 gauss and partially resolved hyperfine structure typical of quinone-type radicals which has been associated with the donor side of PS 2 (Babcock and Sauer 1975). This signal has also been identified in mutants lacking PS 1 (Bishop

1964, Weaver and Corker 1977, Nugent et al 1980), with three distinct kinetic components, slow (due to back-reactions) and fast and very fast (due to donation to P680+) (Babcock and Sauer 1975).

37 The mechanism of electron flow from the initial electron donor

(l^O) to the PS 2 reaction centre is not clear, although some

progress has been made from the work of Joliot and Kok on the kinetics of oxygen evolution (Joliot and Kok 1975). According to

Kok et al (1970) the donor side of PS 2 goes through a light-

induced process consisting of four steps in a cycle with five oxidation states, the 'S-states'. These states were detected by

Joliot et al (1969) who measured the evolution of oxygen from dark pre-treated chloroplasts after giving a sequence of flashes. The maximum yield of 02 was obtained after the third flash, with a subsequent periodicity of four. The model of Kok et al (1970) which has been used to explain these results is presented below. The dark adapted state is assigned the S-state, Sp which Kok envisaged as the accumulation of one positive charge. The S-states may not represent positive charge accumulation in sequence, since protons are also released at certain steps in the cycle which may neutralize the charge.

The S-State Model

Flash 1 Flash 2 Flash 3 DARK Flash 4

S1 ^ S2 S2 ^ S3 S3 ^ S4 S4 ^ S0 S0 ^ S1 r + °2

The oscillations in the evolution of oxygen become damped, the oxygen yield being about equal for all flashes of light after the

25th flash. The damping appears to be partly due to 'double turn- overs' of the same reaction centre during the course of the flash, since flashes of longer duration increase the damping of the osc-

38 illation (Joliot and Kok 1975). Damping of the oscillations may

also be due to back reactions between S-states.

The discovery of the S-states by Joliot has been crucial to the

identification of components of electron transport on the donor

side of PS 2. Many of the light-induced EPR signals and absorption

changes assigned to these components undergo oscillations with a

periodicity of four (Velthuys 1981, Siderer and Dismukes 1981).

Unfortunately the electron transport components identified by their

light-induced EPR or spectral changes have not been biochemically

isolated and their structural identity remains unknown. Much effort

has been spent in recent years in an attempt to identify the struc-

ture of the oxygen-evolving system; a possible role for manganese

has frequently been suggested. Recent advances in the preparation

of oxygen-evolving detergent-extracted particles may lead to the

resolution of this problem (Stewart and Bendall 1979, Berthold et al 1981).

The possible effect of membrane fluidity on the reactions on the oxidizing side of Photosystem 2 will be discussed later, however it is worth noting at this point that PS 2 activity is particularly sensitive to high-temperature stress (above 30^C), and that this sensitivity appears to be due an irreversible denaturation of the water-splitting complex. Recently thermophilic blue-green algae have been isolated that have optimum temperatures for oxygen evolution above 50®C, and can survive in hot springs at temperatures as high as 75°C (Yamaoka et al 1978). These blue-green algae have an unusual membrane system in that the lipids which comprise it are highly saturated, suggesting a rigid membrane at room temperatures. It could be that the stability of the oxygen evolving system in these organisms is dependent on the fatty-acid composi-

tion, and hence the fluidity of the membrane system.

1.6. The Production of ATP

One of the products of the light reactions of photosynthesis is

stored chemical energy in the form of ATP which is subsequently

used in the fixation of carbon dioxide and in other energy-

requiring processes occuring in the cell. The reaction:

ADP + ?± — ATP + H20

requires the input of energy, equivalent to a redox potential of

about 550 mV per molecule of ATP. The inter-system electron

transport chain from Q~~ to P700+ can account for about 400 mV per

electron transferred (see Fig.l). The extra energy needed to

produce one molecule of ATP is probably provided by the electron

transfer events on the oxidising side of PS 2 and on the reducing

side of PS 1. As shown in the scheme below, if less than one ATP

molecule is synthesised per electron transferred by the light

rections, then there will not be enough ATP available to effective-

ly Utilize the NADPH in the fixation of C02.

+ H20 02 + 4H + 4e~——2NADPH2 + 4 ATP 1ATP I (other processes) light 2NADPH2 + 3ATP

C02 " ^ (CH20)n

Extra ATP may be provided by cyclic electron flow around PS 1 which produces ATP without the overall accumulation of NADPH.

The mechanism of phosphorylation of ADP to produce ATP in the chloroplast (and mitochondrion) has been the subject of great

40 controversy over the last twenty years. The hypothesis which has

gained the most credence was first put forward by Peter Mitchell

(1961). In this model the formation of ATP is driven by an electro-

chemical potential gradient which is formed across the thylakoid

membrane by the translocation of hydrogen ions which occurs during

photosynthetic electron flow. As long as the thylakoid membrane has

a low permeability to hydrogen ions the energy stored in this way

will be long lived, and can be harvested by allowing the hydrogen

ion gradient to be dissipated by a specific enzyme, an ATP-synthe-

tase.

Many observations have now been made which support the basic

concepts involved in Mitchell's hypothesis. An ATP-synthetase has

been isolated from chloroplast membranes by McCarty and Racker

(1966). Thylakoid membranes take up hydrogen ions in the light

(reversed in the dark)^and this process is closely linked

with electron transport and ATP synthesis (Jagendorf and Neumann

1964). Jagendorf also showed that an artificially created hydrogen

ion gradient across the thylakoid membrane will induce the form-

ation of ATP (Jagendorf and Uribe 1966). Treatments that make the

thylakoid membrane leaky to hydrogen ions also inhibit ATP synth-

esis, and by removing the mass-action effect of the hydrogen ion

gradient on the electron transport processes, these treatments

accelerate (or uncouple) electron flow.

A small electrical potential also exists across the membrane

(Witt 1975), of the order of 100 mV, or less, but it is not as

large as would be expected from the unequal distribution of hydrogen ions. There is an exchange of cations for hydrogen ions across

41 the thylakoid membrane which tends to neutralize the charge build- up, however a small potential exists because cations do not cross the membrane as rapidly as the hydrogen ions (Barber 1972).

The synthetase requires a stoichiometry of two or three protons per molecule of ATP synthesised, and therefore the number of protons translocated across the thylakoid membrane per electron passed along the chain must be at least two to arrive at one ATP molecule per electron. The hydrogen ion to electron ratio (H+/e") has recently been investigated in thylakoid membranes, with two as the generally accepted value, (Junge and Auslander 1973, Olsen et al

1980b) although some reports have shown ratios as high as three

(Olsen and Cox 1979, Fowler and Kok 1976). The higher H+/e" ratios are interpreted by the suggestion that an extra proton-motive cycle

(Q-cycle) involving plastoquinone is in operation (Crowther and

Hind 1980).

In conclusion it appears that at least half of the hydrogen ion gradient in the chloroplast is created by the diffusion of plastoquinone molecules across the thylakoid membrane. It therefore seems extremely important that an understanding of the effect of the physical properties of the thylakoid membrane on these processes is arrived at.

42 1.7 Lipid Composition and Structure of the Thylakoid Membrane

In 1972 Singer and Nicolson put forward a model, the "fluid-

mosaic model' which has gained considerable acceptance as a working

hypothesis for the general underlying architecture of natural mem-

brane systems. In this model membrane proteins are embedded in a

matrix consisting of a double layer of lipid molecules, with the

polar head-group region of the lipids facing out into the aqueous

medium. Membranes will therefore have a hydrophobic core comprised

of the fatty-acyl residues of the lipids, and a hydrophilic region

on both sides, with proteins penetrating into or through the lipid

bilayer (see Cherry 1976 for a critical review of this model).

The thylakoid membrane is unusual in its lipid composition and

in its high protein content, and the early studies of the membrane by low-angle X-ray diffraction suggested that its structure was not like the lipid bilayer architecture of other membranes studied up to that point (Kreutz 1970). Later studies of the thylakoid membrane using X-ray diffraction, however, were interpreted as showing a usual type of membrane in which protein was embedded to various extents in a lipid matrix (Sadler et al 1973). Sadler and co- workers found a repeat unit of 16.5 nm from X-ray diffraction studies of Euglena stacks, consistent with two membranes separated by a small space in the partition region, and a large space within the sac. Sadler and Worcester have found a repeat unit of 17 nm in the same organism by using neutron diffraction measurements. They concluded that water was able to penetrate inside the sacs and into the partition region (Worcester, 1976).

The thylakoid membrane has an unusual lipid composition, with the majority of the lipids being non-polar, galactolipids with

43 highly unsaturated (double-bond containing) fatty acyl chains (see

Quinn and Williams 1978 for a review).

The effect of the degree of unsaturation of the acyl chains on

the phase-behavior and structure of lipid molecules was first

investigated by Byrne and Chapman (1964). They used differential

scanning calorimetry to look at melting transitions in solid lipid

samples, and found that two transitions were present, the true

melting transition (around 230°C) which was not affected by the

degree of unsaturation in the acyl residues, and a sharp transition

occur.rin^at lower temperatures which was highly dependent on the

chain length and unsaturation. The transition occurringat lower

temperatures is due to a co-operative 'melting' of the acyl chains,

i.e. the packing of the chains changes from a highly ordered,

closely packed state with mainly all-trans conformation (the gel-

state, see Fig.2) at low temperatures, to a less ordered and wider

spaced structure with many cis conformations in the acyl chain (the

liquid-crystalline state). Thus, lipid acyl chains containing doub-

le bonds are not able to pack as closely as saturated chains and

the gel state is not as stable, resulting in a broader gel-liquid

crystalline transition which occurs at lower temperatures (Umemura

et al 1980, Chapman and Wallach 1968). The gel to liquid-crystalline transition has been studied by a variety of techniques in great detail, and it is characterized by a sudden decrease in the ordering of the lipid chains, a change in the permeability of the membrane, and a decreased 'microviscosity' (see Jahnig 1979).

The melting transition temperatures obtained by Byrne and Chap- man (1964) were very high (around 130°C) because very little water

44 GEL STATE

LIQUID-CRYSTALLINE STATE

FIGURE 2 Two-dimensional representations of a lipid bilayer in the gel state (below the phase transition temperature), and in the liquid- crystalline state (above the transition temperature). The fatty-acyl chains are shown as the hatched lines, and the polar head group region is represented as a circle. In the gel state the trans-conformation predominates, whilst in the liquid-crystalline state, cis-conformers also exist (see above). 45 was present in their samples, it was not until later that the

importance of the degree of hydration of the lipid sample was

realized.

Gel to liquid-crystalline phase transitions have been detected

in natural membranes although they are very broad in comparison to

artificial bilayers or pure lipid. The broadness of the transition

is due to the great degree of heterogeneity in natural membranes

which contain many different lipids, proteins, and sterols.

The effect of cholesterol on artificial bilayer systems has been

studied in great depth (Chapman 1973, Umemura 1980, Kawato et al

1978, Jahnig 1979), and it has been shown that cholesterol has a

condensing effect on the lipid acyl chains above the gel to liquid

-crystalline transition temperature, whilst it has a disordering

action in the gel state. The overall effect is to broaden the gel

to liquid-crystalline transition and to reduce the fluidity of the

membrane in the liquid-crystalline state.

Some of the structures of the lipids forming the thylakoid

membrane are shown in Figure 3; also shown are the relative prop-

ortions of the fatty-acyl residues which form the hydrophobic

interior of the membrane. The high content of unsaturated acyl chains suggests that any gel to liquid-crystalline phase transition in the lipids would occur at very low temperatures below 0°C.

Virtually no sterols are present in the thylakoid membrane, and this coupled with the high degree of unsaturation of the acyl chains suggests that the lipid matrix of this membrane will be highly fluid (Benson 1964).

Only a few studies have been directed towards an understanding of the overall physical properties of the thylakoid membrane in

46 FIGURE 3 Structures of the major acyl lipids composing the thylakoid membrane in pea chloroplasts:

The combination of the different polar head groups with the different fatty-acyl chains results in at least 250 possible molecular species, and so the head-groups and chains are drawn separately.

The fatty-acid composition of the pea thylakoid membrane is

* (approximately) :

18:3 18:2 16:0 18:0 16:1 18:1 65% 15% 14% 3% 2% 1%

N.B. Two isomers exist for 16:1, palmitoleic and trans-hexadecan- oic acids with cis and trans double bonds respectively.

The polar head-group composition for pea thylakoids was found to be the following*:

Monogalactosyldiacylglycerol, 33%; digalactosyldiacylglycerol, 24%; phosphatidylcholine, 17%; phosphatidylglycerol, 9%; sulphoquinovosyl-

diacylglycerol, 8%; other phospholipids, 9%.

* The data was averaged over summer and winter, and was taken from

Chapman et al, 1982.

** The hydroxyl group lies below the six-membered ring in sulpho- quinovosoyl diglyceride.

47 POLAR GROUPS:

( a)GALACTOLIPIPG CH2~0-X X= -H (MONOGALACTOSYL DIGLYCERIDE)

OH X=-SO H (SULPHOOUINOVOSYL- DIGLYCERIDE) Hy CH2OH

OH X= OH

OH (DIGALACTOSYL DIGLYCERIDE) (b) PHOSPHOLIPIDS

— P-O— CH CH N(CH ) (PHOSPHATIDYL CHOLINE) i 2 2 3 3 0- 0 II P— 0 — CH_CH(OH)CH_OH (PHOSPHATIDYLGLYCEROL) I 2 2 0-

POLAR GROUP FATTY ACYL CHAINS: I

R = ^^^ Where 2 and 3 are different chains

/ / /

- \ I C=0 C-0 C=r 0 c=o

CH. V CH3 ™3 cis trans Lino 1 eni c Linoleic: Oleic Stearic Palmi tic 16:1 18:3 18:2 18:1 I 8:0 lb:0

48 higher plant chloroplasts, and these have mainly used ESR spin- labels, although some studies have utilized X-ray diffraction measurements. There are also very few reports of the use of fluorescent probes to investigate the physical structure of the thylakoid membrane (see Kraayenhof 1980). Hydrophobic fluorescent probes such as 12-(-9-anthroyl)-stearate (12-AS) have been used to monitor the high-energy state in chloroplasts (Vandermeulen and Govindjee 1974,

Kraayenhof et al 1975), by looking at changes in the fluorescence intensity from the probe. Experiments involving the use of fluorescence polarization of probe molecules introduced into the thylakoid membrane to provide information on the fluidity and structure of the thylakoid lipids have not previously been reported in the literature.

Other techniques have been used to try to probe the structure of the thylakoid membrane, such as the use of chlorophyll a_ fluorescence by Murata and Fork (1975) as an indication of lipid phase transitions in algae and higher plants. These experiments were not based on the known behaviour of other fluorescent molecules dispersed in lipid systems and it seems unlikely that significant amounts of chlorophyll a_ will be present in the lipid matrix of the thylakoid membrane (Thornber and Barber 1979). Nevertheless it may be feasible that protein-bound chlorophyll pigments are in some way sensitive to the physical nature of the thylakoid lipid matrix.

1.7.1 The use of ESR Spin-Labels

The degree of motion of a free-radical group attached to a suitable hydrophobic molecule that will freely partition into natural membranes has been used to provide detailed evidence on the

49 environment of the lipid molecules in natural and artificial mem-

branes. Hiller and Raison (1980) have recently used a variety of

spin-labelled fatty-acids to probe the fluidiy gradient in the

thylakoid membrane. They found that the thylakoid membrane was

similar to other natural and artificial membrane systems in that

the hydrophobic core of the membrane was relatively fluid in compa-

rison to the rigid region close to the polar head-groups. Other

workers have looked at the partitioning of spin-labelled probe

molecules into the thylakoid membranes which is highly dependent on

the fluidity of the membrane (Torres-Pereira et al 1974). They

found that the partitioning of the spin-label 2,2-dimethyl-5,5,

dipentyl-N-oxyloxazolidine (5-doxyl decane) into the thylakoid

membrane was much greater at higher temperatures.

Raison and co-workers have also used EPR spin-label probes to look at possible phase changes in higher plants and algae (Raison They

1973). report that break-points or discontinuities in Arrhenius plots of spin-label motion only occur in chloroplast membranes derived from chill-sensitive species such as tomato and maize.

These discontinuities occur- at temperatures where chilling damage begins in the plants, and were explained in terms of a lateral phase separation of membrane lipids and proteins into two distinct regions. One region separates into the gel-state from which membrane proteins are excluded, and the other region consists of fluid lipid containing the protein. This concept of a lateral phase-separation of membrane components in chill-sensitive plants has been widely accepted, however such separations have only been observed (by freeze-fracture electron microscopy) in algal cell membranes which contain much higher levels of unsaturated lipids than chill-

50 sensitive plants (Verwer et al 1978, Williams et al 1981). Phase

separations in some strains of Anacystis nidulans, a

blue-green alga, have been closely studied, and have been shown to

depend on the growth temperature (Furtado et al 1979).

1.8 The Effect of Temperature

The effect of temperature on the photosynthetic reactions of

chloroplasts and whole plants has now been studied for many years,

with the conclusion that both extremes of temperature have an

inhibitory effect on photosynthesis, but that this inhibition occ-

urs over a broad range of temperatures depending on the plant

species and the growth conditions (see Berry and Bjorkman 1980 for

a review).

Many papers have appeared on the subject of temperature effects

on photosynthetic reactions, some of them reporting xbreak-points'

in the Arrhenius plots of a variety of photosynthetic reactions.

These discontinuities have often been interpreted as being due to phase separations (Raison 1973) in the lipid matrix of the membrane, although the corresponding measurements of membrane fluidity are often not included. Table 1 gives a summary of some of the data relating to the effect of temperature on photosynthetic reactions in higher plants, and Table 2 summarizes some of the recent work on algae which show much more well defined phase separations as described earlier. The large degree of variation in the reported temperatures at which Arrhenius discontinuities or %break-points' occur in the rates of various chloroplast processes in higher plants is apparent from Table 1. In at least one of the studies described in Table 1 the log-plots of various photosynthetic para-

5 1 meters could be interpreted equally well as a smoothly changing

curve, rather than as two straight lines of different slope (see

Addendum in Nolan and Smillie 1976). This reservation about the

interpretation of data presented in an Arrhenius plot coupled with

the fact that few of the studies on higher plants were complemen-

ted by membrane fluidity measurements suggests that the evidence

for lateral phase—separations in the thylakoid membranes of chill-

sensitive higher plants is not convincing.

In thermophilic algae the evidence for lateral phase separations

within the membrane is strong and can be seen with freeze-fracture

electron microscopy. The break-points in the Arrhenius plots of

photosynthetic rates are much more consistent in the temperatures

at which they occur than the reported break-points in higher plants

(see Table 2). The effect of the physical phase and fluidity of the algal membrane on photosynthetic processes seems to be well characterized.

1.9 Other Experiments on Thylakoid Membrane Fluidity

It is important to study the effect of the fluidity of the lipid phase on electron transport processes without the complicating factor of temperature. Most enzymic reactions proceed more slowly at lower temperatures and generally follow the rule that for every ten degrees centigrade rise in temperature the rate of reaction increases by two-fold or more. This law only applies over a limited temperature range for enzymic reactions since at high and low temperatures an irreversible conformational change in the protein will take place leading to an inactivation of the enzyme complex.

The reported break-points in the rate of photosynthetic processes could therefore represent not phase-separations in the lipid phase Table _1 Summary of the reported experiments on the effect of temperature on various photosynthetic processes in higher plants. The observation of Arrhenius discontinuities is recorded.

Key: References: (1) Cox 1975, (2) Graeber and Witt 1974, (3)

Nolan and Smillie 1976, (4) Yamamoto and Nishimura 1976, (5)

Shneyour et al 1973, (6) McEvoy and Lynn 1972, (7) Murata et al

1975

(R) - Chill Resistant Plant

(S) - Chill Sensitive Plant

*- Higher temperature break-point due to irreversible inactivation of PS 2

53 PROCESS SPECIES TEMPERATURE(S) REF, STUDIED OF BREAK-PT(S) (°C)

H20 - DAD Spinach(R) 3

H20 - MV Spinach -13

515nm absorption change Spinach 18

H20 - DCIP Barley(R) 9,20,29'

H20 - DCIP + uncoupler Barley 9,29* * H20 - Fe(CN)6 Barley 9,29 + H efflux Spinach 17 H+ efflux (+PMS) (cyclic phosphorylation) Spinach NONE

H20 - NADP Lettuce(R),Pea(R) NONE

H20 - NADP Bean(S),Tomato(S) 12

H20 - DCIP Lettuce, Pea, Bean, Tomato NONE

H20 - Diquat Lettuce, Pea, Bean, Tomato NONE

H20 - Fe(CN)6 Spinach 17

PS1 - Fe(CN)6 Spinach-digitonin fraction NONE

Phosphoryl- ation (+PMS) Spinach 17

H20 - DCIP Lettuce NONE

PS2 - P700 Lettuce NONE

MgCl2-induced fluorescence rise Lettuce, Spinach NONE

54 Table 2. Summary of experiments on the effect of temperature on photosynthetic processes in algae. Observations of Arrhenius discontinuities are recorded with the growth temperature.

PROCESS SPECIES GROWTH TEMP. OF REF. TEMP.°C BREAK PT(S)°C

P700+ reduc- Anacystis tion nidulans 38 (28) 20 (10) 1

P700+ redn. Anacystis (+ DCMU) nidulans 38 (28) 20 (12) 1

H20 - DCIP Anacystis 38 (28) 24 (13) 1

Statel-State2 Anacystis transition 38 (28) 22 (13) 1

H20 - MV Synechoccus 55 29,11 2

H2O - co2 Synechoccus 55 29,11 2

PQ - cyt553 Synechoccus 55 57,29 2 cyt553 ~ P70° Synechoccus 55 NONE 2

DCIPH2 - P700 Synechoccus 55 NONE 2

H20 - Fe(CN)6 Synechoccus 55 30,9 2

H20 - Fe(CN)6 (+ DCMU) Synechoccus 55 NONE 2

Statel-State2 Anacystis transition nidulans 38 21.5 3

Phase separation Anacystis 38 15-30 (freeze-fracture) 28 5-25 4 18 -5-15

Key: References: (1) Murata et al 1975, (2) Hirano et al 1981, (3)

Williams et al 1980 (4) Furtado et al 1979

55 of the membrane, but conformational changes in protein complexes in

the membrane. Esfahani et al (1971) studied phase-separations in

Escherichia coli with low-angle X-ray diffraction, and also foll-

owed the activity of some membrane-bound processes. They found that break-points in the activity of the membrane-bound proteins occured at much lower temperatures than the phase-separations detected in

the lipid phase.

Thus to clarify the role of lipid fluidity in controlling processes which occur in and across the thylakoid membrane, experiments must be performed at the same temperature, and in some way the manipulation of lipid fluidity must be achieved.

Very few experiments involving the artificial manipulation of lipid fluidity in the thylakoid membrane have been reported. Nolan and Bishop (1978) have studied the effect of the polyene antibiotic

Amphotericin B on membrane-associated photosynthetic reactions in maize chloroplasts. They found that the antibiotic reduced the motion of a spin-labelled probe molecule in the thylakoid membrane and in liposomes prepared from thylakoid lipids (as detected by its

EPR signal). They concluded that its effect on membrane fluidity was responsible for the reduction of the proton gradient and the displacement of plastocyanin from the thylakoids.

Restall et al (1979) used a Rhodium complex catalyst (tris-

(triphenylphosphine)rhodium(I) chloride ) to hydrogenate up to 40% of the double bonds present in the thylakoid lipids in spinach chloroplasts. Although the fluidity of the thylakoid membranes was not monitored during the treatment it seems likely that the hydrogenation process would cause a decrease in the average fluidity of the lipid phase of the membrane by an unknown increment. They found

56 that a 10-20% decrease in Hill activity (water to methyl viologen) in the chloroplasts was produced by an incubation with the catalyst which resulted in up to 40% reduction of the lipid double bonds.

Their conclusion was that membrane fluidity did not greatly affect the capacity of chloroplasts for photosynthetic electron transport.

In conclusion, studies on temperature effects in higher plant chloroplasts, and studies on the artificial manipulation of thylakoid membrane fluidity have not thoroughly clarified its role in controlling photosynthetic processes.

1.10 Summary

A fluid lipid matrix in the thylakoid membrane is predicted from the presence of unsaturated lipids, (section 1.7). A highly fluid lipid phase in the thylakoid membrane may well be required for optimal rates of photosynthetic electron transport, particularly if a lipid-diffusible redox carrier is required for electron flow between Photosystem 2 and Photosystem 1 (see sections 1.5, 1.8 and

1.9). The physical nature of the lipid matrix may also affect the conformation and stability of membrane proteins, and thus have other non-specific effects on photosynthesis.

The electron transport components on the reducing side of Photo- system 2 (plastoquinone, Q^) are expected to be the most sensitive to the fluidity of the thylakoid membrane (see section 1.5), and evidence has recently been presented which suggests that this assumption may be valid (Hirano et al 1981, Yamamoto et al 1981).

It seems likely that at least half of the hydrogen ion gradient in the chloroplast is created by the diffusion of plastoquinone

57 molecules across the thylakoid membrane (see section 1.6), and therefore this process is expected to be highly dependent on the fluidity of the thylakoid membrane (Yamamoto et al 1981).

The stability of Photosystem 2 activity at high temperatures may also be dependent on the fluidity of the thylakoid membrane as discussed in section 1.5.5, although a possible mechanism to explain these observations is not yet clear.

Finally, the fluidity of the thylakoid lipid matrix may play an important role in controlling the lateral reorganization of large pigment-protein complexes within the membrane plane in a way which is necessary to optimise energy-distribution between PS 2 and PS 1

(Rubin et el 1981, Murata et al 1975 and Williams et al 1981).

It is the objective of the work presented in this thesis to investigate the physical nature of the thylakoid membrane lipid matrix and to relate these findings, particularly estimations of fluidity, with the functional activity of the membrane system as a whole.

58 2 Materials and Methods

2.1 Plant Material

The plants used were peas (Pisum sativum, variety: Feltham

First), and lettuce (lactuca sativa). Peas were grown from Micol- dusted seeds in Vermiculite under supplementary lighting conditions

(16 hour day), in a greenhouse throughout the year. In some preparations peas were grown in growth cabinets under warm (17°C day,

14°C night) and cold (7°C day, 4°C night) conditions. The day period was 16 hours and humidity was maintained above 70%, and the lighting was provided by fluorescent tubes at an intensity of 55 to

65 Watts per metre squared. Unless specified in the script all preparations and experiments were performed with peas harvested after 12 to 14 days, however some of the experiments (made in

Denmark) involved the preparation of chloroplasts from lettuce leaves. These plants were purchased at a local supermarket.

2.2 Chloroplast Preparation

Two chloroplast preparations were employed for the experiments to be described later. The preparation used for the majority of the experiments yielded essentially Class 2-type chloroplasts, that is lacking the outer envelope membranes, but capable of maintaining high rates of electron flow in the presence of Hill oxidants

(artificial electron acceptors). The advantage in this type of preparation is that relatively high yields of chloroplasts can be obtained, and so it was used for fractionation procedures.

The second type of preparation used resulted in the isolation of

Class 1 type chloroplasts with the outer envelopes intact which were able to maintain electron flow with C02 as the terminal elec-

59 tron acceptor. These preparations were employed in experiments that investigated flash-induced cytochrome changes which require that the 'chloroplasts contain ferredoxin, are in good condition, and are stable over a long period of time. The two types of chloroplast preparation used have been described in published papers (Nakatani and Barber 1977 - Class 1 preparation, Barber et al 1980 - Class 2 preparation).

2.2.1 Preparation of Class _1 Intact Chloroplasts

Healthy pea leaves were harvested and macerated using a Polytronj

(type PT 35 OD^in ice-cold grinding medium consisting of 0.33 M sorbitol, 0.2 mM MgCl2, 20 mM MES (2(N-morpholino)ethane sulphonic acid) which was brought to pH 6.5 with Tris (tris(hydroxymethyl)- aminomethane) (35g of leaves per 100 ml of grinding medium). The resulting homogenate was filtered through 10 layers of muslin cloth, with the first two layers separated by a thin layer of cotton wool. The filtrate was collected on ice and then subjected to centrifugation at 2200 xg for 30 seconds at A°C. The supernatant was carefully removed by aspiration so that most of the soft pellet associated with the hard pellet at the bottom of the centrifuge tube was discarded with the supernatant. The hard pellet was then resuspended carefully using a paint-brush in a medium containing low levels of cations (0.33 M sorbitol brought to pH 7.5 with Tris) and then subjected to centrifugation at 2200 xg for 20 seconds at

4°C. The supernatant and soft pellet were carefully removed once more by aspiration and the hard pellet was resuspended in the low cation medium at high concentration and stored on ice.

60 2.2.2 Preparation of Class _2 xBroken' Chloroplasts

Broken chloroplasts were prepared essentially as described above for the isolation of intact chloroplasts, except that the hard and soft pellets were pooled after the first centrifugation step, resuspended in a small volume of a low-cation medium containing 100 mM sorbitol, ImM HEPES (N-2-hydroxyethylpiperazine-N'-2-piperazine-

N'-2-ethanesulphonic acid), brought to pH 7.5 with KOH, and then osmotically shocked in ice-cold distilled water for 30 seconds. The suspension was then returned to the desired osmotic strength by the addition of the same volume of doubly concentrated low cation medium. The second centrifugation step was at 5000 xg for 5 minutes at 4°C in order to collect a large yield of chloroplasts. The final pellet was resuspended as previously described in the low cation medium or other desired media and stored on ice.

2.2.3 Determination of Chlorophyll

The amount of chlorophyll present in the chloroplast suspensions prepared as detailed above was determined by the method of Arnon

(1949). A small volume of the chloroplast suspension was added to 5 mis of 80% ice-cold acetone in a 10 ml centrifuge tube so that the final concentration of chlorophyll was between 20 and 100 micrograms per ml. After shaking the tubes were spun in a bench centrifuge at 1000 xg for approximately 3 minutes to remove coagulated protein particles. The light absorption of the supernatant was then measured in a Perkin-Elmer 554 spectrophotometer against a blank at

645, 663, and 750 nanometres. The measurement of the absorption at

750 nm was used to determine the degree of light-scattering in the sample, and if this was significant compared to the absorption at

663 or 645 nm then the sample was subjected to another centrifuga-

61 tion step. The chlorophyll concentration and the ratio of chloro-

phyll a_ to chlorophyll _b in the sample were determined from the

following formulae (Gregory 1971):

Total Chlorophyll = {8.02 x abs(663)} + {20.2 x abs(645)} (micrograms per ml)

Chlorophyll a = {12.7 x abs(663)} - {2.69 x abs(645)} (micrograms per ml)

Chlorophyll b = {22.9 x abs(645)} - {4.68 x abs(663)} (micrograms per ml)

- where the absorption at both wavelengths is corrected for scatte-

ring by subtracting the absorption value at 750nm from both values.

Although this correction makes little difference to the determinat-

ion of the total chlorophyll, it is very important for accurate

measurements of the chlorophyll to Jb ratio.

2.3 Preparation of Thylakoid Membrane Fractions

2.3.1 Incubation Media

Granal and stromal membrane fractions were prepared from Class 2

type chloroplasts prepared as described above. The final washed

chloroplast pellet was resuspended in a high cation medium in order

to bring about the stacking of the thylakoid membranes (see Barber

et al 1980). The high cation medium consisted of 5 mM K^HPO^/KI^PO^

(pH 7.5) plus 130 mM KC1 and 200 mM sorbitol, the membranes were

incubated on ice for at least one hour before mechanical fraction-

ation procedures were employed. The length of time allowed for

incubation in the stacking medium is crucial for the efficiency of

the subsequent separation of the two membrane regions.

Fractions were also prepared from thylakoid membranes which had been unstacked using low cation incubation media, the membranes

62 were then subjected to mechanical fractionation procedures in the

same way as the stacked thylakoid membranes.

These fractions represented control samples since the heavy and

light fractions isolated from unstacked membranes are not enriched

in either photosystem, and seem to represent an average, completely

randomized membrane system (see Barber 1980, Andersson et al 1980).

These fractions were used as controls to determine whether

the mechanical fractionation procedure itself was giving

rise to observed differences, and also if contamination of the

lighter membrane fractions with mitochondrial or envelope material

was occuring.

The unstacking medium consisted of 5 mM K^HPO^/Kl^PO^ (pH 7.5)

plus 330 mM sorbitol (approximately 10 mM K+), and the unstacking

process was carried out under the same conditions as stacking

described above.

2.3.2 Fractionation Procedure

Mechanical fractionation was achieved by two methods; by ultra-

sonic disruption at low power, and by shearing forces produced by

passing the chloroplasts through a narrow aperture at high pressure

using a Yeda pressure cell. Sonication was performed with a xsonip-

robe' sonicator (Dawe Instruments Ltd.) set at low power (setting

3, 1.5 A). Sonication of 15 mis of a concentrated thylakoid suspen-

sion (in a stacked condition) for 70 seconds was found to give a

reasonable yield of stromal^lamellae vesicles. Treatment of thylakoid membranes by two passages through a Yeda pressure cell packed 5 —2

in ice at a pressure of 95 x 10 N m (02-free nitrogen) was found

to give a highly enriched preparation of stromal^lamellae vesicles but with a lower yield than the sonication treatment. 63 The separation of the fragmented membranes was achieved by differential centrifugation using a Beckman J21C high-speed centrifuge. All the fractions were subjected to the same centrifugation steps in an attempt to keep the treatment of each membrane fraction the same (see the flow-diagram, Figure 4). The first high-speed

on step was used to assess the efficiency of the fractionation; if the supernatant (containing the lighter stromal lamellae) was a very light green colour, the pellet was resuspended and the mechanical disruption procedures were repeated.

2.4 Preparation of Cholesterol-Treated Membranes

A method was developed for the manipulation of lipid fluidity in thylakoid membranes which resulted with the incorporation of significant amounts of sterol (cholesterol and cholesteryl hemisuccinate) into the lipid phase of the membrane.

The technique used was based on the method of Shinitzky et al

(1979) who used the blood plasma substitute, polyvinyl—pyrrolidone

(PVP) as a carrier for the cholesterol and cholesterol esters which

_o have extremely low critical micelle concentrations ( about 10 ° M) in aqueous media. By using PVP these workers were able to manipu- late the fluidity of the plasma membranes of mammalian tumour cells growing in tissue culture by the incorporation of higher levels of sterols.

Polyvinylpyrrolidone (Sigma, Mr40,000), at 3% by weight was added to 25 mM Tris-HCl buffer (pH 7.5), and then cholesterol or cholesteryl hemisuccinate was added up to 500 micrograms per ml from a stock solution at 20 milligrams per ml dissolved in tetrahydrofuran (THF). This mixture was then incubated for at least 30 Disrupted Membranes

pellet pellet Granal Fraction Stromal Fraction

FIGURE 4 Flow diagram of the method used to separate stromal and granal membranes. The same procedure was followed when control (unstacked) membranes were fractionated.

65 minutes at 30-40°C, and oxygen-free nitrogen was bubbled through the solution until all trace of THF had disappeared (olfactory test). After cooling to room temperatures, chloroplasts were added to this medium to give a final chlorophyll concentration of 100 micrograms per ml. The chloroplasts were incubated for 2 minutes in the PVP-sterol medium, and then diluted five times with 1 mM Tris-

HC1 buffer (pH 7.5) containing 0.33 M sorbitol prior to centrifugation at 10,000 xg for 10 minutes at 4°C. The pellet containing all the chlorophyll was resuspended in a large volume of the Tris-HCl buffer for washing, and then the treated membranes were pelleted by centrifugation at 10,000 xg for 10 minutes at 4°C. The final pellet was again resuspended in a small volume of the wash buffer.

2.4.1 Determination of Cholesterol

The amount of cholesterol present in the treated membranes was determined on a chlorophyll basis by a simple and relatively rapid chemical method described by Searcy et al (1960), although some modifications of the technique were necessary because of the presence of chlorophyll. Washed membranes equivalent to 200 micrograms of chlorophyll were spotted on to filter paper and then dried in a warm oven at 30-40°C. The green discs on the paper were cut out and treated with a saturated solution of FeSO^ in glacial acetic acid

(2 ml) for at least one hour at room temperature. Using a pasteur pippette, 1.5 ml of solution was removed and 0.5 ml of concentrated sulphuric acid added. In the cholesterol standards, a salmon-pink ed colour develop^ ^and after 10 minutes the absorption at 490 nm read. In the samples containing chlorophyll samples were read at

490 nm against a blank sample which consists of untreated thylakoid

66 membranes assayed in the same way as the treated membranes. Small diff erences in the levels of chlorophyll were corrected for by normalizing the readings of each sample at 490 nm against an absorption value measured at 660 nm.

Despite the very short incubation periods used in this technique for incorporating cholesterol into membranes, it was shown that about 5% of the cholesterol in the incubation medium was associated with the thylakoid membranes. At the higher levels of cholesterol in the incubation medium, the final membrane ratio of cholesterol to chlorophyll on a weight basis was estimated to be about 0.3, corresponding to a sterol to lipid ratio of about 0.1 (see Results section).

2.5 Isolation and Analysis of Chloroplast Lipids

2.5.1 Fatty-Acid Analysis

The analysis of the fatty-acid composition of thylakoid membranes was ach'ieved by a method developed by D.J. Chapman and based on the method of Williams and Merrilees (1970). 3 Thylakoid membrane fractions were centrifuged in 50 cm poly- allomer centrifuge tubes (Sorvall Instruments Ltd., catalogue no.

03147), and the pellet was homogenized with chloroform: methanol

(2:1 vol/vol); 25 cm per milligram chlorophyll was added. The chloroform: methanol mixture was then passed through a Glass- microfibre filter paper (Whatman GF/C) on a Millipore sintered support usingo a slight vacuum and collected in a round-bottomed flask (250 cmJ) containing sufficient Sephadex G-25 beads to absorb the water present in the sample. The residue and filter paper were washed several times with the chloroform: methanol mixture and then

67 the filtrate was evaporated t0 dryness using a rotary evaporator

with the water bath set below 40°C. About 10 cm^ of chloroform was

then added to the extract and then removed by rotary evaporation.

The washing with chloroform was repeated once more and then the final extract (plus Sephadex beads) was resuspended in a small volume of chloroform. The Sephadex beads were removed by passing the extract through a glass-sintered filter (porosity 2) and coll- ecting the filtrate and washings in a round-bottomed flask (50 cm ). The clear extract was again evaporated to dryness and then the extract was taken up in a small volume of chloroform and stored 3 in a small 3cm vial under nitrogen.

All solvents were of xAnalar' grade purchased from BDH Ltd., and butylated hydroxytoluene (0.1 %) purchased from Sigma Chemical Co. was added to the solvents to avoid lipid oxidation by free- radicals .

Quantities of lipids were determined from gas chromatography of fatty-acid methyl esters as described below:

Lipid samples prepared as described above were added to stoppered glass 10 cm glass centrifuge tubes and the chloroform was removed by gassing with oxygen-free nitrogen. Excess boron trifluoride-methanol complex (approximately 14% BF^) purchased from

BDH Ltd. was added and then the tubes were heated in an oven at

100°C for 30

minutes after thorough flushing with nitrogen. The tubes were cooled to room temperature and then methyl pentadecan- oate (purchased from BDH Ltd.) which was used as the internal standard was added, and a drop of distilled water was also added to aid the phases separation procedure. The fatty-acid methyl esters were then separated from the boron trifluoride-methanol complex by 68 3 adding 5 cm of hexane and shaking vigorously. The separation of the two phases was accelerated by centrifugation at low speed in a bench centrifuge. The top (hexane) phase was then removed and the phase-separation procedure was repeated. The hexane phases containing the chloroplast fatty-acid methyl esters and the internal standard were pooled and then the hexane was removed by gassing with nitrogen.

The sample was taken up in a small known volume of hexane and flushed with nitrogen before analysis on the gas chromatography equipment. The samples were injected into a column of 15% Reoplex

400 on Chromosorb 100/120 mesh inside a Perkin-Elmer Sigma 2B gas chromatograph. The peaks due to the fatty-acid methyl esters were then integrated by a Perkin-Elmer 10B data handling station and the total quantities of lipid were determined.

2.5.2 Isolation of Chloroplast Lipids

Monogalactosyl diacylglyceride (MGDG) and digalactosyl diacylglyceride (DGDG) were prepared by High-Performance Liquid Chromato- graphy by Drs. D.J. Chapman and P.A. Millner at Imperial College, and the fatty-acid composition was analysed by gas chromatography as described above.

A small contamination due to carote noid (approximately 1.5% by weight) was present in these samples, and therefore for the measurement of the fluorescent lifetimes of DPH in the lipids, a further purification using thin-layer chromatography was applied.

Thin-layer plates were prepared on glass using silica-gel

(Kieselgel G) purchased from Macherey Nagel & Co., Duren, which contained 5% by weight ammonium sulphate. The samples were deve-

69 loped with an acetone:benzene:water mixture (90:31:8) and after drying were viewed under ultra-violet light using dichlorofluor- escein in methanol as a stain.

2.6 Preparation of Lipid Vesicles

2.6.1 Chloroplast Lipids

Purified chloroplast lipids in chloroform were mixed under nitrogen in glass-stoppered tubes and the chloroform was removed by rotary evaporation an(j then thorough flushing with nitrogen. After all trace of chloroform had been removed, 2 cm of basic medium which had been purged with nitrogen was added to the tube and the suspension was rapidly mixed for 1 minute. The sample was then sonicated under nitrogen in a Kerry bath-type sonicator at 20°C for

20 minutes. The resulting dispersion of lipids was transparent but visibly scattering light, and therefore a population of small multi-lamellar vesicles is expected.

2.6.2 Other Lipids

Liposomes prepared from soya-lipid and cholesterol were prepared by mixing the lipids dissolved in chloroform to the required ratio and then removing the solvent as described above. After the addition of the nitrogen-purged basic medium, the samples were sonicated at high power on ice and under nitrogen using a xsoniprobe' sonicator (Dawe Instruments Ltd.) at setting 4, (2

Amperes! The vesicles were sonicated in 30 second bursts for a total of 5 minutes, and a transparent suspension resulted.

Vesicles prepared from dipalmitoyl phosphatidylcholine (purchased from Sigma Chemical Co.) were prepared as described above for chloroplast lipids except that the water in the bath sonicator was kept above 40°C.

70 The preparation of liposomes from soya-lipid for lipid-enrich- —3 ment experiments where high levels of lipid were used (100 mg cm )

was achieved by using the Kerry bath-type sonicator, and the basic

medium contained 0,1 mM EDTA.

2.6.3 Lipid Enrichment Experiments _ o 3

Liposomes at 100 mg cm , prepared as described above (5 cm )

were added to a stirred chloroplast suspension containing approx-

imately 6 milligrams chlorophyll in a buffer consisting of 0.5 mM

EDTA, 10 mM KC1, 1 mM HEPES and 100 mM sorbitol. The chloroplasts

had previously been incubated in the HEPES buffer for 60 minutes, and so were in the unstacked state. The pH of the medium was then

lowered to pH 6 by adding small quantities of 1 M HC1 with a 0.1 cm syringe. The liposome and chloroplast mixture was allowed to incubate at pH 6 for a further 30 minutes (without stirring), and then the suspensio3n was carefully layered on to a sucrose density gradient in 15 cm centrifuge tubes. Four steps were present in the density gradient, consisting of 40%, 30%, 20% and 10% sucrose.

Different fractions containing increasing amounts of soya lipid were separated by centrifugation for one hour at 25,000 revolutions per minute in a Beckman ultracentrifuge using a swing-out rotor.

Six fractions with different buoyant densities were obtained, and the liposomes separated into a single band with the lowest buoyant density which contained no chlorophyll.

2.7 Protein Analysis

Protein levels in various thylakoid membrane fractions were assayed essentially by the method of Lowry et al (1951). Samples equivalent to 10 to 20 micrograms chlorophyll were added to 2 cm 3

71 of ice-cold 80% acetone containing 50 mM NaCl in glass centrifuge

tubes. After mixing^ the protein was precipitated by centrifugation

for 10 minutes at 5,000 xg at 4°C. The acetone supernatant contain-

ing the chloroplast pigments was decanted off and the chlorophyll

levels were determined for a second time. The protein pellet was

then mixed with 100 microlitres of 1 N NaOH and 15 microlitres of

10% Triton X-100 and rapidly mixed using a Vortex mixer. After one

hour of incubation at room temperature the total volume was made up 3 to 1 cm with water and again rapidly mixed. Reagent A was freshly 3 3 prepared from 50 cm of 2% sodium carbonate in 0.1 N NaOH, 0.5 cm 3 of 1% copper sulphate in 0.1 N NaOH and 0.5 cm of 2% sodium/pot- 3 assium tartrate in 0.1 N NaOH. Reagent A (5 cm ) was added to the

protein suspension and rapidly mixed. After exactly 10 minutes, 0.5

cm of Folin-Ciocalteau's phenol reagent (purchased from Sigma

Chemical Co.) diluted by half with water was added and the contents

rapidly mixed. The colour was allowed to develop for 30 minutes,

and then the absorption at 750 nm was measured in a spectrophoto-

meter against a blank sample containing no protein. The amount of

protein in each sample was determined by constructing a calibration

curve with bovine gamma-globulin (Cohne fraction 2) purchased from

Sigma Chemical Co.

2.8 Electron Transport Measurements

2.8.1 Steady-State Electron Transport

Steady-state rates of electron transport in broken pea chloroplasts were measured using a Rank Brothers (Clark-type) oxygen electrode. The standard assay medium used consisted of 0.33 M sorbitol, 50 mM HEPES, 1 mM MgCl2, 1 mM MnCl2 and 2 mM EDTA

(disodium salt) at pH 7.6, into which chloroplasts at a final

72 o concentration of 50 micrograms per cm were suspended.

Photosystem 2 activity was measured at saturating light

intensities in the presence of benzoquinone (0.5 mM) with ammonium

chloride (5mM) as an uncoupler. Photosystem 1 activity was then

measured by adding DCMU (10 pM), D-isoascorbate-sodium salt (1 mM),

DCIP (0.15 mM) and methyl viologen (20 pM).

Rates of electron transport through both photosystems were meas_

ured in the presence of ammonium chloride (5 mM) and with methyl viologen (20 pM), or potassium ferricyanide (1 mM) as the terminal electron acceptor.

2.8.2 Flash-Induced Electron Transport

The rate of electron transport after a short (10 microsecond) pulse of saturating light can be measured from absorption changes occurring in the millisecond time scale associated with cytochrome f_, cytochrome b^gg and the reaction centre of Photosystem 1 (P700).

Rapid absorption measurements were made with a single-beam flash apparatus constructed by Applied Photophysics Ltd., and a block diagram of the instrument is shown in Figure 5. The measuring beam was provided by a 100 W tungsten halogen lamp and passed through an M300 high-radiance monochromator (slit widths 1.25 mm) and the intensity of the measuring beam was less than 0.2 W m at 550 nm.

After passing through a 10 mm path length cuvette containing the sample, the measuring beam was guided to the photomultiplier using an optical fibre 'light-pipe' that was placed at the side of the cuvette to minimize the loss of light due to scattering from the sample. Saturating light pulses of duration 10 microseconds full width at half maximum were provided by a xenon flash tube at right

73 SAMPLE

Delayed Trigger

FIGURE 5 Block diagram of the single-beam flash spectrophotometer, and the rapid recording devices used in experiments on cytochrome absorption changes. angles to the measuring beam.

Measurements of cytochrome absorption changes were made with the

light flash filtered through a 2mm Schott RG 665 filter and the

photomultiplier was protected by a 4 mm Corning 4-96 filter. The

signal from the photomultiplier was biased against a 10 V direct

current output and then fed into a signal averager (Nuclear Meas-

urements, model 546C) and viewed on an oscilloscope (Telequipment

model S51B).

Intact (Class 1) chloroplasts (equivalent to 50 mM chlorophyll)

were used in these experiments and were osmotically shocked in the

cuvette on ice for 30 seconds in distilled water containing MgCl2

(5 mM). Buffer was then added consisting of 0.33 M sorbitol, 50 mM

Tricine/KOH (pH 8.3), made up to double strength. The medium was

maintained in an anaerobic state for improved stability of the _ rs

thylakoids by adding glucose (5 mM), glucose oxidase (0.1 mg cm )

and catalase (0.2 rag cm ).

For experiments involving cyclic electron flow around Photosys-

tem 1, DCMU was added (10 pM) with ferredoxin (5 pM) and NADPH (0.5 mM). Valinomycin (6 pM), nigericin (6 pM) and KC1 (38mM) wereaiso present in measurements of cytochrome absorption changes to elimi- nate the large contribution of the electrochromic shift centred around 520 nm. Conditions of psuedo-cyclic electron flow were identical to the above except that dithionite (1 mM) was added instead of NADPH and ferredoxin.

Non-cyclic electron flow to methyl viologen (0.5 mM) or potassium ferricyanide (0.5 mM) was also studied by flash spectroscopy under identical conditions as above, but in the absence of DCMU and artificial electron donors.

75 2.9 Chlorophyll Fluorescence Measurements

2.9.1 Salt-Induced Chlorophyll Fluorescence Changes

Type 2, broken chloroplast suspensions were incubated in basic

medium containing 10 mM KC1 for 5 minutes at room temperature and

DCMU (10 pM) was added. Chlorophyll fluorescence was excited

through a filter combination of one Schott BG 18 (2 mm) and one

Schott BG 38 (2 mm) filter by a direct current tungsten-halogen _ o

light source, resulting in a light intensity of 30 W m . The

fluorescence was detected at right-angles by an EMI 9558B S20

photomultiplier shielded by a Balzer B-40 693 interference filter

and a Schott RG 695 cut-off filter, and the output from the photo-

multiplier was displayed directly on a RikaDenki two-pen chart

recorder. Salt-induced chlorophyll fluorescence changes were brought about by adding MgCl2 (5 mM) to the chloroplast suspension 3 in a 3 cm glass cuvette and rapidly mixing.

2.9.2 Modulated Chlorophyll Fluorescence

The relative chlorophyll fluorescence yield in pea leaves

excited by modulated blue-green light chopped at 110 Hz was

detected by a photomultiplier linked to a lock-in amplifier as

shown in the block diagram in Figure 6. Far-red light at 710 nm

could also be applied to the system, although the chlorophyll

fluorescence arising from this light source was not detected since it was not modulated. Pea leaves attached to the intact plant were placed on an aluminium block suspended in a temperature-controlled water bath, and the leaf was covered by a glass microscope slide which served to maintain a more uniform temperature over the leaf,

76 FIGURE 6 Block diagram of the apparatus used to measure modulated chlorophyll fluorescence from pea leaves. The optical filters were selected so that the leaves could be studied in State 1 or State 2.

77 and held a thermocouple close to the surface of the leaf.

The modulated blue-green light at 1.3 Wm was produced with

the filter combination of one Schott BG 18 (4mm), and one Schott BG

38 (2 mm) filter. The non-modulated far-red light at an intensity

of 7.5 Wm , was produced by a 706 nm Balzers interference filter.

Under conditions where just the blue-green light is supplied,

the light is preferentially absorbed by Photosystem 2 and the leaf

adapts to the imbalance by slowly allowing some of the excitation

energy to be transferred (possibly by Forster-type resonance

processes) to Photosystem 1. Therefore under prolonged blue-green

light excitation conditions the leaf is said to move into the

maximum spill-over state, termed 'State 2'.

When the illumination excites predominantly Photosystem 1

reaction centres, as is the case when the supplementary far-red

light is supplied, the plant adapts to maintain the minimum amount

of excitation energy transfer from Photosystem 2 to Photosystem 1,

and this situation is known as 'State 1' (see Chow et al 1981).

2.10 ESR Measurements: Spin-Labels

2.10.1 Measurement of First-Derivative ESR Spectra

The measurements of the electron spin resonance spectra of the spin-labels were made using a Varian E 104A spectrometer fitted with a variable-temperature accessory. The spin-labels, dissolved in ethanol, were added to small glass test tubes, and dried down under nitrogen gas. Membranes equivalent to 2 mg cm chlorophyll suspended in basic medium were added to the tube and gently shaken at room temperature for five to ten seconds. The amount of spin- label added was usually 5 micrograms per milligram of chlorophyll,

78 and the chlorophyll-to-probe molar ratio is given in figure leg-

ends. DCMU (5 pM) was added to avoid the light-induced reduction of

spin-labels, and the samples were protected from direct light as

much as possible. The ethanol concentration in the final suspension

of membranes was always 0.25%. Incorporation of the probe was

achieved after about thirty seconds incubation at room temperature

as judged by the ESR signal intensities at longer incubation times.

When the spin-labels 5-doxyl decane and 12-doxyl stearate were

used, the spin-broadening agent, tris(oxalato)chromate (III), was

added (35 mM) in order to remove the probe-in-water signal as

described by Berg and Nesbitt (1979).

Samples were placed in a Varian variable^temperature aqueous

cell, and the ESR spectra were recorded over a time period of four

to sixteen minutes depending on the intensity of the signal from

the probe in use, although the intensity of the signals from the

same probe in different thylakoid membrane preparations were simi-

lar. The magnetic field strength was set at 3280 gauss (microwave

frequency was 9.18 GHz), with a scan range of 80 gauss. The micro-

wave pOu>e.r was set at 5 mW, and the modulation frequency and

amplitude were 100 kHz and 1.0 gauss respectively. Where not indi-

cated measurements were made at room temperature.

The spin-label 5-doxyl decane was obtained from Molecular

Probes, Inc., (Piano, Texas) and 5-doxyl stearate and 12-doxyl stearate were obtained from Syva Ltd. (Palo Alto, California).

2.10.2 Calculation of Rotational Correlation Times

The apparent rotational correlation times for the spin-labels 5- doxyl decane and 12-doxyl stearate were calculated from the

79 linewidths and the ratios of the three lineheights derived from

the ESR spectra according to Marsh (1981) (see Figure 7A).

The equations below are not strictly valid in the anisotropic

membrane systems used, but they give a quantitative measure of the

relative rate of tumbling of the spin-label.

The relative heights of the three peaks of the first derivative

electron spin resonance spectrum (low field h+p centre hQ and high

field and the width of the centre line in gauss ( A Hq) were

used to calculate the linewidth parameters B and C using the

following equations:

B = 1/2 Aiyo^/h^)1/2 - O^/h^)1/2]

c = 1/2 Aiya^/h^)1/2 - oyh^)1/2 - 2]

These parameters were then used to calculate the apparent ro-

tational correlation times for isotropic motion of the spin-labels using the equations:

Tb = -1.22 B Tc = 1.19 C where Tg and Tq are in nanoseconds.

2.10.3 Calculation of Order Parameters

The rate of tumbling motion of the spin-label 5-doxyl stearate is too slow to be analysed by the above procedure, but instead information on the degree of order of the lipid acyl chains can be obtained from this probe. The method adopted for the estimation of order parameters by the spin-labelling technique was the method of

Gaffney and McConnel (1974).

The theory of these measurements is presented in more detail in the results section (see Figure 22 in particular), and is discussed

80 in great depth in "Spin Labelling, Theory and Applications", L.J.

Berliner (1976).

The splittings in gauss of the outer and inner lines of the first derivative ESR spectrum of 5-doxyl stearate were measured

(see Figure 7B).

The measured outer line splitting (2Amax) is a good approximation for the high spin to low spin line splitting (2A^j) for the parallel orientation of the applied magnetic field and the nitroxide TT-bond orbital shown in Figure 22A. The measured inner line splitting (2Am^n) is a fair to poor approximation for the low to high spin line splitting of the perpendicular orientation (2A^) shown in Figure 22B. Thus:

Amax = AII (Sood assumption)

= Am^n Aj (fair assumption)

An approximate order parameter (Sflpp) was calculated initially from the following expression:

Sapp "

A better approximation for A^ was obtained from the following expression:

AX • V„ + !-32 + !-86 losO - sapp> The polarity correction was also calculated from the following

83 (B)

FIGURE 7 Diagramatic representations of first-derivative ESR spectra of a fast moving (A) and slow moving (B) spin-label.

The parameters marked are used for the calculation of order param-. eters (S) for slow motions, and rotational correlation times (T. ii and T_.) for rapid motions. D

84 equations:

Aq = 1/3(AI]; + 2Aj)

Ac = 1/3(A$X + A^y + A^z)

k = polarity correction = Aq/Ac

The order parameter was finally calculated from the expression:

S = (Au - A1)/[A=z - 1/2(A$X + A|y)l k

When the values for the single crystal line splittings are

inserted the expression simplifies to:

S = 0.568 (k1T - AI)/[1/3(AII + 2Aj)]

2.11 Fluorescence Polarization Measurements

2.11.1 Preparation of DPH-labelled membranes

Class 2 chloroplasts (thylakoid membranes) were labelled with

the fluorescent probe molecule l,6-diphenyl-l,3,5-hexatriene accor-

ding to a protocol developed for these membranes. The initial

studies using fluorescence polarization were performed with a lipid

to probe ratio of about 150 to 1 (by weight), although this ratio

was later doubled to 300 to 1. Studies on the effect of the lipid

to probe ratio are presented in section 3. The studies on the rate

and degree of incorporation of the probe into thylakoid membranes

are also presented in section 3.

Thylakoid membranes equivalent to 100 micrograms per ml Chi.

were added to a thoroughly cleaned test-tube before the addition of

7.5 pM DPH from a 3 mM stock solution in tetrahydrofuran (2.5 pi

per ml of chloroplast suspension). The incorporation of DPH was

allowed to proceed over an incubation period of at least 30 minutes

at room temperature, and in many cases the thylakoid membranes were

10 7 10 7 unstacked by the presence of low cation concentrations since it was found that the incorporation of DPH proceeded at a faster rate under these conditions (see section 3).

It is important that all the glassware is thoroughly clean and free of grease in experiments involving highly hydrophobic compounds such as DPH since it will readily partition into any hydrophobic substances present. All test-tubes and cuvettes were therefore scrubbed in strong detergent solution and then rinsed several times in double distilled water, followed by several rinsings in absolute ethanol. This processes repeated at least twice and after drying, the glassware was rinsed in distilled water once more.

The thylakoid membranes were separated by centrifugation

(10,000xg, 10 minutes) from any DPH which had not partitioned into the membranes, but which may have been present in non-membrane material or in micellar form. The pellet was resuspended in the desired medium, and stored on ice.

2.1 1 .2 Steady-State Fluorescence Polarization Measurements

Thylakoid membranes which had been labelled with DPH were diluted to 10 micrograms per ml chlorophyll in the resuspension medium desired (generally this was the low-cation HEPES buffer described in 2.2.2). Unlabelled membranes were also prepared and used as a blank to compensate for scattering and intrinsic fluorescence from the thylakoid membranes and the resuspension medium, generally the background intensities from the unlabelled samples were less than

10% of the fluorescence from DPH in the labelled sample.

Fluorescence was measured in a Perkin-Elmer MPF44-A fluorescence spectrophotometer fitted with a polarization accessory. Film polarizers were used (Polaroid type HN-P'B), and when oriented perpendicular to each other the transmission of light by the filters was

less than 0.2% of the transmission with the filters oriented para-

llel. The excitation monochromator was set at 360 nm (slit 20 nm),

and any fluorescence arising from the polarizing polaroid filter

itself was removed by the presence of a Schott UG1 cut-off filter.

Fluorescence from the sample in a three ml quartz cuvette was

detected at right angles through the polaroid analyzer, a 390 nm

cut-off filter and a monochromator set at 460 nm (slit 20nm). The

photomultiplier used was a red-sensitive type, Hamamatsu R777.

Fluorescence polarization was calculated from the corrected fluor-

escence intensities ( I ) with the polarizer and analyser in various positions using the following expression:

P = Ivv ~ ^h*2 Iw + Iv h, .Z where Z = / and the first and second subscripts represent the positions of the polarizer and analyzer respectively. The factor Z compensates for slightly unequal horizontal and vertical excitation intensities, and although this parameter usually remains constant over a period of time, it was determined for each reading of fluorescence polarization.

Steady-state fluorescence anisotropy values are related to polarization by the following expressions: anisotropy = r = Iv v — Ivh * Z Iw + v(2 1v h. .Z7 ) r = 2P 3 - P

Where I + (21 ..Z) is the total fluorescence intensity.

10 7 2.12 Time-Resolved Fluorescence Measurements

Time-resolved fluorescence decays were recorded at 25°C by using

the single-photon counting technique with a nanosecond fluorescence

spectrophotometer (Applied Photophysics Ltd.) which is described in

Appendix 1. Excitation light at 360nm was provided by a thyratron-

gated nitrogen-filled flash lamp set to run at 50 kHz and at a

voltage of 5 kV in experiments with intact thylakoid membranes and

20 kHz and 4 kV in experiments with chloroplast lipid dispersions.

Fluorescence decays were recorded at 460nm with a counting frequen-

cy which was 2% of the lamp flash frequency, under these conditions

99% of the photons detected are single photons. Corrections for

scattering and intrinsic fluorescence from the thylakoid suspension

were made by subtracting counts from an unlabelled sample for the

same length of time. The background fluorescence and scattering

accounted for about 10% of the total counts for labelled thylakoid

membranes and less than 3% for chloroplast lipid dispersions.

In experiments to determine the decay of anisotropy of DPH

fluorescence in thylakoid membranes, the data was collected over

several hours and the samples were changed every fifteen minutes

from a concentrated stock solution stored on ice. The lamp profile

was also collected at regular intervals to correct for any drifts

in the lamp intensity and pulse width over the course of the

experiment.

The intensity of the fluorescence from DPH in experiments invol-

ving thylakoid membranes was low since very dilute suspensions were used to avoid the re-absorption of the DPH fluorescence by the

efficient light-trapping system of the chloroplast membranes. The

10 7 10 7 intensity of the flash-lamp was therefore adjusted to be as high as

possible by using a nitrogen-filled lamp with a broad electrode

gap; relatively broad pulses were obtained with a full-width at

half maximum (FWHM) of about 6 nanoseconds. The difficulty in the

deconvolution procedures for anisotropy decays becomes more intense

as the duration of the lamp pulse becomes longer, and therefore it

was necessary to take a value of 0.39 as the time-zero anisotropy

(rQ) value as has usually been the case in this type of experiment

(Kawato et al 1978, Chen et al 1977).

The lamp profile (plus instrument response function), and the

resulting convoluted fluorescence decay were recorded on a multi-

channel analyzer, and then double or single exponential deconvolu-

tion procedures were performed with a Vector Graphic micro-computer

using a non-linear regression type analysis with four or two para-

meter fitting.

Anisotropy decay data was obtained from fluorescence decay pro-

files by essentially the same method as Kawato et al 1981b and Chen et al 1977. The lamp pulse was passed through a Glan-Thompson polarizing prism and the fluorescence from the sample was detected at 90° through a second prism oriented parallel or perpendicular to the polarizing prism. The recorded decay profiles with polarizer and analyzer parallel and perpendicular were then used to create the total ( FT(t) ) and difference ( Fp(t) ) fluorescence decay data:

FT(t) = Fn(t) + 2.FI(t).G

FD(t) = Fn(t) - FI(t).G

where G is a correction factor for partially polarized incident excitation light, and was calculated so that : t=oo t=°° r s -/FD(t)//FT(t) yt= o yt= 0

The total fluorescence decay data was analyzed by a double exponential deconvolution procedure and the best-fit pre-exponential ( A^ ) and lifetime ( T^) parameters obtained for the decay were then used in a second deconvolution procedure to analyze the difference decay data. Thus the fluorescence and anisotropy decays were assumed to have the following forms:

(t) = A1 expC-t/T^ + A2 exp(-t/x2)

d r (t) = (rQ - roo) expC-t/^) + rm where the superscript xd' represents the response to an infinitely short (delta-function) pulse of light, is the rotational correlation time of the probe, and rQ and rTO are the time-zero and limiting (time infinity) anisotropy values. Therefore the parameters A^,

Ti> roo anc* ^ were so determined that when convoluted with the lamp profile, the calculated total and difference decays best fitted the recorded total and difference decays. A more detailed explanation of the theory of the fluorescence polarization measurements is given in section 3. A description of the computer programs used to calculate the best-fit parameters of the decay of fluorescence anisotropy from DPH, is presented in Appendix 2.

10 7 10 7 3 RESULTS

3.1 Lipid Fluidity Measurements

The measurement of lipid fluidity in the thylakoid membrane is a

central part of the experiments to be presented in this thesis, and

therefore at the onset of the studies it was necessary to develop

rapid and relatively simple assays for the estimation of the order-

ing and viscosity of the lipids in this membrane. To this end, lipophilic probe molecules (fluorescence and ESR spin-label probes) were employed.

3.1.1 .A Fluorescent Probe; DPH

The probe molecule most often employed in this work was the highly fluorescent compound 1,6-diphenyl-l,3,5-hexatriene (DPH).

This probe has been widely used to estimate the lipid fluidity of a variety of artificial and biological membranes, and its use was pioneered by Shinitzky and Barenholz (1974).

The major advantages of DPH as a fluorescent probe are its very high fluorescence yield (in a hydrophobic environment), and a large

Stoke's shift which minimizes the need for scattering corrections in the measurement of fluorescence. The compound has a low fluorescence yield in a highly polar environment, and thus DPH fluorescence in an aqueous membrane dispersion arises almost entirely from DPH molecules embedded in the hydrocarbon region of the membranes (see Figure 15).

At the beginning of this study, no reports of the use of DPH in the thylakoid membrane had appeared, and therefore it was necessary to attempt a thorough characterization of the behaviour of DPH in this membrane system. Recently studies by Kinosita et al (1981a,

1981b) have been published which confirm the use of DPH as a probe of lipid fluidity in other pigmented membrane systems; bacteriorho-

dopsin purple membrane sheets and in cytochrome oxidase-containing

liposomes. They showed that the fluorescence lifetime of the probe

was considerably reduced in these systems which may have resulted

from energy transfer from DPH to the retinal or heme moieties bound

to the membrane proteins. A relatively short lifetime for DPH

fluorescence was also found in the thylakoid membrane, as will be

presented and discussed later.

Figure 8 shows the excitation and emission fluorescence spectra

of DPH embedded in various membrane and non-membrane environments.

The overall shape of the spectra are very similar with the main

excitation peak at 360 nm and with emission peaks at 430 nm and

460 nm.

The excitation and emission spectrum of DPH embedded in thylakoid membranes compared with the fluorescence spectra of DPH in non-pigmented soya lipid vesicles is shown in Figure 9. The most notable differences between the fluorescence emission spectra of

DPH in the two different membrane systems are that in thylakoid membranes, DPH seems to have a larger xtail' into the green region

(500-580 nm), and that the 460 nm peak is larger in comparison to the 430 nm peak. Similar features can be produced in the emission spectrum of DPH in soya-lipid vesicles by placing two "screens' consisting of chloroplast suspensions (10 pg cm in 0.5 cm path length cuvettes) across the excitation and emission beams (Figure

9). This feature of the DPH emission spectrum in thylakoid membranes could possibly arise, therefore, from the re-absorption of

DPH fluorescence by the light-harvesting pigments which do not

10 7 EXCITATION B EMISSION

ethanol

soya - pl liposomes

hepes-buffer

250 300 350 400 400 450 500 550 600 X (nm)

(1.5 FIM) FIGURE 8 Corrected excitation and emission spectra of DPH in various environments. Excitation spectra (Slit width 10 nm) were recorded for emission at 460 nm (Slit width 20 nm), and emission spectra were excited at 360 nm with the same slit widths.

10 7 10 7 FIGURE 9 Corrected excitation and emission spectra of DPH in

thylakoid membranes and in soya-phospholipid liposomes (dotted line). The effect of a chlorophyll 'screen' on the latter spectra is also shown (solid line). Other conditions as in Figure 8. 10 7 absorb strongly in the 500-600 nm region (sometimes referred to as

the 'chlorophyll window').

The excitation spectra of DPH in soya-lipid vesicles and in

thylakoid membranes are very similar, however a small peak at about

290 nm is apparent in thylakoid membranes and this may suggest that

energy transfer from some membrane component (possibly aromatic

amino acids) to DPH is occurring. Since all fluorescence polariza-

tion measurements with DPH were made with excitation light at 360

nm any depolarization of fluorescence due to energy transfer from

this component would be negligible.

The fluorescence properties of DPH in the thylakoid membrane,

and the possibility that energy transfer was occurring from DPH to

other chromophores was investigated using several approaches:

a) Measurement of the excitation and emission spectra of chlorophyll fluorescence (in the presence of DCMU).

If significant energy transfer can occur from DPH embedded in the thylakoid membrane to the light-harvesting pigments, then this should be reflected by an increase in chlorophyll fluorescence, particularly at an excitation wavelength where DPH absorbs maximally (360 nm). As shown in Figure 10, the excitation and emission spectra of chlorophyll fluorescence are identical in the presence or absence of DPH. Since the light-harvesting pigments are by far the largest population of chromophores present in the thylakoid membrane this result argues against the possibility of efficient excitation energy transfer from DPH to these pigments. b) Increasing the level of DPH in the thylakoid membrane.

The possibility of energy transfer from DPH to the light- harvesting pigments was further investigated by monitoring the chlorophyll EMISSION fluorescence (b) *

DPH fluorescence * J[ 400 500 600 700 800 A(nm)

FIGURE 10 (a) Corrected excitation spectrum of chlorophyll fluorescence (at 685 nm) in the presence (solid line) and absence (dotted line) of DPH (1.5 pM). Chloroplasts were suspended at 10yg ml * Chi.

(b) As above but the emission spectra of chlorophyll (and DPH) is shown excited at 360 nm. Slit widths as in Figures 8 and 9.

10 7 intensity of chlorophyll fluorescence when excited at 360 nm with

increasing amounts of DPH present.

The results presented in Figure 11 show that chlorophyll fluor-

escence at 685 nm (arising mainly from Photosystem 2) and at 720 nm

(which results partly from Photosystem 1) is not preferentially

excited at 360 nm when DPH is present, even at relatively high

levels of eight times the normal incubation levels; in fact the

intensity of chlorophyll fluorescence is slightly reduced at high

DPH levels. This result argues against the existence of energy

transfer from DPH to chlorophyll, although it does not rule out the

possibility of transfer to pigments which do not show a significant

fluorescence yield (for example non-fluorescent forms of chlorophyll and carote noids). c) Measurement of the fluorescence lifetime of DPH incorporated

into thylakoid membranes.

When a fluorescent molecule is excited by a short pulse of

light, the fluorescence will decay in an exponential manner:

F(t) = Fq exp( -t /T)

Where T is the time (t) taken for the fluorescence intensity to fall to 1/exp times its initial level (FQ). In practice, different populations of fluorescent molecules can exist, and then the fluorescence decay is described by the equation:

F(t) = £ A^exp ( -t/Ti)

Where Ai is the pre-exponential factor describing the relative proportion of the fluorophores in the i th population.

The quantum yield of fluorescence ($ ) is given by:

0 = V ( ke + k±)

10 7 i i 70 t—t—* — • i—• x 60 EMISSION at 685 nm

m E 00 50 cz so G> CNI 40 L_* O O ZD =3 30 :—t—t QJ 10 -53 20 EMISSION at 720 nm

10 H5

0 2 3 4 [DPH] JJM

11 Effect of increasing incubation levels of DPH on chlorophyll fluorescence in pea thylakoids excited at

Chloroplasts were suspended at 10 yg ml \ Lower trace- emission at 720 nm, upper trace, emission at 685 r Where kg is the rate of fluorescence emission due to transitions

from the singlet excited state (S^) to the ground state (Sq) and k^

is the rate of all other non-radiative processes which de-populate

the S^ level. The lifetime is related to the fluorescence yield by

the following relationships:

$ = ke t and 1/ x = kg + k^

Thus, the greater the rate of emission from the S^ level, the

shorter the lifetime.

The existence of energy transfer between DPH and another chromo-

phore should lead to an increased k^ which would lead to a shor-

ter fluorescence lifetime dependent on the statistical distribution

of the distances between DPH and acceptor molecules. A complication

with the use of this approach to test for energy transfer is that

the fluorescence lifetime is also highly dependent on several other

factors which can de-populate the S^ level. The physical properties

of the local environment of the fluorophore such as the viscosity

and the polarity can influence non-radiative processes such as

thermal quenching of the fluorescence. Nevertheless, an investiga-

tion of the fluorescence lifetime of DPH in thylakoid membrane

systems was carried out and in particular, a comparison of the

fluorescence properties of DPH incorpoated into aqueous dispersions

of the major thylakoid membrane lipids (MGDG and DGDG) and in

thylakoid membranes was made. The fluorescence lifetime of DPH

incorporated into stromal lamellae and granal stack membranes were

also compared since in these two fractions the average distance between DPH and the light-harvesting pigments may be different (see section 3.3.2).

The results of these experiments are summarized in Table 3, and a typical decay profile of DPH incorporated into thylakoid mem-

branes is shown in Figure 12. Fluorescence decay profiles of DPH in

stromal and granal membranes (plotted on a linear scale for com-

parison with Figure 12) are shown in Figure 13 with the results of

the deconvolution procedure for these two particular experiments.

The decays of DPH fluorescence were almost always found to be best-

fitted by the deconvolution procedure by assuming a double, rather

than a single exponential decay, although this probably represents

only an approximation to a more complex situation which may exist.

Nevertheless, the goodness of fit of the data to the assumed double exponential decay was often excellent (see Figure 12), and in general when poor fitting was obtained it was a reflection of

instrumental factors (e.g. radio-frequency pick-up by the photomul-

tiplier giving rise to a ripple on the decay profile).

The lifetime of DPH in the chloroplast lipid dispersions at 25°C of about 6 nanoseconds is expected from previous studies of other unsaturated lipid systems, where it has been shown that the rate of

DPH fluorescence decay increases with higher levels of fatty-acid unsaturation (Stubbs et al 1981). The shortening of the fluorescence lifetime may arise from increased xthermal quenching' in the more fluid environment associated with higher levels of fatty-acid unsaturation.

The results summarized in Table 3 show that in all of the thylakoid membrane fractions the proportion of the short-lifetime component of DPH fluorescence is larger than in the pigment-free thylakoid lipid dispersions. The lifetime of the T2 component in the thylakoid fractions is longer than in the purified lipid system,

10 7 Table _3 Fluorescence Lifetimes of DPH in Thylakoid Membrane Systems

at 25°C.

a H T 2 Membrane No. of l 4 «2 2 X i- 3 System Expts. C O (ns) (ns)

Intact Thylakoid 6 0.79 1.1 0.21 7.1 5.1 1.5-4.9 (.04) (.4) (.04) (.25) (.1)

DGDG ** vesicles 2 0.68 0.7 0.32 5.5 4.4 2.0-2.6

MGDG ** dispersion 1 0.72 0.5 0.28 5.1 4.4 3.1

TLC-pure MGDG:DGDG 2 0.33 1.2 0.67 6.4 5.9 2.4-2.5 2:1

Granal Membranes 4 0.75 1.4 0.25 7.1 5.0 1.4-4.5 (.04) (.1) (.04) (.3) (.3)

Stromal Membranes 7* 0.56 1.7 0.44 7.6 6.3 1.3-4.3 (.05) (.2) (.05) (.1) (.2)

= average fluorescence lifetime = £ (A^ T^ )/E (A^T.)

(from Chen et al 1977).

a V i= IA.1 * data from one experiment best-fitted a single exponential decay with a lifetime of 6.4 ns.

** - these fractions contained carotenoid impurities (1.5%), which may explain the shorter lifetime. 2

The range of X values for the non-linear regression analysis are given. The figures in brackets represent the standard error of the mean for the measurements. 10 7 FIGURE 12 (A) Decay of DPH fluorescence in thylakoid membranes

(dots) and the best-fit line calculated from deconvolution procedures by assuming a double exponential approximation (aj=0.9,

Tj=0.3 ns, o^O.l, 1^=6.1 ns). The lamp profile is also shown (B)

The insert shows the weighted residuals and autocorrelation funct for the non-linear regression analysis.

The time-scale is given by 0.163 ns per channel.

10 7 FIGURE 13 Decay of DPH fluorescence in stromal and granal fractions of pea thylakoid membranes plotted on a linear scale for comparison with Figure 12. When deconvoluted from the lamp profile the best-fit approximations were double exponentials:

Granal - ai=0.65, Ti=1.4 ns, a2=0.35, T2=6.5 ns.

Stromal - ai=0.57, T!=2.5 ns, A2=0.43, T2=8.0 ns TIME (nanoseconds) 10 7 /a and this balances the effect of greater proportion of the short-

lifetime component so that the average lifetime of DPH in the

pigmented and non-pigmented systems is similar, Grana stack mem-

branes (where the fatty-acid composition is similar to stromal

membranes but the chlorophyll and protein content relative to lipid

are different, see later) show a shorter average lifetime and a

greater proportion of the short lifetime component for DPH fluor-

escence.

These results emphasise the complexity of the thylakoid mem-

brane, and suggests that it is not feasible to draw direct correla-

tions between the fluorescence lifetime of DPH and (possible)

energy transfer in this system, d) Lipid enrichment experiments:

A second investigation of the behaviour of DPH in the thylakoid membrane with fluorecence lifetime measurements has involved the artificial enrichment and increase in the size of the lipid matrix with soya lipid (as described in Section 2.6.3). This approach has been used by Hackenbrock and co-workers (Hackenbrock 1981, Schn- eider 1980) in experiments on the inner mitochondrial membrane.

The average lifetime of DPH in soya lipid vesicles is longer than in the thylakoid and purified chloroplast lipid systems (see

Figure 14) and thus the lifetime of DPH is expected to increase in the lipid-enriched membranes because of this factor. As can be seen in Figure 14 the lifetime of DPH in the lipid-enriched membranes does indeed increase with the level of soya lipid present. An unusual element in these studies, however, was that the lifetime of

DPH in the lightest chlorophyll-containing fraction (with a lipid composition which consists of 95 % soya lipid) was still signifi- SOYA LIPID ENRICHED CONTROL LIPOSOMES FRACTIONS

o CTN

0-3 0-4 0 0-1 0-2 0-3 0-4 Chi ./lipid rafio

FIGURE 14 Variation of the lifetime of DPH fluorescence in soya-lipid enriched

thylakoid membrane fractions with a decreasing chlorophyll:lipid ratio and reduced

buoyant density. The long and short lifetimes (T2 andTi) are shown with the average

lifetime, , calculated as in Table 3. cantly shorter than in the soya-lipid liposomes, and this observa-

tion has been repeatedly observed in these experiments. Again one

possible explanation of this result may be the existence of energy

transfer from DPH molecules, although a further analysis of this

complicated experiment is required, ideally with the use of chloroplast lipids.

The general conclusion from these experiments was that within

the matrix of the thylakoid membrane processes occur which tend to reduce the radiative properties of DPH. Energy transfer from DPH to other chromophores present in the membrane seems to be one possible candidate for this process, although no evidence exists for chlorophyll being the acceptor as judged by chlorophyll fluorescence measurements. This is perhaps an unexpected conclusion when considering the large quantity of chlorophyll pigments present in this system, and the broad overlap of the DPH emission with the chlorophyll absorption spectrum, and it probably reflects the fact that the chlorophyll pigments are located in large pigment-protein complexes, rather than in the lipid matrix (see section 1.3).

Further characterizations of the use of DPH in the thylakoid membrane were also carried out. The time-course of entry of DPH into the thylakoid membrane was detected by measuring the increase in fluorescence from DPH which occurs as it is allowed to partition into the lipid matrix (Figure 15). The rate of entry of the molecule is relatively rapid when compared with previous reports for other membrane systems (Van Hoeven et al 1979), and this may be a reflection of the relatively fluid nature of the membrane. A dependence on the salt conditions was found for the entry of the probe

10 7 ^^vlOmM KCl DPH / 1 /

QLJ // / / V) 1 CD I (_J I ZD 1 MgCl2 «4- 1

"aj I yS c 1 X

i 1 5min

FIGURE 15 Time-course of entry of DPH into thylakoid membranes in

unstacking (lOniM KC1) and stacking (5mM MgC^) media. Initial levels of-fluorescence arise from the thylakoid membranes which were suspended at lOyg ml \ DPH (0.75 yM) was added rapidly through a syringe and the

suspension was immediately mixed. The fluorescence was monitored

continuously.

108 as illustrated in Figure 15. Under low-salt conditions the rate of

entry of the probe was found to be much faster, and under these conditions the thylakoids will be in an unstacked state with a relatively large area of membrane exposed to the medium. Thus, utrastructural differences between the stacked and unstacked condi-

tions may explain these findings.

3.1.1.1 Fluorescence Polarization Measurements with DPH

When DPH molecules in a membrane system are excited with highly polarized light, the fluorescence emitted will also be initially polarized since the absorption and emission dipoles of the molecule lie along the same direction (Shinitzky and Barenholz 1978). If the probe can move out of the plane of polarization of the excitation light within its own fluorescence lifetime (4 to 12 ns for DPH), then the fluorescence emitted will be depolarized to an extent which will depend on the motion of the probe. Since the mobility of the probe is highly dependent on its immediate environment, fluorescence polarization measurements give an estimate of the fluidity of the hydrocarbon region of the membrane. This is the basis of the fluorescence polarization technique, although complications arise with membrane systems since the probe cannot take up all possible orientations with equal probability because of the constraints imposed on its motion by the lining-up of the lipid acyl chains.

Thus, the term 'membrane fluidity' is used here as a generality which does not differentiate between the rate of motion of the probe and the degree of constraint imposed upon that motion. As will be presented later, time-resolved fluorescence polarization measurements can resolve this problem, and provide information on

109 the dynamic motion of the probe and hence the local viscosity as

well as the degree of order of the lipid acyl chains.

3.1.1.2 Steady-State Fluorescence Polarization Measurements

The polarization of DPH fluorescence during the time-course of

entry into the thylakoid membrane reaches a constant value rela-

tively quickly as shown in Figure 16, however a dependence on the

ultrastructure of the thylakoid membranes is again observed. The

possible significance of these results will be discussed later in

connection with fluidity measurements of the stromal lamellae and

granal stack membranes of the thylakoid system.

Fluorescence polarization and anisotropy measurements of DPH

fluorescence were usually recorded at 460nm with excitation light at 360nm, however, the dependence of these measurements on the emission wavelength selected was investigated (Figure 17). The fluorescence polarization values for DPH in thylakoid membranes are relatively independent of the emission wavelength as expected

(Shinitzky and Barenholz 1978), and this behaviour compares with the pigment-free liposomes prepared from soya-lipid and cholesterol

(Figure 17).

The steady-state polarization values determined for DPH in freshly isolated thylakoid membranes typically fell in the range 0.20 to 0.23 (anisotropy values of 0.15 to 0.18). During the course of the day, the polarization values slowly increase and this may be an ageing phenomenon as discussed later in section 3.2.3. These polarization values are typical of a relatively fluid membrane lipid environment compared to other biological membranes, particularly when taking into account the shorter fluorescence lifetime of the probe in the thylakoid system (Van Hoeven et al 1979, Kinosita et

110 i—i—I—r~i—i—i—i—i—i—i—i—i—i—i—i i i

unstacked 0-28 C O thylakoids % 0-26 - —' N

I 0-24 - ^.stacked CL - • thylakoids S 0-22

0-20 i i i i i i i i i i i i i i i i i i 0 10 20 30 TIME (minutes)

FIGURE 16 Steady-state polarization of DPH fluorescence from thylakoid

membranes during the time-course of entry of the probe (see Figure 15).

Stacked thylakoids are suspended in basic medium plus 5 mM MgCl , and

unstacked membranes, basic medium plus 10 mM KC1.

111 0-4 Q

Soya-lipid/Cholest. Liposomes

cz o 0-3- "ru M

ru o CL \ 0-2- Thylakoid Membranes

0-1 n. 420 440 . 460 480 A (nm)

FIGURE 17 Wavelength-dependence of the steady-state fluorescence polarization values of DPH incorporated into thylakoid membranes and soya-lipid/cholesterol (1:1) liposomes.

1 12 al 1981b)-see Table 14. This is not surprising when considering the

high degree of unsaturation of the thylakoid lipids. It is possible

to calculate raicroviscosity parameters for the thylakoid membrane

lipid matrix by using the steady-state polarization values as

described by Shinitzky and Barenholz (1978). The microviscosity

values obtained in this way are usually high (about 1.0 poise)

because the degree of orientational constraint on the motion of the

probe is not taken into account.

3.1.1.3 Time-resolved Fluorescence Polarization Measurements

When a population of fluorescent molecules in an isotropic

medium are excited by a very short (delta) pulse of polarized

light, the time-dependent fluorescence anisotropy ( r(t) ) will

initially be at a maximum ( rQ ), but will undergo a rapid exponen-

tial decay because of the Brownian rotational motion of the fluoro-

phores. This decay can be described by the following relationship (Weber, 1953):

r(t) = rQ exp( -t/^ )

In this equation, t is the time, and ^ is the rotational

correlation time of the molecules given by l/(kT/r. v e) where k is the Boltzmann constant, T is the absolute temperature, n is the

viscosity of the isotropic medium and v is the effective volume of e

the fluorophore (Perrin 1929). With DPH, the rotational correlation

time is essentially a measure of motions about the long axis of the molecule along which the absorption and emission dipoles are oriented.

In artificial and biological membranes the environment of fluorescent probes such as DPH has been shown to be anisotropic, and the decay of anisotropy was found to fit the following form:

113 r(t) = (rQ - r^ ) exp( -t/ ) + r^

Where r^ represents the final anisotropic distribution of the

emitting dipoles.

The steady-state anisotropy ( r ) is related to the time-

dependent anisotropy ( r(t) ) and the total fluorescence ( FT )

decays by the following integral (also see Appendix 2)

t=°° t=°°

rs = /r(t) FT(t) dt j I FT(t) dt

t=0 t=0 Where r(t) is given above and F,j,(t) follows the exponential decay

law-

FT(t) = yiiexp( -t/ T.)

Which was presented earlier in section 3.1.1. Integration of the

above equations yields the following solution (Van Blitterswijk et

al 1981):

r r rs = o " + roo 1 + t/(}>

Where x is the average fluorescence lifetime defined in Table 3 as

< T>. When roo approaches zero, (when an isotropic environment

exists), then the equation becomes:

rg = rQ / (1 + */ )

This is the original form of the Perrin equation (Perrin 1929).

The treatment of the data presented in this section has

therefore been based on a model for the rotation of DPH which is

described by a rapid wobbling motion within a restricted cone (see

Figure 18a). In this theory, the average half-angle of the cone,

) , is decided by the order of the lipid chains, whilst the rate of c wobbling within the cone is an indication of the viscosity of the

114 hydrocarbon region surrounding the probe. The final, limiting anisotropy value, r^ , is therefore related to the the average half- angle of the cones in which the DPH molecules are wobbling:

r / r = [1/2 cose_ (1 + cose )]2 00 O t- (2 The ratio, r / r , is termed the 'degree of orientational con- oo O straint', and has been related to the order parameter, S,^ which is often determined in experiments with spin-labelprobes (see later):

2 s = rj r0

The wobbling-within-cone diffusion constant, Dw, is calculated from both the structural ( r^ , 0C ), and the rotational ( ) parameters derived from the anisotropy decay measurements (Kinosita et al 1981b):

D4(r - r )/ r = -x2(l + x)2[ln{(l -f x)/2} + (1 - x)/2] + ... W ° 00 ° 2(1 - x)

(1 - x)(6 + 8x - x2 - 12x3 - 7X4)/24

Where x = cos0 . The D values give a much better idea of the rate c w of movement of the probe rather than using the reciprocal of the rotational correlation time for this purpose which may give a misleading picture. For instance, the rotational correlation times of DPH in a highly ordered lipid system and in a less ordered membrane may be similar because in the latter case, the probe must move through a much larger angle to reach the limiting anisotropy value (r0 0 ). The Dw values in the less ordered membrane system will be higher since the structural parameters are taken into account.

The viscosity in the cone ( n ) can be calculated from the D c w value, and this gives a much more accurate estimation of the viscosity of the lipid than the 'microviscosity' values calculated from

115 the steady-state polarization values as described earlier.

n c = kT/6DwVe f

Where v is the effective volume of the fluorophore, and f is the e shape factor. The parameter, v f, has been determined for DPH in e liquid paraffin by Kawato et al (1977), and has an average value of

17 x 10~23 cm3.

Estimates of the average half cone angle, and the xviscosity-

within-cone' can thus be obtained from the fluorescence anisotropy

decay data by applying this model, although it should also be noted

that it is possible to fit the data to a different scheme in which

two populations of fluorophores exist; one rapidly rotating, and

the other consisting of very slowly moving molecules (with a rota-

tional correlation time of several hundred nanoseconds- Parola et

al 1979). In fact the wobbling-with in-cone model has become the

more widely accepted, and most of the analysis of fluorescence

anisotropy decay data which has been published has been based on

this theory (Jahnig, 1979).

The decay of anisotropy of DPH fluorescence in thylakoid mem-

branes after a pulse of polarized light is shown in Figure 18b. As discussed earlier in section 2.12, the deconvolution proced^ure becomes more difficult as the pulse width of the lamp becomes broader. In Figure 18b, the FWHM of the lamp is about 6 ns, and the rotational correlation time derived from the deconvolution procedure was about 1.3 ns. Thus, the fluorescence anisotropy of a population of DPH molecules will decay to close to the limiting anisotropy value within the duration of the pulse . Similar experiments were also performed with dispersions of the major thylakoid lipids labelled with DPH, although in these cases the

1 16 FIGURE 18

(a) The motion of DPH within a lipid bilayer is thought to be

described by a rapid wobbling within a restricted cone, thus

the probe cannot take up all possible orientations, and at

long times after a pulse of light the fluorescence remains aniso-

tropic (see (b)). The drawing shows the average half-cone angle

(0 ) for the wobbling motion of the probe.

(b) Decay of fluorescence anisotropy from DPH in thylakoid

membranes. The recorded data (dots), and the calculated decay

using the best-fit parameters from the deconvolution procedure

are shown. The lamp profile is represented by the triangles.

The anisotropy does not decay to zero, but reaches a limiting

value (roo) of 0.12.

117 (a)

lipids

(b) r(f)

1 \ ^^v1 •^••''•••.-•.y:-. •vq 4- .Q .i 00 4— tz ZD O 3-

3 2

100 200 300 400 500 channels ( 0.083 ns per channel)

118 problems of the re-absorption of DPH fluorescence by pigments did not arise, and therefore a weaker lamp pulse with a FWHM of 2 ns was used to excite the molecules.

Various parameters describing the motion of DPH in the thylakoid membrane are presented in Table 4. The accuracy of the deconvolution procedure was assessed by calculating the steady- state anisotropy values using the modified Perrin equation given earlier (Van Blitterswijk et al 1981). The calculated value for rg was then compared with the measured value (see Table 4), and in general the agreement between the two values was within the experimental error involved in the technique.

Table Motional parameters of DPH inorporated into thylakoid membrane systems.

Membrane r r r D S s TO (ns ) e (cafc) c (ns-1) (P) (order param Intact Thylakoids 0.18 0.18 0.12 1.4 48 0.12 0.34 0.55

DGDG vesicles 0.11 0.11 0.01 1.5 77 0.20 0.20 0.16

MGDG/DGDG 2:1 0.13 0.13 0.05 1.5 61 0.16 0.25 0.36 dispersion

Large differences exist in the degree of ordering of the lipid chains between thylakoid membrane and extracted lipid systems. This is particularly true of the DGDG dispersions which showed an extremely low ordering of the lipid chains as probed by DPH, and this is manifested in the decay of the difference fluorescence (F^)

119 to almost zero (see Figure 20). In contrast, the decay of the

difference fluorescence data from DPH in thylakoid membranes

becomes parallel (on a log scale) to the total fluorescence decay

(F,p) at long times when the limiting anisotropy value of about 0.12

has been reached (see Figure 19). The DGDG purified lipids form

lamellar bilayer structures when dispersed in water whilst the

MGDG/DGDG 2:1 mixture has been shown to form unusual non-bilayer

forms (possibly inverted micelle strucures), and this may explain

the differences observed between the two purified systems shown in

Table 4.

To summarize, Table 4 shows that the rate and amplitude of the

wobbling motions of the probe are reduced in the thylakoid membrane

compared to the extracted lipid systems. Thus, the probe can be

said to diffuse more slowly through a much narrower cone in the

thylakoid membranes, and these results will be discussed later in

terms of the possible ordering effect of the intrinsic membrane

proteins on the thylakoid lipids. Although the apparent fluidity of

the intact thylakoid membrane is lower than the extracted acyl

lipid, the parameters shown in Table 4 suggest that compared to

other biological membranes, a relatively fluid lipid environment

exists for DPH (see later Table 15).

3.1.2 ESR Spin-Label Probes

Introduction

The use of spin-labels to estimate the fluidity of the thylakoid membrane has been examined in the introduction to this thesis

(Section 1.7.1) and in the materials and methods section (2.10).

The spin-labelling technique is based on the paramagnetic

120 -I 1 1 f- 10 20 30 40 TIME (ns)

FIGURE 19 Total (F^,) and difference (F^) fluorescence decays, and the calculated best-fit lines for DPH in thylakoid membranes at 25°C. The lamp profile is represented by the triangle symbols

121 TIME (ns)

FIGURE 20 Total (F,^) and difference (P^) fluorescence decays of

DPH in digalactosyl diacyl glycerol (DGDG) vesicles at 25°C (dots).

The best-fit lines for the total fluorescence and the subsequent difference decays after deconvolution are also shown,with the lamp

profile.

1 22 properties of nitroxide radicals derived from five or six-membered rings. The structures of the particular spin-labels used in this work are shown in Figure 21. The features of the ESR spectra of these molecules are largely influenced by their mobility, and a simplified explanation of this phenomenon is presented below.

Consider the extreme cases shown in Figures 22A and B, where the free radical electron of the N*4 atom has three possible spin quantum numbers of +1, 0 and -1 which results in three values for the local magnetic field and hence three resonance positions with the applied magnetic field. If the nitroxide radical is oriented so that the 77-orbital is parallel with the applied magnetic field, the resonance positions are as shown in Figure 22A with a splitting

(Ajj) of approximately 30 gauss. If the TT -orbital is oriented perpendicular to the applied magnetic field, then the three resonance positions are much closer together with a splitting (A^-) of approximately 6 gauss as shown in Figure 22B. In reality the probe is randomly oriented and can occupy positions intermediate to the extremes of Figure 22A and B, and when the motion of the probe is slow enough (with a rotational correlation time of around 100 ns), these intermediate states are resolved so that the spectrum now takes up the idealized form shown in Figure 22C. The actual shape of the spectrum recorded will be similar to Figure 22D, giving rise to a first derivative spectrum shown in Figure 22E.

For fast motion of the spin probe (with a rotational correlation time between 1 and 10 ns), the rate at which the probe moves from one resonance position to another is greater than the difference in the resonance frequency of the two positions. Thus, there

123 COOH COOH

5-DOXYL STEARATE 12-DOXYL- 5-DOXYL DECANE STEARATE

FIGURE 21 Structures of the spin-labels used in ESR measurements of thylakoid membrane fluidity. The molecules are drawn in the position they are expected to occupy in the membrane, with the centre of the membrane at the bottom of the page.

124 is no time for resonance to occur at each position and the resulting resonance frequency gives rise to a single peak at the average of all the resonance positions. For tumbling of the probe with a rotational correlation time less than about 10 ns, the idealized spectrum is shown in Figure 22C' which is detected as single reso- nances at the average positions shown in Figure 22F, resulting in the first-derivative spectrum shown in Figure 22G.

The two ESR spectra represented in Figures 22E and G represent the limits of the time-scale of motion probed by the ESR spin- f or labelling technique, which is useful'detecting tumbling motions of the spin-probes occurring between 1 and 100 ns. The ESR spectra presented in this work are within this time-scale, and therefore show intermediate states between the spectra represented in Figures

22E and G.

As described earlier, for slow tumbling motions, an order parameter was calculated from the splittings of the the outer and inner lines of the first-derivative ESR spectrum, whilst for fast motions a rotational correlation time was determined using the relative peak heights and the centre line splitting.

The control value for the order parameter shown in Table 7 for lettuce thylakoid membranes was typical of the values obtained with

5-doxyl stearate for freshly isolated thylakoids and were found to fall in the range 0.705 - 0.719, the average order parameter being

0.712 +0.005 (standard deviation). These order parameters are lower than ones obtained by Hiller and Raison (1980) for a similar probe

(6-doxyl stearate), and using the same method for calculating the order parameters. They found that the order parameters in barley thylakoids were between 0.76 and 0.77, and for spinach thylakoids

125 /s A

2A,

/N /1\ 4s

B 2AL L

/M*t<

TtTTTTT ^^TT^* T T ^^

E TV

1444444 IH I4-W4-W c'

FIGURE 22 A and B - low, centre and high line splittings of a rigid (single crystal) spin label with the 7T-orbital oriented parallel

(A) and perpendicular to the applied magnetic field (B). i C to E - randomly oriented spin-label rotating slowly i and fast(C -G)

126 order parameters of around 0.73 to 0.74 were reported. The possible

discrepancy between the results obtained in this work and the

values published by Hiller and Raison are discussed below. The

experiments described were used to check the behaviour of the spin-

label in the thylakoid membrane system.

A slow disappearance of the ESR signal from nitroxide probes

is often observed because of the chemical reduction of the nitrox-

ide radical (ascorbate, for example is often used to completely

remove the probe-in-water signal from aqueous suspensions of spin- labelled membranes). In lettuce thylakoids this reduction of the probe was found to be slow (requiring several hours for a noticeable loss in the ESR signal) in the presence of DCMU (5 pM) and in the dark, but the signal was rapidly lost when no DCMU was present and the sample was exposed to room light (see Figure 23). When the labelled thylakoids were illuminated with strong red light the removal of the probe signal occured in less than two minutes, and no spectrum could be obtained. As presented in Figure 23, the reduction of the nitroxide probe is accompanied by an apparent increase in the order parameters obtained from the ESR spectra. A possible explanation for this effect may be that a small population of the probe molecules exist in a highly rigid environment which allows some protection against chemical reduction.

In the paper by Hiller and Raison it is not mentioned whether

DCMU was present, and this may possibly explain the higher order parameters that they obtained for barley and spinach thylakoids.

A second complication in the use of the spin-label probes is the importance of the probe to chlorophyll ratio since at decreasing

127 10 Gauss

2-5 min. s = 0.747

10-5min.s = °-785

36min. s - 0.9

FIGURE 23 Disappearance of the spin-label ESR signal from 5-doxyl stearate in thylakoid membranes, in the absence of DCMU, and under room lighting conditions. The estimated order parameters (S) are shown. probe levels it was found that the order parameter increased until

it was impossible to estimate the order parameter (see

Table 5). The order parameter also increases at the low probe

levels when scans are made at longer periods after the initial

labelling which again suggests that the larger order parameters

arise from the chemical reduction of the probe which is more

noticeable at lower probe to chlorophyll ratios.

Hiller and Raison quote that the lipid to probe ratio in their

experiments was always greater than 100:1 (chlorophyll:probe

~30:1), and so it is difficult to assess whether the

probe:chlorophyll ratio could be an important factor in explaining

the differences in the results. No spectra are reproduced in their

report and thus comparisons between the data are extremely

difficult.

For the reasons presented above, DCMU (5 jiM) was always present

in the samples when fluidity measurements involving spin-label probes were taken and the labelled samples were handled in the

dark. The chlorophyll:probe ratio in the experiments was 50:1 or

less, and is specified in section 2.10.

Spin-labelling experiments with the probes, 12-doxyl stearate and 5-doxyl decane will be presented later in section 3.3.1. These compounds were used to give estimates of various membrane fractions prepared by sonication and Yeda press treatments, and with these probes a rotational correlation time was calculated rather than an order parameter.

129 Table 5 Effect of the spin-label:chlorophyll ratio on the order parameters derived from 5-doxyl stearate in lettuce thylakoids.

Probe:Chi. Time after Ama x \±n S ratio labelling (order (minutes) (gauss) (gauss) parameter)

1:12.5 8 28.4 9.0 0.712

1:25 4 28.4 8.9 0.719

1:25 16 28.4 9.0 0.712

1:50 8 28.5 9.0 0.715

1:100 8 28.4 8.9 0.719

1:140 2.5 28.8 8.6 0.747

1:140 6.5 28.1 8.1 0.768

1:140 10.5 28.5 8.0 0.785

1:200 8 N.D. N.D. ~0.9

1:400 8 N.D. N.D. ~0.9

3.2 Lipid Fluidity and Function

The experiments presented below have used steady-state fluorescence polarization measurements and ESR spin-labels to follow overall changes in the fluidity of thylakoid membranes. These changes were brought about by altering the temperature, allowing ageing of the membranes and by adding sterols to the membrane. The effect of these treatments on the various processes occurringin the thylakoid membrane has also been determined in order to establish any links which may exist between lipid fluidity and function in this membrane.

130 3.2.1 Effect of Temperature

The effect of temperature on the steady-state polarization of fluorescence from DPH incorporated into thylakoid membranes and dipalmitoyl phosphatidyl choline (DPPC) vesicles is compared in

Figure 24. The physical structure of the DPPC fatty-acyl chains changes abruptly at 40°C from the gel to liquid-crystalline state, and this change is characterized by a sudden decrease in the polarization of DPH fluorescence. It is clear from Figure 24 that no such transition can be detected in the pea thylakoid membrane by this technique, rather, a small but progressive change in the polarization values is observed.

A similar temperature dependency of fluorescence polarization values to that observed for thylakoid membranes was observed in liposomes formed from soya lipid. The soya lipid (azolectin) contains a variety of different lipids with many unsaturated acyl chains^ and phase transitions of the type observed in DPPC vesicles were not detected above 0°C

In many of the experiments presented, apparent fluidity changes measured by DPH fluorescence polarization have been linked with the ability of the thylakoid membrane components to undergo large changes in their organization in the membrane in response to changes in the salt levels. The changes in organization cause a change in the degree of spill-over of excitation energy from Photo- system 2 to Photosystem 1 and thus can be monitored using chlorophyll fluorescence measurements (Barber 1980). The effect of temperature on the salt-induced spill-over change in the thylakoid membrane has been investigated by Murata et al 1975 and Rubin et al

131 .. DPPC vesicles 0-4 h O «

-g 0-3 Q O thylakoids a . 0-2 JI V. O• U) N> H— 0-1 h

0 10 20 30, £0 50 60 temp C

FIGURE 24 Steady-state polarization values of DPH fluorescence from thylakoid membranes

and dipalmitoyl phosphatidyl choline (DPPC) vesicles at various temperatures. The

buffer used in both cases was the Hepes-basic medium described in section 2. 1981, both showing that a dependency on temperature existed. Spill- over changes associated with Statel-State2 transitions in Anacystis nidulans have also been found to be highly temperature-dependent, and discontinuities in the temperature-dependency of the transition occur at the same temperature as phase separations in the membrane are observed (Williams et al 1981 - see Table 2).

The effect of temperature on photosynthetic electron transport reactions has been extensively studied, and is reviewed in section

1.8., although it has not been particularly studied in this thesis.

In some of the experiments presented below, flash-induced changes in the absorption of cytochromes f_ and b^g^ were followed as a way of determining the rate of electron transfer in the thylakoid membrane. The effect of temperature on these changes has been investigated and the results are presented in Figure 26. Figure 25 shows the flash-induced difference spectra of the two cytochromes.

In the following section the effect of cholesterol on electron transport reactions in the thylakoid membrane is investigated, and in particular the flash-induced changes in cytochromes f_ and b^g absorption are compared in the normal fluid membrane and in the sterol-treated (more rigid) membranes.

3.2.2 Effect of Cholesterol

Above the phase transition temperature^ cholesterol tends to have an ordering effect on lipid chains in both artificial and biological membranes (Jahnig 1979, VanBlitterswijk et al 1980). This effect is dependent on the rigid steroid nucleus and therefore the ordering of the lipid chains is not so pronounced in the region of the terminal methyl groups at the centre of the lipid bilayer. Cholesterol has been shown to have the opposite effect in

133 540 550 560 570 A(nm)

FIGURE 25 Difference absorption spectra of cytochrome f and cytochrome h^^ at 20°C in pea thylakoids. Conditions are as described in section 2 for 'pseudo-cyclic1 electron flow.

Absorption changes after 2ms and 20ms are shown.

134 cyth redn. _ -1 CD 28 kJ mol ro

l-o-

cxi o

31 kJ mol

3-3 3-4 3-5 3-6 1/T(K ) xio3

FIGURE 26 Arrhenius plot of the reduction of cytochrome b,.^ and cytochrome f in pea thylakoid membranes under conditions of cyclic electron flow. For cytochrome f_ ,the reciprocal of the half reduction time is plotted, and for cytochrome b,.^, the reciprocal half rise time is shown.

Measurements were made in the presence of DCMU, NADPH and ferredoxin as described in section 2.8.

135 the polar head-group region of artificial bilayers formed from

DPPC, and was found to allow a greater mobility of the phospho-

choline moieties (Shepherd and BUldt 1979). These workers suggested

that this effect was caused by the lateral expansion of the hydro-

carbon region by the bulky steroid nucleus, with little steric

contribution to the polar region by the hydroxyl group of the

sterol. Although not investigated in this work, this may be an

important effect in thylakoid membranes where the polar head-group

region is occupied by a high proportion of large galactose resi-

dues.

The effect of cholesterol on the fluorescence polarization

values for DPH in soya-lipid /cholesterol liposomes is shown in

Figure 27. A similar experiment using the Sigma Type II-S soya

lipid has been reported by Van Hoeven et al (1979) and the values

presented in Figure 27 agree closely with their results.

A comparable approach has been used to increase the rigidity of

the thylakoid membrane lipid phase except that the cholesterol is

added exogenously to a thylakoid membrane suspension. The techni-

que described in Section 2.4 resulted in a rapid incorporation of

cholesterol or cholesteryl hemisuccinate into the thylakoid mem-

branes which was found to be dependent on the initial levels of

cholesterol in the incubation medium. This relationship is shown in

Figure 28 (for cholesteryl hemisuccinate) and in Figure 29 (for

cholesterol). The levels of cholesteryl hemisuccinate present in

the final washed pellet begins to level off at high incubation levels where the cholesteryl hemisuccinate accounts for about one tenth of the total lipid phase (by weight). Similar values were

136 £ 0-26-

0-0 0-1 0-2 03 0-4 0-5 Choi./ Lipid Ratio

FIGURE 27 Effect of cholesterol to soya lipid weight ratio on the steady-state fluorescence polarization values of DPH incorporated into the vesicles.

137 03

O ro 0-2 C—

0-0

0 1 2 3 4 5 Choi/Chi. ratio (incubation)

FIGURE 28 Incorporation of cholesteryl hemisuccinate into thylakoid membranes (as determined by the method of Searcy et al 1960) at various initial incubation levels. Polyvinyl pyrrolidone was present in the incubation medium (3.5%). Error bars show the standard error

of the mean from five seperate determinations.

138 i r t 1 1 r

CHOL/CHL pellet

0 • ' • I I 0 5 10 CHOL/CHL incubation

FIGURE 29 Determination of the levels of cholesterol associated

with the washed pellet after incubation with (closed) and without

(open circles) polyvinyl pyrrolidone (PVP).

In the latter case, cholesterol was precipitated in micellar form

with the thylakoid membranes at high incubation levels.

139 found for the cholesterol-polyvinyl pyrrolidone (PVP) system, but

Figure 29 also shows that in the absence of PVP the levels of

cholesterol associated with the washed pellet suddenly increase at an incubation concentration of about 200 jig cm . The sudden in-

crease is due to the precipitation of large micelles of cholesterol

with the thylakoid membranes.

The effect of cholesteryl hemisuccinate treatment on DPH fluor-

escence polarization in thylakoid membranes is shown in Figure 30.

The relatively small levels of the sterol present in the membrane

produce a large response in the DPH polarization values when com-

pared with the results with soya lipid / cholesterol liposomes

shown in Figure 27. Sterol levels producing changes in the polari-

zation values from 0.26 to 0.35 in thylakoid membranes only result

in a change in polarization from 0.18 to 0.20 in the soya lipid

system. The efficiency of cholesteryl hemisuccinate in the thyla-

koid system may well be a reflection of the high levels of protein

compared to lipid present in the membrane, which, as discussed

later, already exert a considerable ordering effect on the lipid

phase.

In Figure 30 the effect of a reduction in the fluidity of the

thylakoid membrane is compared with the salt-induced change in the

chlorophyll fluorescence yield which as stated earlier is suggested

to arise from the lateral reorganization of pigment-protein com-

plexes within the thylakoid membrane. A smaller change in the salt-

induced chlorophyll fluorescence yield may arise from the restrict-

ion of the motion of pigment-protein complexes within the thylakoid

membrane as the order of the lipid phase is increased by the intro-

duction of cholesteryl hemisuccinate. Although not presentd here,

140 B^S

CWO <3 S PK S3 O w H

12 3 4 5 CHOL/CHL. RATIO (INCUBATION)

FIGURE 30 Effect of cholesteryl hemisuccinate on the steady-state fluorescence polarization values from DPH incorporated into the thylakoid membrane (closed circles), and the percentage increase in chlorophyll fluorescence upon addition of MgC^ (5 mM). Error bars shown are the standard error of the mean for four separate experiments.

141 essentially the same results have been obtained when cholesterol was used instead of cholesteryl hemisuccinate to increase the rigidity of the thylakoid membrane.

Cholesterol was found to cause an increase in the order parameter of the spin-label 5-doxyl stearate in soya lipid / cholesterol vesicles as shown in Table 6.

Table 6 Order parameters (S) of 5-doxyl stearate in soya lipid / cholesterol vesicles.

% CHOLESTEROL A A , S ^ , \ max rain (by weight) (order (gauss) (gauss) parameter)

0 26.8 8.9 0.684

20 27.3 8.9 0.694

50 28.2 8.9 0.715

The changes in the order parameter presented in Table 6 are significantly different because the maximum order parameter for this probe in a biological membrane would be about 0.9, whilst the minimum would be about 0.6 and so the changes in the order parameter observed show relatively large differences in the fluidity of the membrane lipids.

The treatment of thylakoid membranes with increasing levels of cholesteryl hemisuccinate also results in the reduction of lipid fluidity as probed by 5-doxyl stearate. The first-derivative ESR spectra of 5-doxyl stearate in control membranes and in membranes

142 containing the highest levels of cholesteryl hemisuccinate are

shown for comparison in Figure 31 and the order parameters calcu-

lated from the spectra are tabulated in Table 7. The cholesteryl hemisuccinate caused a broadening of the outer splitting (2AIucl X) , and a narrowing of inner line splitting (2Am^n).

Table 7 Order parameters of cholesteryl hemisuccinate-treated lettuce thylakoids probed with 5-doxyl stearate.

Cholesterol: Aaax Vin S Chlorophyll (order (incubation) (gauss) (gauss) parameter)

CONTROL 28.2 9.0 0.708

1:1 28.5 8.6 0.747

2:1 29.3 8.6 0.758

4:1 29.6 8.6 0.763

The effect of cholesterol in reducing the fluidity of the thylakoid membrane was therefore concluded from these studies, and the possible result of this effect on electron transport reactions in the thylakoid membrane was investigated.

The inhibitory effect of cholesterol treatment on the steady- state rate of photosynthetic electron transport 0^0 to FeCNj? ) by thylakoid membranes (measured with an oxygen electrode) is shown in

Figure 32. Uncoupled rates are shown in Figure 32, although it was found that the cholesterol treatment tended to uncouple electron transport, an effect which may be related to the xfluidizing'

1 43 10 Gauss

i x5 magnification

FIGURE 31 First-derivative ESR spectrum of 5-doxyl stearate incorporated into cholesteryl- hemisuccinate treated membranes (dotted line) with an initial incubation level of 4:1 sterol .the to chlorophyll (see Figure 28). The solid line shown is the same probe incorporated into control 1 1 BOO J 1 1 1 L 3_ H2O - FeCN, o • l_J cn V E s • • ,200 s — N ZD cr • ^ X • QJ \ N. • S

V. QJ \ • ^ V fU • \ _ 100 \

•

1 1 1 1 l l l 0 1 2 3 4 5 6 CHOL/CHL ratio (incubation)

FIGURE 32 Effect of cholesterol on the steady-state rates of linear electron flow in pea thylakoid membranes. The rate was measured by monitoring the evolution of oxygen in a Clarke-type oxygen electrode at saturating light intensities.

145 action of cholesterol in the head-group region discussed earlier.

In these experiments it was difficult to decide whether the reduction in the rates of electron transport by cholesterol treatment was due to the reduction in fluidity in the membrane, or whether the inhibition was due instead to a non-specific chaotropic action by the sterols (for instance a denaturation of intrinsic membrane proteins).

In an effort to resolve this problem, the rate of electron transport after a short, saturating flash of light was measured, as described in section 2.8.2. With this approach chaotropic action of the sterols should be detected since the resulting amplitude of the absorption changes due to the oxidation and reduction reactions will be reduced. If chaotropic action occurs more specifically around PS 2 then cytochrome f_ would not be expected to be fully reduced after several short flashes. Electron transport (uncoupled) from H2O to methyl viologen or ferricyanide was measured by this technique by following the rapid oxidation (a few microseconds) and slower re-reduction ( 10-20 milliseconds) of cytochrome f_ by monitoring absorption changes at 554 nm (with 540 nm as the reference wavelength). Under these conditions, after a few saturating flashes the rate of reduction of cytochrome £ will depend mainly on the rate of diffusion of plastoquinol to the

Rieske centre from Photosystem 2, and thus this may give a good indication of possible effects arising from lipid fluidity changes, particularly if the cytochrome b/f complex is located at a relatively long distance from the appressed membrane regions (Olsen etal 1980, Barber 1980, Anderson 1980). Thus, when DCMU is added to the thylakoid membranes under non-cyclic conditions, no absorption

146 change at 554 nm is detected since cytochrome f does not become re- reduced by Photosystem 2 as the reduction of plastoquinone is blocked by this inhibitor (see Figure 33), Figure 33 clearly shows that the absorption change at 554 nm is bi-phasic under non-cyclic conditions, and both phases are inhibited by the presence of DCMU.

The effect of cholesteryl hemisuccinate on the rate of reduction of cytochrome f_ is shown in Figures 34 and 35. Figure 34 shows that the amplitude and kinetics of the absorption changes at 554 nm are very similar when either potassium ferricyanide or methylviologen are used as the terminal electron acceptors. However, in both cases the rate of reduction of cytochrome f_ is slower in the presence of cholesteryl hemisuccinate, whereas the amplitude of the change remains relatively unchanged. Figure 35 shows the effect of cholesteryl hemisuccinate at various levels in the incubation medium, in general a progressive reduction in the rate of cytochrome f_ reduction was found, although at the higher levels a small decrease in the amplitude of the absorption change was detected, possibly suggesting some chaotropic action by the sterol. Figures 34 and 35 show the absorption changes on different time scales to emphasise the biphasic nature of this change. The signal-to-noise ratio in these experiments is relatively good (c.f. Olsen et al 1980), but nevertheless it is difficult to assess whether the effect of cholesterol is more marked on the slow or the fast components of the reduction from this data; a further improvement in the signal to noise ratio in these type of measurements may resolve this question.

Cholesteryl hemisuccinate treatment was found to have no inhi-

147 FIGURE 33 Flash-induced absorption changes at 554nm with 540mn as

the reference wavelength (henceforth termed cytochrome _f absorption

changes). Two phases are present under conditions of linear electron

flow, both are removed by the presence of DCMU (5 yM). The oxida-

tion of cytochrome f is too rapid to be resolved even on this fast

timescale. The half-reduction time (Tx) in the non-inhibited case 2 is 5 ms. "3 The reaction medium (2 cm ) consisted of chloroplasts (at 50 yg 3

per cm chlorophyll), MgCl2 5mM, KCl 38mMi valinomycin 6yM, niger-

icin 6yM, methyl viologen (or potassium ferricyanide) 0.5iriM.

The buffer used was the one described in section 2.8 at pH 8.3.

(50mM Tricine, 0.33M sorbitol)

FIGURE 34 Effect of cholesteryl hemisuccinate (incubation ratio 0.5:1

with chlorophyll) on the reduction kinetics of cytochrome f_ with

different electron acceptors from Photosystem 1. Other conditions as above. On the slower time-scale the slow and fast components of the reduction are more clearly resolved in the control traces.

The half reduction times for these traces are (from top to bottom): 3_

T^ control (H20 to FeCN^ ) 8 ms, +cholesteryl hemisuccinate- 24 ms.

TA control (E 0 to methyl viologen) 6 ms, +cholest. 24 ms.

148 control

T IAI/I= 3-6-10 JL H20- MV

DCMU A 10ms

CONTROL H20 - FeCNg"

•CHOL.

CONTROL H20- MV

CHOL.

T -4 A 40ms A I/I =7-2* 10 i.

149 H20-MV

control

T -4 A I/±I = 3-6x|0

chol: chl 0-5 : l

chol: chl 2 : i

10ms

FIGURE 35 Effect of cholesteryl hemisuccinate at various initial incubation levels on the rate of cytochrome f reduction at 20°C.

Cytochrome f^ reduction was followed at 554-540nm, and methyl - viologen (MV) was used as the electron acceptor from Photosystem 1.

Half reduction times were measured as (top to bottom), 4ms (control)

10 ms, 14 ms.

150 bitory effect on the rate of cytochrome f_ reduction under ^psuedo- cyclic' conditions (Figure 36b). Under these conditions the plastoquinone pool is kept fully reduced by the presence of sodium dithionite (1 mM) in the medium. As before, some chaotropic action of the sterol is observed at high levels, where the amplitude of the absorption change is smaller than control levels, but in this case the rates of reduction remain rapid, and appear to be even faster than control rates. Under these conditions the reduction and subsequent re-oxidation of cytochrome b^^ can be detected, and the results shown in Figure 36a suggest that again there is no detect- able effect of cholesterol on this process even at the relatively high levels which cause some decrease in the amplitude of cytochrome _f oxidation.

The effect of cholesterol and cholesteryl hemisuccinate on electron transport in the thylakoid membrane are probably complex, and will be discussed in greater detail later, however at low levels, the main effect of the compounds seems to be an inhibition in the rate of plastoquinol oxidation under linear electron flow from PS 2 to PS 1.

3.2.3 Effect of Ageing on Lipid Fluidity

If freshly isolated thylakoid membranes are allowed to age at room temperature for several hours, an increase in the DPH fluorescence polarization values is detected (see Figure 37), and as observed with the sterol treatments, the apparent reduction in membrane fluidity is accompanied by an inhibition of the salt- induced changes in chlorophyll fluorescence.

151 554-540

FIGURE 36 Effect of cholesteryl hemisuccinate at various initial incubation levels on the rate of (a) cytochrome b,.^ reduction and oxidation, and (b) cytochrome f reduction. The measurements are at

20°C under conditions of psuedo-cyclic electron flow.

The reaction medium was similar to that shown in Figure 33 except that DCMU was added (lOpM), and dithionite (ImM) was used as an electron donor. No electron acceptor was added. Anaerobic conditions -3 were maintained with a glucose (5mM), glucose oxidase (0.1 mg cm ) -3 and catalase (0.2 mg cm ) system.

152 The mechanism of ageing (in terras of chemical changes) in the thylakoid membranes has not been investigated in this work, although experiments by D.J. Chapman (unpublished results) have shown that a small loss in the acyl lipid occurs, and that MGDG seems to be particularly sensitive to this process. Henry et al

(1982) have shown a similar loss of acyl lipids during ageing of spinach chloroplasts at room temperature, with a reduction in the amount of MGDG by 25%. These small changes in the acyl lipid content may possibly explain the rise in DPH polarization values, since as discussed later, a reduction in the size of the lipid matrix leading to increased protein to lipid ratios may be associated with increased ordering of the acyl chains. An increased protein to lipid ratio has been associated with ageing in other membrane systems (Bartosz 1981), although many other processes may also be occuring during this phenomenon.

153 150 030 „

CZ o O ZJ roN dd 100 0-25

QJ O to ID ro QJ L_ LJ £c 50 0-20 CL Q

0 1 2 .3,. * ... 5 6 7 ageing rime (hr)

FIGURE 37 Effect of ageing at room temperature and in the dark on the steady-state polarization of

fluorescence from DPH in thylakoid membranes (squares). The percentage increase in chlorophyll fluores

after the addition of MgCl~ to destacked thylakoids is also shown (circles) 3.3 Lateral Heterogeneity in the Thylakoid Membrane

The thylakoid membrane of higher plant chloroplasts normally

consists of appressed membrane regions (grana) with interconnecting

lamellae which are not appressed (stroma lamellae), and recently

evidence has been presented that the two regions are distinguished

by the pigment-protein complexes they contain (see section 1.5).

There seem to be four major intrinsic protein complexes in the

thylakoid membrane, Photosystem 2 (and light-harvesting Chl. a/b

proteins); Photosystem 1 and the associated light-harvesting

antennae proteins; the cytochrome b^/f complex (with the Rieske

centre) and part of the ATP-synthetase complex (termed CFQ) Ander-

son 1982. The location of the Photosystem 2 units seems to be in

the appressed membrane regions while the stromal lamellae contain

the ATP-ase and Photosystem 1 protein complexes. The location of

the cytochrome b^/f complex is not clear at the present time,

however, since conflicting evidence exists that: a) the complex is

distributed evenly between the two regions (Cox and Andersson 1981,

Anderson 1982), and: b) that the complex is not present in the

appressed membranes (Henry and Moller, 1981).

A knowledge of the location of the b^/f complex is vital for the

completion of the new, structural model of the photosynthetic

electron transport chain which requires that a mobile electron

carrier exists between Photosystem 2 and 1 units. Abg/f complex

that was located in the stromal lamellae would receive electrons

from plastoquinol molecules diffusing from the appressed membrane

regions under conditions where linear electron flow between Photo- system 2 and Photosystem 1 was occuring. On the other hand, if the bg/f complex is evenly distributed between the two membrane regions

155 then the role of the mobile electron carrier may also be fulfilled

by plastocyanin which would probably diffuse along the inner thyla-

koid surface.

3.3.1 Characterization of the Thylakoid Membrane Fractions

Details of some of the properties of the membrane fragments

obtained from Yeda press disruption are given in Table 8.

Table 8^ Photosystem One and Two Activities of Pea Thylakoid

Membrane Fragments. (Figures shown are the average of three sep-

arate preparations and represent micro-equivalents (O2) per mg

chlorophyll per hour.)

Fraction Chi a/b PS2 activity PS1 activity ratio (H20 - DCIP/Ascorbate- Benzoquinone) Methylviologen

Intact Thylakoids 3.2 250 670

High Salt

Granal 2.9 170 350

High Salt

Stromal 6.0 <10 680

Low Salt

Heavy Fraction 3.1 140 710

LighLow tSal Fractiot n 3.3 170 510

The stromal lamellae fractions obtained by mechanical fractionation in high-salt buffer had high chlorophyll a/b ratios and showed essentially only Photosystem 1 activity. In contrast the granal fraction had lower chlorophyll a/b ratios than the intact

156 thylakoid membranes and had reduced Photosystem 1 activity. To obtain membranes with a high Photosystem 2 to Photosystem 1 ratio phase-partition methods have recently been developed by Andersson et al (1976), but this method is lengthy, and the stromal and granal membrane fractions are subjected to different external conditions which may induce changes in the physical properties of the different membrane fractions. Membrane fractions derived from thylakoids which had been incubated with low-salt buffer (which induces unstacking and a randomization of the pigment-protein complexes-

Andersson et al 1980) were not enriched in either Photosystem 1 or

Photosystem 2 activities as expected.

3.3.2 Fluidity Measurements of Stromal and Granal Membranes

The steady-state fluorescence polarization values of DPH incorporated into granal and stromal thylakoid membranes prepared by Yeda press and sonication treatments are shown in Figure 38. Granal membrane fractions were always found to have much higher polarization values than the stromal lamellae fraction possibly suggesting a less fluid environment for DPH. In general it was found that sonication produced stromal fractions which were less enriched in

Photosystem 1 (on a chlorophyll a/b ratio basis) and that the granal fractions were more enriched in Photosystem 2 than the Yeda press-prepared fractions. This relationship is shown in Figure 38 where the membrane fractions most enriched in chlorophyll Jb_ have the highest DPH fluorescence polarization values.

Fractions prepared from thylakoids incubated in low-salt conditions (xrandomized' membranes) showed DPH fluorescence polarization values which were intermediate to the extremes of the stromal and granal membranes (see Table 9). Intact thylakoids which

157 DPH •

0-30- GRANAL

A ¥0 Pt o

0-25- • • Q STROMAL

- o

Chi a/b ratio

FIGURE 38 Polarization values (P) at 20°C of diphenylhexatriene fluorescence in stromal and granal lamellae prepared by sonication (closed circles) and Yeda press (closed circles) treatments of pea chloroplasts. The Chi a/Chl b ratio is plotted on a reciprocal scale.

158 had not been subjected to the fractionation and centrifugation

forces were found to have slightly lower polarization values when

labelled with DPH than the ^randomized' membrane fractions,

possibly because less ageing had occured in the intact system (see

section 3.2.3).

Table DPH Fluorescence Polarization Values of Pea Thylakoid

Membrane Fractions. (Fractions were prepared by Yeda press and

the measurements were made at 20°C. The data are from four separate

experiments.)

Fraction P values (+ S.E.) Chl. a/b ratio (+ S.E.)

Intact Thylakoids 0.262 + 0.002 3.23 + 0.14

High Salt Granal 0.290 + 0.005 2.77 + 0.06

High Salt Stromal 0.250 + 0.004 5.70 + 0.32

Low Salt Heavy Fraction 0.285 + 0.004 3.01 + 0.11

Low Salt Light Fraction 0.273 + 0.001 3.28 + 0.05

The differences in fluorescence polarization between the granal and stromal fractions are much larger than changes that could arise simply from the difference in the fluorescence lifetime of

DPH in the two membrane systems (see Table 3). A reduction in the average fluorescence lifetime from 6.3 to 5.0 ns would only account for a small change in the fluorecence polarization (from 0.25 to

159 0.26) in stromal lamellae membranes. The lack of relationship

between the fluorescence lifetime and the steady-state DPH polar-

ization values is also demonstrated in the intact thylakoids where

the fluorescence lifetime is comparable with granal membranes, but

the polarization values measured with DPH are much lower than in

the granal membranes.

The temperature-dependency of the steady-state polarization values of DPH embedded in stromal and granal membranes is shown in

Figure 39. The difference between the polarization values of the

two membranes is maintained over a wide temperature range, and no distinct phase transitions could be observed using this technique in accordance with the observations on intact thylakoid membranes.

Time-resolved fluorescence depolarization measurements of DPH in stromal and granal membranes have also been taken, although the results presented are initial studies with lower total counts than was obtained in the measurements on total thylakoid membranes presented in Table 4, and so the final parameters obtained are not as accurate. However, large differences in the ordering of the lipids in the two membrane fractions is suggested from the results of these experiments shown in Table 10.

Table 10 Motional parameters of DPH embedded in granal and stromal thylakoid membrane fractions obtained from pea chloroplasts.

- — Fraction r r r (j) 9 ^c S S (cllc) °° (ns) C (ns"1) (P)

Granal Membranes 0.22 0.23 0.145 2.7 45 0.05 0.76 0.61

MembraneStromal s 0.16 0.15 0.057 2.6 60 0.08 0.46 0.38

160 temp °C

FIGURE 39 Effect of temperature on the steady-state polarization of fluorescence

from DPH embedded in stromal (squares) and granal (triangles) membranes. The fractions were suspended in 33% ethanediol to maintain a fluid medium below 0°C. ESR spin-label measurements were also made to extend the fluor-

escence polarization measurements. The apparent rotational correla-

tion times of the spin-labels 5-doxyl decane and 12-doxyl stearate

embedded in the thylakoid membrane were calculated from the ESR

first-derivative spectra as described in section 2.10.2 (see Figure

7). Figure 40 shows the first-derivative ESR spectra of 5-doxyl

decane embedded in stromal and granal membranes prepared by sonica-

tion of lettuce chloroplasts with a four-fold expansion of the

centre line width which was used for more accurate measurements.

The ESR spectra of 12-doxyl decane in stromal and granal membranes

is shown in Figure 41. The stromal membranes appear to have a more

fluid lipid matrix than the granal membranes using both spin-labels

as judged at the first approximation by the broader centre line

width and smaller outer line heights in the granal membranes (Fig-

ures 40 and 41). Less distinct differences can be observed between

the first-derivative ESR spectra of 5-doxyl stearate embedded in

the two membrane regions (see Figure 42) but the order parameters

calculated from these spectra again indicate a less ordered lipid

environment for the probe in the stromal lamellae fraction.

The calculated rotational correlation times (Tj andx q) of the

spin-labels 5-doxyl decane and 12-doxyl stearate incorporated into

the various thylakoid membrane fractions is shown in Figure 43a and b. The data clearly shows that in the fraction derived from the stromal lamellae membranes both spin-labels are able to tumble more rapidly than in the granal membrane fraction, in agreement with the

DPH fluorescence polarization measurements. The rotational correlation times of the control, low-salt treated membranes which represent the randomized membrane system with no stromal or granal

162 FIGURE 40 First derivative ESR spectra at 20°C of granal and stromal membranes prepared from lettuce chloroplasts by sonication and labelled with 5-doxyl decane. Gain setting is the same for both spectra. Also shown is a four-fold expansion of the field sweep to give a more accurate estimate of the centre line width (AH ). o

FIGURE 41 As in Figure 40, but using the spin-label,

12-doxyl*stearate. As before, the granal membrane spectrum is represented by the dashed line.

FIGURE 42 As in Figures 40 and 41, but using the spin-label

5-doxyl stearate. The estimated order parameters from these spectra were: S=0.697 (stromal membranes)

S=0.713 (granal membranes)

163 FIGURE 42 fa] [b] 5-DD • 12-DS 1-8- • 1 1 • -7

1-6-

-6 K X ' GRANAL GRANAL Nsec • • Nsec • • 1-2- -5 • • • O U 8 10- STROMAL STROMAL -I*

o 0-8- <8 o o o 1 6 5 I 3 Chi a/5 ratio

FIGURE 43 (a) Rotational correlation times (T), at room temperature, of 5-doxyl decane in stromal and granal lamellae prepared by sonication of lettuce chloroplasts. The parameters T

(open circles) and T^, (closed circles) are plotted.

(b) As in (a), but using 12-doxyl stearate. The Chi a/

Chi b ratio is plotted on a reciprocal scale.

165 regions were found to be intermediate to the extremes of the stromal and granal systems in agreement with the DPH measurements.

The temperature-dependence of the tumbling motion of the spin- label 5-doxyl decane in stromal and granal membrane fractions was investigated and the results, plotted on a logarithmic scale are shown in Figure 44. In both stromal and granal membranes the slope of the log-plot is linear over a range of physiological temperatures with an activation energy of about 11 kJ mol""1 but a steady deviation from the straight line begins at around 0°C in granal membranes, and at about -8°C in the stromal lamellae vesicles. At all temperatures the spin-label tumbles more slowly in the membrane fraction derived from the granal stacks.

3.3.3 Composition of the Stromal and Granal Fractions

The protein to lipid ratio in the thylakoid membrane fractions studied was obtained by measuring the lipid to chlorophyll ratio and the protein to chlorophyll ratio for each fraction as described in section 2. Large differences in the protein to chlorophyll and lipid to chlorophyll ratios existed between the stromal and granal membranes, the stromal fraction containing much higher ratios in both cases (particularly lipid to chlorophyll which was about double the ratio found for granal membranes). Differences also existed between the protein to chlorophyll and lipid to chlorophyll ratios in all the membrane fractions isolated from greenhouse pea plants and plants grown in a growth cabinet. The lipid to chlorophyll and protein to chlorophyll ratios of greenhouse grown and growth cabinet pea thylakoids are presented in Table 11. The complete results for the protein to lipid ratios of the thylakoid

166 3-4 3-5 3-6 3-7 3-8 3-9 temp1 (K"1) *io3

FIGURE 44 Arrhenius-type plot of the effect of temperature on the rate of rotation of the spin-label 5-doxyl decane in granal and stromal membrane fractions prepared by sonication of lettuce chloroplasts.

167 membrane fractions for six sets of experiments with pea chloroplasts are presented in Table 12.

Table 11 Relative levels of Protein, Acyl Lipid and Chlorophyll in

Pea Thylakoid Membrane Fractions Obtained from Greenhouse (GH) and

Growth Cabinet (GC).

Fraction Protein:Chl Lipid:Chl Protein:Lipid ratio ratio ratio

Intact Thylakoid (GH) 6.6 3.7 1.8 (GC) 5.0 3.1 1.6

High Salt Granal (GH) 5.3 3.2 1.7 (GC) 4.2 2.6 1.6

High Salt Stromal (GH) 9.6 7.7 1.2 (GC) 5.3 4.3 1.2

Table 12 Relative Levels of Protein and Acyl Lipid in Thylakoid

Membrane Fractions. (Ratios given are by weight, + Standard Error, for six experiments, except where stated. Pea thylakoid membranes were used and fractionation was acheived using a Yeda press.)

Fraction Protein:Lipid ratio

TotalThylakoid (n=4) 1.98 + 0.33

High Salt Granal 1.84 + 0.16

High Salt Stromal 1.22 + 0.11

Low Salt Heavy Fraction 1.65 + 0.13

Low Salt Light Fraction 1.70 + 0.24

168 The protein to lipid ratios in the stromal fractions is significantly different from those of the granal membrane fractions

(P < 0.002). Intact thylakoid membranes which were not subjected to the mechanical disruption procedure were found to have protein to lipid ratios which were not significantly different from the granal membrane fraction and the unstacked (randomized) membrane systems.

The fatty-acid composition of the thylakoid membrane fractions was also investigated (see Table 13):

Table 13 Fatty-Acid Composition of the Thylakoid Membrane Frac- tions. (Percentage composition + standard error, in brackets, are shown for 3 experiments with pea thylakoid membrane fractions prepared by Yeda press treatment.)

Fraction Fatty-Acid Type

16 ;0 18;0 18; 1 18; 2 18; 3

Total Thylakoid 10.2(0. 2) 1.6(0.1) 1.6(0.1) 8.8(0.9) 77 .8(0.7)

High Salt Granal 9.3(0. 1) 1.8(0.2) 1.6(0.1) 8.0(0.8) 79 .3(0.6)

High Salt Stromal 11.8(0.7 ) 2.4(0.5) 1.8(0.2) 11.1(0.1) 72 .9(1.4)

Low Salt Heavy 9.5(0. 2) 1.8(0.1) 2.3(0.4) 9.5(0.5) 76 .9(0.3) Fraction

Low Salt 10.0(0. 3) 1.8(0.1) 1.6(0.2) 8.4(0.8) 78 .2(1.1) Light Fraction

169 Small differences exist between the fatty-acid composition of the stromal membrane fraction and the other membrane fractions, although the differences shown (a reduction in the level of unsaturated fatty-acids) would not be expected to account for an increase in the lipid fluidity in this region, in fact the opposite would be expected. Thus it seems as though the increased lipid fluidity in the stromal membranes may arise because of the lower levels of protein compared to acyl lipid in this region. These results will be discussed in detail later, and the possibilty of contamination of the stromal membrane fraction with non-thylakoid lipids will be examined.

3.3.4 Lipid Fluidity and Temperature Sensitivity of Photosystem 2

The results presented in the previous section would suggest that a more rigid lipid environment exists for Photosystem 2 than for Photosystem 1, and that unstacking of the thylakoids and the subsequent randomization of the particles within the membrane may reduce the stability of the Photosystem 2 environment.

Inactivation of Photosystem 2 occurs at temperatures above 30°C, and this inactivation is followed by a sudden increase in the chlorophyll fluorescence yield (Schreiber and Berry 1977, Raison and Berry 1980). Raison and Berry (1980) found that the temperature at which the mid-point of the heat-denaturation of Photosystem 2 occurred was dependent on the growth conditions of the plant that they studied (Nerium oleander). Inactivation of Photosystem 2 occurred at higher temperatures in plants grown in elevated temperatures (in Death Valley, California). The plants resistant to the higher temperatures were found to have a much less fluid thylakoid

170 lipid matrix, as determined by the ESR spin-labelling technique.

Thus it seems feasible that the stability of Photosystem 2 activity

at high temperatures is dependent on the existence of a relatively

non-fluid, ordered state of the thylakoid lipid matrix as discussed

in section 1.5.5.

Figure 46 shows the increase in modulated chlorophyll fluoresc-

ence in pea leaves (grown in the growth cabinet) heated from 20°C under different illumination conditions. Leaves were illuminated under the conditions described in section 2.9.2 for five minutes before heating was commenced so that the plants were either in

State 1 or State 2 prior to heating. Figure 45 shows that for the pair of leaves studied in this experiment the denaturation of

Photosystem 2 occurs at higher temperatures when the plants are in

State 1 (minimum spillover state). This effect was found to be variable depending on the leaves selected. The temperature was measured by a thermocouple held against the upper surface of the /by leaf' a glass slide, and so it was expected that factors such as leaf thickness would produce large variations. The results of nine pairs of experiments were analysed and the denaturation temperature for leaves in State 1 was found to be 40.7 + 0.5 °C (standard error), and for leaves in State 2 the denaturation temperature was found to be 38.0 + 0.6 °C. The differences between the two sets of data are significant at the 0.5% level (Students xt' test).

Similar heating experiments were also performed with Class 2 chloroplasts in the stacked and unstacked state. In these experiments the temperature could be followed accurately by a thermocouple in the cuvette, and variations were found to be small. Low levels of light were used to excite the chlorophyll fluorescence

171 and potassium ferricyanide was used as the electron acceptor in

these experiments. Figure 46 shows typical heating curves of chl-

oroplasts in the stacked (5mM MgC^), or unstacked (10 mM KCl plus

0.1 mM EDTA) conditions. Stacking seems to confer extra stability

to the Photosystem 2 units at high temperatures. Less variation was

observed in these experiments, and the final temperature at which

half the total fluorescence increase occured was 44.7 + 0.2°C

(standard error) for the stacked state, and 39.8 + 0.8°C for the unstacked state. The differences observed were significant at the

0.1% level.

The possible relationship between lipid fluidity and the stabi-

lity of Photosystem 2 will be discussed later.

172 FIGURE 45 Percentage increase in chlorophyll fluorescence from pea leaves heated at approximately 2°C per minute illuminated with State 1 and State 2 light regimes (see section 2).

FIGURE 46 Chlorophyll fluorescence increase as a percentage of the maximum during heating of isolated pea thylakoids in the unstacked or stacked condition. Heating rates were as for Figure 45. Membranes were prepared and suspended in low cation medium (basic medium), and _3

then diluted to lOyg cm in a medium containing 10 mM KC1 (for un-

stacking), or 5mM MgC^ (for stacking). The salt-induced fluorescence

changes were allowed to stabilize before the heating was begun.

173 Chi. fluor. increase (%of total)

ro o^ oo Q o o>

% increase in Chi. fluor. 4 Discussion

In the previous section the results of various experiments on

the thylakoid membrane were presented, and three main points can be

concluded:

1) That the thylakoid membrane seems to have a relatively fluid

lipid matrix, (although not as fluid as extracted thylakoid

lipids).

2) That treatments which were shown to reduce the fluid nature of

the lipids were also found to reduce the rate of linear electron

flow.

3) That the physical properties of the hydrocarbon matrix in the

stromal and granal regions of the membrane appeared to be different.

A more detailed examination of these conclusions and of the techniques used to estimate lipid fluidity will be discussed in this section.

4.1 Comparisons With Other Biological Membranes

The fluorescent probe DPH has been widely employed to probe the lipid structure of many biological membranes and therefore the thylakoid membrane can be compared with other membrane systems.

In principle, steady-state polarization values are not completely reliable as comparative measures of membrane fluidity for the reasons previously discussed, but as a rough guide, polarization values for freshly isolated thylakoids of about 0.20 to 0.23 would tend to place them at the xfluid' end of a league of biological membranes (see Table 14). However it must be born in mind, that since the lifetime of DPH is shorter in thylakoid membranes than in many other biological systems, the steady-state polarization values

175 may in fact be higher than expected.

Table 14 A comparison of the steady-state fluorescence polarization

values of DPH embedded in various biological membranes at 25°C.

Membrane System Polarization Value Ref.

Purple Membrane (Halobacterium halobium) 0.41 1

Human Eye Lens Cortex 0.40 2

Rat Novikoff Ascites Hepatoma 0.35 2

Rat Hepatoma 484A 0.34 2

Human Erythrocyte Plasma Membranes 0.33 2,3

Rat Liver Plasma Membranes 0.32 2

Mouse Thymocytes 0.31 2

Ascites Tumour Cell Plasma Membranes 0.28 3

Mitochondrial Membranes 0.24 1

Sarcoplasmic Reticulum Membranes 0.22 1

Pea Thylakoid Membranes 0.20-0.23

Rat Liver Endomembranes 0.21 2

Human Milk Fat Globules 0.12 2,3

REFERENCES: (1) Kinosita et al 1981b. (2) VanBlitterswijk et al

1981. (3) VanHoeven et al 1979

More accurate comparisons between the thylakoid membrane and other biological membranes can be made with the time-resolved fluorescence polarization data presented in section 3.1. The data again indicates a relatively fluid membrane (see Table 15) compared to many other biological lamellae.

176 KEY:

D 17 STU-mouse fibroblast tumour cells

BHK 21... Baby hamster kidney cells

N-Egg avian strain of influenza virus

NDBK-MDBK ...Newcastle disease virus grown in MDBK cells

HeLa human tumour cells

BR bacteriorhodopsin (purple membrane fragments)

REFERENCES:

(1) Kinosita et al 1981b. (2) Hildenbrand and Nicolau 1979 (3) Sen£ et al 1978. Table 15 Time-resolved fluorescence depolarization studies of biological membranes probed with 1,6-diphenyl-l,3,5-hexatriene: Dynamic and structural (order) parameters.

Membrane Temp r r S D Ref, s 00 0 4> w System ( C) c (ns)

BR purple 35 0.30 0.28 30 0.81 1.9 0.05 0.87 1 25 0.32 erythrocyte (human) 35 0.22 0.20 37 0.72 1.1 0.10 0.42 1 25 0.25

NDV-MDBK 25 0.25 0.23 30 0.77 1.1 0.06 0.69 2

N-Egg 25 0.24 0.22 32 0.74 1.8 0.04 0.97 2

HeLa 25 0.20 0.16 41 0.64 2.1 0.06 0.73 2

LM fibroblasts 25 0.20 0.15 42 0.62 2.1 0.06 0.69 2

BHK 21 25 0.17 0.13 45 0.57 1.9 0.07 0.55 2

D 17 25 0.17 0.12 47 0.54 1.9 0.08 0.51 2 mitochondrial 35 0.14 0.09 53 0.48 1.3 0.14 0.29 1 25 0.17 sarcoplasmic reticulum 35 0.13 0.09 53 0.48 1.3 0.14 0.30 1 25 0.16 pea thylakoid 25 0.15 0.12 48 0.56 1.3 0.12 0.34

L 1210 mouse leukemic 25 0.11 0.07 47 0.43 2.6 0.05 0.82 3 The properties of the thylakoid membrane are similar to the highly

fluid mouse leukemic L 1210 cell membranes and the D^y mouse fibro-

blast membranes. At 25°C, the thylakoid system also seems to have simi-

lar properties to the mitochondrial and sarcoplasmic reticulum

membranes (at 35°C) studied by Kinosita et al (1981b), and thus a

slightly more fluid environment for DPH in the thylakoid membrane

would be expected if mitochondrial and thylakoid membranes were

compared at the same temperature. The steady-state anisotropy

values of the mitochondrial membranes (rg = 0.17) are slightly

higher at 25°C than the thylakoid membranes (r = 0.15) as shown in O Table 15.

In Table 15, the biological systems which have been studied by

the time-resolved fluorescence depolarization technique are listed

in descending anisotropy values. As can be seen, the steady-state

anisotropy (and hence polarization) of fluorescence from DPH is

mainly decided by the structural (order) parameters. In contrast

the viscosity (within cone) values show no strict relationship to

the steady-state anisotropy values. This conclusion has also been

reached by Van Blitterswijk et al 1981 who have used steady-state anisotropy values to provide order parameters rather than micro- viscosities which were originally obtained in this way (Shinitzky and Barenholz 1978). For the thylakoid membranes, a low order parameter (0.56) and a low viscosity (0.34 poise) are indicated from the time-resolved measurements with DPH, and thus at least in this system, the steady-state anisotropy and polarization values seem to give a less ambiguous estimate of x fluidity'.

The lipid matrix of the thylakoid membrane would therefore seem

179 to have physical properties similar to the more fluid biological

membranes, although most of the membranes studied so far by the

fluorescence polarization technique seem to be much more rigid

(particularly the plasma membranes).

The lipid composition of the membrane is unusual in that no

sterols are present and the fatty-acid residues are highly unsat-

urated (see Figure 3). The unusual composition of the thylakoid

membrane does not fit very easily into the current models of a

typical biological membrane with a complement of sterols, phospho-

lipids and proteins. For instance, Rhomer et al (1979) have sugg-

ested that in the molecular evolution of all biological membranes

the presence of ^stiffening' molecules such as sterols (in euka-

ryotes) or their structural equivalents, tri- and tetra-terpenoids

(in prokaryotes) was required to maintain a stable bilayer stru-

cture. The thylakoid membrane seems to lack a likely Nstructural

equivalent' of the sterols and terpenoids as a candidate for this

role.

Kinosita et al (1981b) have suggested that as far as motions

within the hydrocarbon layer are concerned a biological membrane could be represented by the equation:

biological membrane = unsaturated lipid + protein + cholesterol

They suggested that the precise compositions within each term on the right-hand side of the equation are not as important as the relative amounts of the three components.

From the data presented in the previous section it would seem feasible that in thylakoid membranes the absence of sterol molecules may be compensated by the very high levels of protein mole-

180 cules present in the membrane which could exert a Stiffening' or

ordering effect on the lipid chains. Thus the simplistic rela~

tionship above may be applied to the thylakoid system if the abs-

ence of sterols is balanced by the presence of high levels of

protein (2:1 by weight with lipid). In other membrane systems where

very high protein to lipid ratios exist, (such as the purple mem-

brane and the mitochondrial membranes- see Kinosita et al 1981b) a

similar ordering phenomenon has been suggested. The similarities

between the physical properties of the (inner) mitochondrial mem-

brane and the thylakoid system are particularly interesting since both participate in electron flow and phosphorylation. In both cases only trace amounts of sterols can be detected in the membranes which contain many lipid molecules with highly unsaturated acyl chains (cardiolipin in mitochondria, galactolipids in thylakoid membranes), Quinn and Chapman 1980, Tanford 1980. It remains to be seen whether the unusual lipid composition of these two specialized membrane systems plays a significant role in factors such as charge stabilization or proton translocation, although the highly fluid nature of both membranes may well be a requirement for the lateral movement of electron carriers (Hackenbrock 1981, Barber

1980,1982).

Some experiments that support the application of the simple equation presented above to the thylakoid lipid matrix are summarized below. a) Preparations of thylakoid lipids (MGDG and DGDG) which consti- tute about 60% of the total acyl lipid were found to have a much lower ordering when extracted and purified than in the intact

181 membrane (see Table 4). Hiller and Raison (1980) observed a similar

difference between the isolated lipid and the intact membrane using

ESR spin-labels. Comparisons between the intact membrane system and

isolated galactolipids are, however, complicated by the fact that

the purified lipid dispersions form unusual non-bilayer structures

as discussed earlier in section 3.

b) Less ordering of the lipid chains was found in stromal membranes

(xwobbling' cone angle 60°) than in granal membranes (cone angle

45°) where protein to lipid ratios were consistently higher. The

degree of ordering of the lipid chains in stromal lamellae appears

to be almost as low as in the extracted galactolipid mixture,

possibly suggesting that the ordering effect of protein is only

detected by DPH depolarization measurements at the elevated protein

to lipid ratios found in the granal membranes.

c) Stralka and Subcynski (1981) have shown that spin-labels are

more freely mobile in thylakoids extracted from plants treated with

protein synthesis inhibitors, and in immature thylakoids where the

protein levels are reduced, and few grana are present.

d) Finally, the increased ordering of lipid acyl chains by intrin-

sic membrane proteins (at high protein:lipid ratios) has been

frequently observed in model membrane systems ( Kinosita et al

1981a, Jahnig 1979 ) and has been treated theoretically by Jahnig

(1981). In some cases a disordering action of protein has been

detected (especially below the phase transition temperature) in

artificial membrane systems composed of saturated fatty-acids

(Chapman et al 1979).

It would therefore seem as though the major factor in deciding whether an ordering or disordering action is detected when large

182 amounts of protein are present in a lipid matrix is the relative

order of the protein compared to lipid, and this has been examined

in detail by Jahnig (1979,1981a and 1981b). He has suggested that

an order parameter of about 0.8 can be estimated from fluorescence

anisotropy measurements of dansylated protein segments in a

membrane, and thus no ordering effect of membrane protein should

arise when the lipid ordering approaches this value (for instance

when the lipid is in the gel-state). This hypothesis would also

predict that the ordering effect of protein would be considerably

reduced in the hydrocarbon region close to the polar head-groups

where the lipid chains are already highly ordered (see for

instance, Table 7 where 5-doxyl stearate gives approximate order parameters of 0.7 for the thylakoid membrane. Thus, the effect of high levels of intrinsic membrane proteins on acyl lipid should be most pronounced at the fluid centre of the bilayer (the region probed by DPH and 5-doxyl decane), and it is interesting to note that this hypothesis seems to be supported by the data presented by

Hiller and Raison (1980). They found that the order parameters were only 14% higher in the chloroplast than in the extracted lipids for the probe 6-doxyl stearate, whilst a 33% increase in the order parameter was detected for the more deeply embedded nitroxideprobe

(12-doxyl stearate). Further evidence that would suggest that this hypothesis may apply to the thylakoid membrane comes from this work where it was found that the differences between stromal and granal membrane fractions were most easily observed when the probes 12- doxyl stearate and 5-doxyl decane were employed to estimate lipid fluidity rather than the spin-label 5-doxyl stearate (for instance,

183 compare Figures 40,41 with 42).

In conclusion, it seems feasible that in the thylakoid membrane of some higher plants the fluidity of the lipid matrix may be at least partly controlled by the relative bulk levels of unsaturated lipid compared to protein. In particular, this hypothesis may have direct relevance to the study of the mechanisms of adaptation by plants to varying temperature regimes (winter and summer, for example). Initial reports seem to indicate an increase in the lipid matrix relative to protein levels in thylakoids isolated from cold-grown pea plants (Chapman et al 1982). The conclusions presented here may also have relevance to studies of the greening process in higher plant chloroplasts. Stralka and Subcynski (1981) have found that immature (greening) thylakoids which contain few grana have a higher lipid fluidity than mature, fully developed chloroplasts. They suggested that the differences observed were due to an increase in the proteinrlipid ratio during greening which they did not associate with the formation of grana stacks in the mature chloroplasts. The work presented in this thesis, however, would emphasise the close relationship between grana formation, the proteinilipid ratio and the fluidity of the thylakoid lipid matrix.

The interpretation of the fluidity measurements of the thylakoid membrane which have been discussed above has been based to some degree on the assumption that an even distribution of the probes exist in the lipid matrix. The validity of this assumption is discussed below.

184 4.2 Distribution of the Probes

A knowledge of the location of the spin-label and fluorescence

probes within the thylakoid lipid matrix is important in the

interpretation of the results presented in this thesis. On this

question, the experiments presented in Figures 15 and 16 on the

rate of entry of the probe, DPH may be examined. The incorporation

of DPH into the membranes shows a marked dependence on the salt

conditions in the suspension, and in an ^nstacking' medium the

incorporation of DPH is relatively rapid compared to the stacked situation with an apparent half-incorporation time of less than 0.5 minutes. In the stacked state, the incorporation is much slower,

(half-incorporation time of 2.5 minutes), and a possible explana-

tion for the differences observed could be that initially the DPH molecules penetrate into the exposed surfaces of the grana stacks and into the stromal lamellae before diffusing into the appressed membrane regions. It is therefore interesting to note that in the stacked condition the steady-state polarization values of DPH fluorescence initially start at a low value before reaching the same level as in the unstacked membranes where the polarization values immediately stabilize to a constant value. These results would be consistent with a model where DPH molecules incubated with stacked membranes at first penetrate mainly into the more fluid lipid matrix in the stromal lamellae.

The results shown in Figures 15 and 16 are from slightly aged membranes, and it was found that with both DPH and the spin-label

5-doxyl decane, the partitioning of the probe into the thylakoid membrane was lower in aged membranes and at lower temperatures. It would seem as though the probes initially partition into fluid

185 lipid areas, and at early times the main contribution of the fluorescence will come from the probe molecules in these areas. At long times, though it seems that a more even distribution of the probe exists, as suggested from the results presented in Figure 16 where the polarization values of DPH fluorescence are not dependent on stacking after about 10 minutes of incubation. Raison and Hiller

(1980) also found that order parameters derived from ESR spin-label measurements were identical in stacked and unstacked membranes.

According to Figure 16, the effect of unstacking would therefore seem to cause a randomization of fluid and rigid lipid areas as predicted from the results presented on the fluidity of stromal and granal regions.

During the course of the work presented in this thesis, an attempt has also been made to investigate the distribution of DPH across the thylakoid lipid bilayer, and this has involved the use of DPH-analogues which have a polar group attached at one end. The rationale behind this approach was that the DPH analogues are expected to be oriented so that the polar group is located in or close to the head-group region of the bilayer. The fluorescence properties and polarization values measured with these analogues of

DPH were then compared those of DPH.

The analogues used were trimethylamine-DPH (TMA-DPH), and dime- thylamine-DPH (DMA-DPH), where the nitrogen atom is attached to the para-position of one aromatic ring and three or two methyl groups respectively. Thus, TMA-DPH is positively charged at neutral pH, whilst DMA-DPH is neutral (pKa =3. 9 for the positively charged form of DMA-DPH). It was found that both probes showed much higher

186 polarization values when incorporated into thylakoid membranes, as

is shown below:

PROBE DPH TMA-DPH DMA-DPH

Polarization Value (P) 0.232 0.313 0.448

These results are consistent with the idea that DPH is located

towards the centre of the lipid bilayer which as discussed earlier has a much more fluid environment than the region close to the polar head-groups (see Hiller and Raison 1980). The very high polarization value given for DMA-DPH is probably an anomalous one, since it was found that this probe was rapidly broken down in thylakoid membrane systems so that the fluorescence excitation and emission spectra were grossly changed.

The location of DPH in artificial membrane systems has often been suggested to be towards the centre of the bilayer (Shinitzky and Barenholz 1978) with an average orientation in the same direction as the lipid chains (Andrich and Vanderkooi, 1976) in agreement with the conclusions of these experiments with DPH and DPH analogues.

The problem of relating the fluidity measurements presented in this thesis to the rate of lateral diffusion of components in the thylakoid lipid matrix is discussed below.

4.3 Possible Links Between Lipid Fluidity and Lateral Diffusion

The lipid fluidity measurements using fluorescence polarization and ESR techniques described earlier were indications of the rate and the degree of restriction of the rotational motion of DPH and the spin-labels, and it has been shown that the rotational

187 properties of a molecule are dependent on the viscosity and order

of the lipid matrix. The question of whether the rate of lateral

diffusion of protein or lipid components is sensitive to these same

factors has been investigated by several workers, and recently, the

study of the lateral diffusion of membrane-bound components has

been advanced by a new technique called "fluorescence recovery

after photobleaching" (FRAP). This technique involves the rapid

bleaching by a laser beam of a population of fluorescent molecules

situated in a discrete area in the membrane. The beam from the

laser is immediately attenuated several thousand-fold after bleaching and is used to excite fluorescence from the bleached area which is initially low, but fluorescence is ^recovered' from the bleached segment of membrane as fluorescent molecules from the non- bleached areas diffuse in. In this way a simple analysis of the

fluorescence recovery yields a lateral diffusion co-efficient for the fluorescent-labelled compound within the membrane system.

Saffman and Delbruck (1975) predicted that, to a first approximation, diffusion coefficients would be inversely proportional to the viscosity of the membrane and the vertical length of the diffusing molecule, and in their theory the diffusion co-efficient was relatively unaffected by the cross-sectional area of the molecule.

On the other hand, Schindler et al (1980) have studied the lateral diffusion of lipopolysaccharides, and phospholipids in a reconstituted membrane system by the FRAP technique, and could not recon- cile their data with the theory of Saffman and Delbruck. They found that the lipopolysaccharide molecules with similar vertical dimensions as the phospholipids, but with a much larger cross-sectional area did not diffuse at the same rate. Moreover the temperature

188 dependence of the diffusion coefficients of the two molecular

species were different as well as the response to high protein

levels in the reconstituted system. These workers suggested that

the lateral diffusion of membrane components may be approximated

to the ^hopping' motion of substances through a polymeric network

where a %hole' in the matrix must be created before the component

can move. It seems likely that this theory would only apply direct-

ly to the lateral motion of small molecules of the same dimensions

as the lipids since the formation of protein-sized ^holes' in the

lipid matrix would be highly unfavourable. Clearly, comparisons can

be drawn between the relative rates of rotational and lateral

motion of components within a membrane system, and it seems likely

that in an increasingly viscous environment both types of motion

will be slower. However, when specific lipid-lipid or protein-lipid

interactions exist in the membrane, then a polymeric structure may be formed, and in this case rotational motion may be relatively unaffected whilst lateral diffusion is severely reduced.

In some cases, neither the theory of Saffman and Delbruck nor the model of Schindler et al would seem to apply, for instance in many cell membranes (particularly plasma membranes) it has often been found that the rate of lateral diffusion of membrane proteins is highly dependent on interactions between the cell cytoskeleton and intrinsic membrane proteins (Jacobson and Wojcie- szyn,1981). For the thylakoid membrane, no influence due to the cytoskeleton is expected, and in this particular system the physical properties of the lipid matrix should play an important role in controlling the lateral diffusion of membrane components.

189 The possibility that a relationship between lipid fluidity and

lateral protein movements can exist in the thylakoid system has

been explored, particularly in section 3.2. where possible links

between DPH fluorescence polarization values and the salt-induced

chlorophyll fluorescence changes have been investigated.

When thylakoid membranes are prepared in a medium containing low

salt levels and EDTA, the appressed membrane regions unstack and

this process is concomitant with a randomization of the pigment-

protein complexes, Photosystem 2 and Photosystem 1. Upon the

addition of cations, (100 mM monovalent or 5 mM divalent), the

negative charges on the thylakoid membrane surface are more

effectively screened and this allows a closer approach of the

lamellae. At the same time, the lateral separation of the protein

complexes into areas of low and high charge density (the appressed

and stromal lamellae regions respectively),occurs and chlorophyll

fluorescence rises because of the reduction of energy spill-over

from PS 2 to PS 1. If stacking of the randomized lamellae is

brought about by lowering the pH to the isoelectric point of the

membrane (about pH 4.3-Nakatani et al 1978), then charge

neutralization rapidly occurs, the photosysterns remain randomly

distributed, and thus no chlorophyll fluorescence increase is obs-

erved (Barber et al 1980, Andersson et al 1981).

In this thesis, therefore, the chlorophyll fluorescence increase was employed as a sensitive indication of the dynamic properties of

the thylakoid membrane, and in many of the experiments where a

reduction in the fluidity of the lipid phase was observed, a concomitant inhibition of the salt-induced chlorophyll fluorescence increase occurred. Evidence that the lateral movements of pigment-

190 protein complexes are involved in salt-induced chlorophyll fluor-

escence and stacking changes has also come from experiments I have

conducted in collaboration with Dr. W.S. Chow and Prof. J. Barber.

We found that a continuous thylakoid membrane connecting the granal and stromal regions is a requirement for the salt-induced chlorophyll fluorescence changes and this further emphasises the close relationship between these events and the properties of the membrane (Chow et al 1981).

A complex theoretical analysis of the temperature sensitivity of the kinetics of the salt-induced chlorophyll fluorescence rise has been presented by Rubin et al (1981), and diffusion co-efficients 11 o of 3 x 10 cm /s at 30°C were calculated for the lateral mobility of PS 2 complexes. This value is not inconsistent with other experimental determinations of lateral protein diffusion co-efficients

(Schindler et al 1980, Flanagan 1980).

4.2 The Effect of Lipid Fluidity on Electron Flow

A reduction in the fluidity of the lipid matrix of the thylakoid membrane may be manifested in increased packing and ordering and decreased mobility of the lipid acyl chains. The effect of this change on protein-protein and protein-lipid interactions and on the diffusion of lipid-soluble compounds may well cause a reduction in the rate of photosynthetic electron flow.

In this thesis exogenously supplied sterols have been used to artificially increase the rigidity of the thylakoid membrane,

(these compounds must also have a secondary effect in slightly increasing the size of the lipid matrix). The ordering action of

191 the sterols cholesteryl hemisuccinate and cholesterol in the thyl-

akoid membrane appears to be similar to their effect in other

biological and model membrane systems (i.e. an overall reduction in

lipid fluidity by the condensing of the acyl chains- see Kawato et

al 1978,) however since the protein levels are very high in this

membrane, the action of cholesterol may not be quantitatively

identical to model systems such as the soya-lipid - cholesterol

system. For instance, cholesteryl hemisuccinate and cholesterol have a large affect on lipid fluidity in the thylakoid system

despite the relatively small levels ( 10% of the total lipid) which are incorporated (compare Figure 27 with Figure 30 and Table 6 with

Table 7).

The reason for the greater efficiency of the sterols in the

thylakoid membrane compared to model systems is not clear, but a possible explanation may involve the fact that high levels of proteins are already present in this membrane, and therefore the presence of small levels of an extra ordering group may push the

system from a fluid to a more densely packed and ordered state.

Cholesteryl hemisuccinate was used in many experiments rather than cholesterol because it has been found to incorporate more effectively into biological membranes, although its effect seems to be identical to that of cholesterol (Shinitzky et al 1979).

The results presented in this thesis on the possible link between lipid fluidity and electron flow were part of a more extensive research programme carried out in collaboration with Dr. Yasusi

Yamamoto and Prof. James Barber. The overall aim of this project was to investigate the effect of lipid fluidity on many processes occuring in the membrane including electron flow, proton flux and

192 lateral protein movements (connected with changes in the distribu-

tion of light energy between different pigment-protein complexes).

The results of some of these experiments have already been published (Yamamoto et al 1981).

In the paper mentioned above it was shown that cholesteryl hemisuccinate inhibited the flow of electrons between water and ferricyanide (under continuous illumination), whilst the rates of both proton uptake and release were reduced. A double reciprocal analysis of the initial rates of ferricyanide reduction versus light intensity showed that the efficiency of the photoreactions was relatively unaffected by the sterol treatment, however, the rate-constant of the dark rate-limiting step at infinite light intensity showed a 33% reduction with a cholesterol: chlorophyll ratio of 0.3 in the membranes.

To a large extent, therefore, the results presented in this thesis on electron transport in sterol-treated membranes are complementary to my earlier collaborative studies, but for reasons previously discussed, the measurements of flash-induced electron transport provide information on possible non-specific chaotropic action of the sterols.

A more detailed discussion of these results must examine the possible role of plastoquinone as a long-range electron carrier between the electron transport components located in the appressed membrane regions and those found in the stromal lamellae.

As discussed earlier the rate-limiting step of uncoupled linear electron flow appears to be the oxidation of plastoquinone by the

Rieske iron-sulphur centre, and thus if this process is dependent

193 on the diffusion of plastoquinol through the lipid matrix, then changes in the fluidity of the membrane may be expected to be manifested in the overall rate of electron flow. The evidence supporting the concept of plastoquinone as a lipid-soluble mobile electron carrier is summarized below: a) There seems to be strong evidence that a large proportion of the plastoquinone pool is located in the lipid matrix. At least 90% of the total plastoquinone can be extracted by organic solvents resulting in the loss of electron transport activity, which can be reconstituted by the re-addition of the plastoquinone (see section

1.5.2.1). b) Some of the plastoquinone present in the membrane seems to be bound to protein and in the case of the binding of the plastoquinone Q^ by the DCMU-binding protein of Photosystem 2 this association is reversible when the quinone is fully reduced, suggesting that the plastoquinol molecule is then released into the larger plastoquinone pool (see section 1.5.2.2). Plastoquinone has also been found associated with the cytochrome bg/f complex (Hurt and

Hauska 1981), although it is not clear whether this binding is also reversible. c) Plastoquinone appears to be located throughout the thylakoid membrane (as would be expected of a mobile electron carrier), and as stated above, it is often extracted with both Photosystem 2 and cytochrome b^/f complex preparations. On the other hand if plastocyanin was the main mobile electron carrier, then the b^/f complex should be located close to Photosystem 2 in the appressed membrane regions, and the requirement for plastoquinone to diffuse through the lipid matrix over relatively long distances would be

194 removed. On this point there is conflicting evidence: i) Cox and Andersson (1981) have shown that the cytochrome complex is not enriched in either stromal lamellae vesicles or inside-out vesicles (from the apressed membrane regions). ii) Henry and Moller (1982) have prepared inside-out vesicles which were very highly purified and contained virtually no Photosystem 1 complexes, and reported that they found only very small amounts of cytochrome b^g or cytochrome f_ in these preparations. iii) The cytochromes are also absent from Triton-prepared Photo- system 2 particles which are oxygen-evolving and contain cytochrome t>559 and plastoquinone (Berthold et al 1981, Lam and Malkin 1982).

Evidently this question of the distribution of cytochromes b^g and f_ is not yet resolved, but it is important for this discussion, since if plastoquinone is responsible for the long-range tranport of electrons, then the bg/f complex may be expected to be located in the stromal lamellae and margins of the grana, close to Photo- system 1. The speed of oxidation of cytochrome f_ by Photosystem 1

(a few microseconds - see Figure 33) would certainly argue that the bg/f complex and the Photosystem 1 complex are located close together. On the other hand, if the b^/f complex is evenly distributed between the stromal and granal regions of the membrane, as argued by Cox and Andersson (1981), and Anderson (1980), then it seems likely that plastocyanin must communicate between cytochrome f and Photosystem 1 with extremely rapid kinetics. It must be pointed out that in the experiments of Cox and Andersson, cytochromes b^g and f_ were also found in the stromal lamellae fractions, and so at least this population of cytochromes is situated at

195 a relatively long distance from Photosystem 2 complexes, and thus if this population is to participate in linear electron flow the existence of a prior mobile electron carrier must be postulated, d) Results presented in this thesis: When comparing the effect of cholesteryl hemisuccinate on linear and psuedo-cyclic electron flow presented in Figures 34 to 36, it can be seen that cholesteryl hemisuccinate treatment only results in a reduction in the rate of cytochrome f_ reduction under conditions where the electrons are coming from Photosystem 2. Under pseudo-cyclic conditions where electrons are supplied from dithionite to the plastoquinone pool, no inhibition of the rate of cytochrome f_ reduction is observed,

(in fact the rate may be stimulated slightly). If the effect of cholesteryl hemisuccinate in the thylakoid membrane is purely to decrease the fluidity of the lipid phase, as indicated by the DPH polarization and ESR spin-labelling measurements, then these observations would argue that the function of plastoquinone is dependent on the fluidity of the lipid matrix only when it is functioning to shuttle reducing equivalents from PS 2 to PS 1. When plastoquinone is reduced by dithionite its ability to reduce cytochrome _f and

^563 seems t0 be relatively unaffected by lipid fluidity. In this case plastoquinone in close proximity to, or bound to the cytochrome complex could be directly reduced by the chemical reductant.

It should also be mentioned that a mechanism of electron flow incorporating plastoquinone molecules has been suggested which allows a dynamic electron transfer process not necessarily requiring the presence of tightly (protein) bound plastoquinol molecules

(Rich and Bendall 1979).

Previous explanations for the mechanism of electron transfer

196 from plastoquinol (PQ^), which is a two-electron donor, to the

bg/f complex, which accepts one electron at a time, have invoked

the presence of a binding site where the intermediate plastosemi-

quinone radical (PQH*)> which is unstable, can be preserved for

long enough to allow the transfer of the second electron. In the

model of Rich and Bendall, electrons and protons would be rapidly

passed between plastoquinone molecules and the bg/f complex with a

bimolecular collision mechanism occuring within the lipid matrix.

This process is thermodynamically unfavourable under equilibrium

conditions since the PQ^/PQH* redox couple is high compared to

the acceptor potential. However the model predicts that the rea-

ction would be allowed to proceed by the rapid removal of product

from the plastoquinone pool by the b^/f complex (and also, ultima-

tely, by the light-driven reactions of Photosystem 1). Thus it

seems feasible that, at least in theory, the concept of a dynamic,

collisional process of electron transfer between the plastoquinone

pool and the bg/f complex is possible, and therefore the role of

lipid fluidity would be particularly emphasised in this model.

4.3 Stromal and Granal Membrane Fractions

Early studies of the protein to lipid ratios in thylakoid membrane fractions obtained from spinach by Allen et al (1972) suggested that no significant differences existed between the granal and stromal lamellae. Their estimates of the relative protein and lipid levels included chlorophyll as part of the total lipid, but, as discussed earlier, the light-harvesting molecules are widely be- lieved to be tightly bound in the pigment-protein complexes of the

197 photosystems. When the weight due to chlorophyll is subtracted from

the data presented by these workers, the protein to acyl lipid

weight ratios in granal and stromal membranes become approximately

1.9 and 1.7 respectively, with the value for unfractionated thyla-

koids being 1.9. These values for protein to lipid ratios are very

similar to the the ones for pea thylakoid fractions presented in

Table 12 except that a higher protein to lipid ratio was found by

Allen et al for their stromal membrane fractions compared to the

pea stromal lamellae used in this study. Even in this earlier

study, however, a lower protein to lipid ratio was indicated for

stromal lamellae compared to granal membranes.

Allen et al also reported that a much greater ratio of

chlorophyll to galactolipid was found in the granal membranes in broad agreement with the results presented in Table 11. This observation had been made previously by Wintermans (1971) (also for spinach sub-chloroplast fragments). High ratios of galactolipid to chlorophyll were also reported by Bishop et al (1971) in the chloroplasts of the bundle sheath cells of maize and sorghum which lack appressed membrane regions and are highly enriched in Photo- system 1 activity. In contrast they found that chloroplasts extracted from the mesophyll cells of these plants had much lower levels of galactolipid compared to chlorophyll. The mesophyll cells are enriched in Photosystem 2 activity and contain chloroplasts with extensively stacked lamellae and low chlorophyll a/b ratios.

Unfortunately data on the protein to lipid ratios of bundle sheath and mesophyll chloroplasts is not given in the paper by Bishop et al, and it seems that an investigation of the lipid fluidity and

198 protein to lipid levels in these unusual chloroplasts is worth attempting.

In the reports on sub-chloroplast fractions discussed above the

differences in galactolipid to chlorophyll ratios were discussed in

terms of a model in which the chlorophyll molecules were localized

in the lipid bilayer rather than in protein complexes. At the time,

therefore, it would seem that the relative levels of chlorophyll to

galactolipid were considered more important than the protein to

galactolipid levels.

The fatty-acid composition of the stromal membranes appeared to

have some small but consistent differences from the other membrane

fractions as shown in Table 13. According to this data, the stromal

membrane fraction contains slightly more saturated fatty acid resi-

dues, and these differences are most noticeable in the relative

amounts of 18:3 and 18:0. These differences in the fatty-acid

composition should not be expected to explain the higher lipid

fluidity in the stromal regions since, as discussed earlier, a

lower degree of unsaturation is generally associated with a red-

uction in lipid fluidity.

The data presented in Table 13 should also be discussed in terms

of a possible contamination of the stromal fraction by mitochondria

and chloroplast envelope material (both would cause a lower degree

of fatty acid unsaturation). If we apply the (possibly incorrect) assumption that the actual fatty acid composition of stromal membranes is identical to granal membranes, then the contamination of

the stromal fraction by non-thylakoid material can be estimated

from the percentage of 18:3 present and is calculated to be about

10%. Contamination of the stromal fraction by this level of mito-

199 chondrial material would probably give rise to a higher estimate of

the protein to lipid levels since the ratio by weight for the

mitochondrial inner membrane is about 3:1 and about 2:1 in the

outer membrane (Quinn and Chapman, 1980). Furthermore, contamina-

tion by mitochondrial membranes would not be expected to cause an

increase in the lipid fluidity (see Table 14 in this section). The

actual level of contamination of the stromal fraction is difficult

to assess and may well be lower than 10% for the following reasons:

a) The assumption that the fatty-acid composition is similar in

stromal and granal membranes may not be correct (for instance, different lipid compositions have been identified in the different chlorophyll protein complexes isolated by detergent treatments

(Rawyler et al 1980). b) If the differences in the fatty acid composition observed in

the stromal membrane fraction were arising from contamination similar changes should also be observed in the light membrane fraction obtained in the same way as the stromal membranes, but from unstacked membranes. As shown in Table 13, the fatty acid composition of this control fraction is similar to the other fractions and does not show a lower level of fatty-acid unsaturation. c) The fatty acid residues of the stromal fraction were often found to be more susceptible to break-down during extraction than the heavy (granal) membrane fraction which contained most of the membrane material. The breakdown of lipid during extraction generally leads to a dramatic loss in the highly unsaturatedfatty-acids such as 18:3 which are more susceptible to oxidation reactions. The reason for this extra sensitivity of the stromal fraction is not

200 clear, but this factor may partly explain the small differences in

fatty-acid composition observed.

In conclusion it would seem as though a large contamination of

the stromal fraction by non-thylakoid material is unlikely, and the

small differences in the fatty-acid compositon of the granal and

stromal membrane fractions may be due to either a geniune lateral heterogeneity in the lipid matrix, or to a selective break-down of

the stromal fraction. In order to answer this question carefully a

thorough investigation of the lipid composition of the two membrane regions is necessary, with an analysis of the fatty-acid composition within each lipid class. Initial results from this kind of study have indicated that, within experimental error, no differences exist between the lipid composition of the stromal and granal lamellae isolated by Yeda press treatment of pea thylakoids (D.J.

Chapman, unpublished results). These findings would further emphasise that the fluidity differences observed between the stromal and granal fractions seem to arise mainly from the different protein: lipid ratios in the two regions, rather than differences in the composition of the lipid matrix.

The results discussed in this section would therefore suggest that the fluidity of the lipid environment in the stromal lamellae is significantly higher than in the granal regions, and whilst this observation is interesting in itself, the possible significance of this lateral heterogeneity in lipid fluidity has been investigated.

For instance, a randomization of the protein:lipid ratio throughout the thylakoid membrane can be acheived by unstacking, and one result of this process should be a less ordered environment for

PS 2. Possible effects of this change in environment on the fun-

201 ctioning of PS 2 are discussed below.

As discussed earlier, the stability of PS 2 at elevated temperatures may depend, to some extent, on its lipid environment, and this suggestion has been advanced by Schreiber and Berry (1977) and

Raison and Berry (1980) from studies of the high-temperature denaturation of Photosystem 2 in leaves of plants adapted to different temperature regimes. The levels of saturated fatty-acids present in thylakoids isolated from the plants adapted to high temperature were much greater than in thylakoids extracted from plants grown at normal temperatures (20°C). The change in the lipid environment of

PS 2 due to unstacking could therefore lead to a significant reduction in the conformational stability of the complexes, particularly at high temperatures, and and an attempt has been made to investigate this possibility by following the chlorophyll fluorescence yield of the PS 2 complexes. As shown in Figure 46, the rise in chlorophyll fluorescence associated with the irreversible inactivation of PS 2 occurs at lower temperatures when the thylakoids are in the unstacked condition.

The results presented in Figures 45, show that a similar xdesta- bilization' of the PS 2 units occurs when leaves are subjected to blue-green lighting conditions which induces the maximum spillover state (State 2). In recent models, a close relationship between the salt-induced (in vitro) spillover changes and the in vivo Statel-

State2 changes has been suggested (Barber 1982), and a reduction in the degree of stacking is associated with a transition from Statel to State2 (Bennoun and Jupin 1974). The similarities between the results in Figures 45 and 46 are perhaps to be expected, although

202 the extremes of the salt-level manipulations are unlikely to be

reproduced in the plant, and indeed the differences in temperature

sensitivities of leaves in State 2 and State 1 are smaller than the

differences observed between the (in-vitro) stacked and unstacked

conditions.

It must be emphasised that these results only refer to the

overall chlorophyll fluorescence emission from the system, and more

detailed investigations of the temperature sensitivity of the sta-

bility of the Photosystem 2 units are required to resolve which

component(s) of the protein complex are most susceptible to the

high temperatures.

The chlorophyll fluorescence increase during heating to elevated

temperatures in pea chloroplasts is characterized by a mid-transi-

tion point at a temperature of about 40-43°C. These values compare

closely to the mid-point temperatures at which a broad endothermic

transition was detected (by differential scanning calorimetry) in

spinach chloroplasts by Cramer et al (1981). This transition was

associated with a loss in oxygen evolution, a release of manganese

from the membrane and a drop in the potential of the hydroquinone-

reducible form of cytochrome b^^^.

Other indirect evidence for the possible role of high protein:

lipid ratios and stacking being important for the stability of PS 2 at elevated temperatures comes from studies by Alberte et al (1974) on temperature-sensitive mutants of higher plants. They found that temperature-sensitive mutants often lacked the light-harvesting chlorophyll a/b protein (LHCP), which is associated with PS 2 and thylakoid stacking (Mullet and Arntzen, 1980), and is one of the major polypeptides present in the grana membranes (Thornber and

203 Thornber, 1980).

It seems feasible that in the future, an awareness of the impor-

tance of the link between the thylakoid lipid matrix and the con-

formational stability of the Photosystem 2 complex may throw some

light on this little-understood area of photosynthesis.

Future Studies

In this section, investigations of the interaction of acyl lipid

and protein within the thylakoid membrane of higher plants have

been given particular attention, and with these studies in mind,

new lines of approach towards a better understanding of photosyn-

thetic processes in higher plants may be discussed.

In this thesis the extremes of the natural membrane and the

extracted lipid have been examined, however, it is now possible to

enrich the lipid content of the membrane (with thylakoid lipids

rather than soya lipid) and this may prove to be a powerful approach in the study of lipid-protein interactions. In this way it should be possible to gradually xdilute-out' the effect of protein on the lipids, and a time-resolved fluorescence anisotropy study of this process (possibly with different probes) may yield very interesting results. Hiller and Raison (1980) have estimated that at least 50% of the lipid in spinach thylakoids lies within two annuli of molecules around protein complexes, and the lipid-protein interactions in this region may be strong.

The effect of lipid order and viscosity on the motional properties of the probes employed has been particularly emphasised, and the time-resolved fluorescence measurements have proved useful in

204 the resolution of the two components. The temperature-dependency of

these two factors in the thylakoid membrane should now be investi-

gated, although some indications of the possible outcome of this

study may be inferred from steady-state polarization measurements.

For instance the differences in the lipid fluidity of stromal and

granal membrane regions seems to be strongly reflected in the

ordering of the lipid chains (see Table 10), and in this respect it

is interesting to note that at very low temperatures ( 0 to -20°C )

the differences in the steady-state DPH fluorescence polarization

measurements between the two fractions becomes much smaller (see

Figure 39). As discussed earlier, the possible ordering effect of

intrinsic membrane proteins on their lipid environment is most

pronounced when the constituent lipid (examined in isolation) has a

low order parameter, whilst this effect is reversed in some artifi-

cial membrane systems where the strucure of highly ordered lipids

may be disrupted by the presence of protein (Chapman et al 1979).

Thus, at very low temperatures (close to 0°C) where the intrinsic

ordering of the thylakoid lipids may approach that of the proteins,

differences between the stromal and granal fractions could be

reduced. In this situation at very low temperatures it would seem

unlikely that any increase in lipid fluidity could be gained by a

reduction in protein levels, a factor which may have relevance to

the earlier discussion on temperature adaptation within the thylakoid membrane. For this reason, future studies of temperature effects on lipid fluidity in the thylakoid membrane should include time-dependent fluorescence anisotropy measurements, and by exam- ining a variety of chill-sensitive and chill-resistant plant species in this way, new insights into the mechanisms of temperature adaptation in higher plants may be gained.

An understanding of protein-lipid interactions in the thylakoid system may also be important in the successful reconstitution of intrinsic membrane proteins into purified lipid systems were they can be studied in isolation. The thylakoid lipid phase appears to be able to support very high levels of protein whilst maintaining a relatively fluid hydrocarbon region, and this emphasises the need to use the natural lipids whenever possible in these types of experiments. Recently, experiments from Malkin's laboratory with detergent-extracted particles have shown that it is possible to re- assemble electron transport from PS 2 to NADP+ although no oxygen- evolving activity was detected, and no ATP was produced (Lara and

Malkin, 1982). Very concentrated suspensions of the particles were required for electron transport along the complete chain, and although lipid and plastoquinone are present in the complexes, a further addition of lipid is not required. Thus it is possible that in the future the reconstitution of oxygen evolution, NADP+ reduction and ATP synthesis in an artificial membrane will be accompl- ished. An understanding of protein-lipid interactions in the thylakoid membrane may well be a pre-requisite of this achievement.

In this thesis an attempt has been made to extend the concept of the function of lipids in the thylakoid membrane beyond the idea of simply providing a framework in which protein-associated processes take place. The fluid properties of the lipid matrix appear to play an important role in the maintenance of rapid rates of electron flow between PS2 and PS1 and in the control of other important processes occuring in the membrane. Although specific functions may

206 exist for some of the lipids in this membrane, the general conclusion from this work is that the relative bulk levels of protein and lipid in the thylakoid membrane seems to be the major factor which influences its overall lipid fluidity.

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234 Appendix _1

Description of the Nanosecond Fluorescence Spectrophotmeter and the

Single Photon Counting Technique

a) Single Photon Counting

The single photon counting technique requires that the fluoresc-

ence signal is greatly attenuated so that after each flash, zero or one photons are counted. In this mode the probability that a photon

is detected by the photomultiplier at a time (t) after the flash is proportional to the fluorescence intensity at that instant. With multiple photon events only the first photon is recorded since the multi-channel analyser has a ^dead-time' of a few microseconds before the next photon can be recorded, thus the fluorescence decay profile will be distorted in favour of the early channels if many multiple-photon events occur.

The probability of detecting two or more photons can be calculated from the Poisson relationship:

_ mnexp(-m) Pn~ n! where Pn is the probability of detecting n photons per pulse when an average of m photons are detected per pulse (m is determined using a ratemeter).

If the average count rate is 2% of the flash frequency as used in the experiments described in this thesis then:

PQ = 0.98 = exp(-m) and thus, m = 0.0202 _o

therefore P^ = m exp(-m) = 1.98 x 10

4 finally, P>: = 1 - (PQ + Px) = 2.04 x 10~

Therefore in the experiments described earlier, about 1% of the photon counts are due to two or more photons. This is a very low

235 rate of detection of multiple photon events, and generally it is

possible to record at up to 5% of the flash frequency before it is

necessary to apply corrections to compensate for the distortion of

the decay curve by the preferential selection of earlier photons.

Precision of the Measurements

The count, N^ in the i th channel produced by exciting pulses

is:

Ni = FsNe At T)/ T

Where At is the time per channel, t^ is the time corresponding to

the i th channel and Fg is the fraction of pulses producing a photon at the photomultiplier. For a precision of 10%, (N^ = 100) at the channel where the fluorescence is 1% of the original intensity, then the number of exciting pulses (Ne) required is calculated to be about 30 x 10^ for DPH in the thylakoid membrane system. At a flash frequency of 50 kHz this would take about 10 minutes to accumulate. Generally, to determine the fluorescence lifetime with a precision of 1%, about 2,000 counts in the peak channel are required, but ten times this number are needed for accurate estimations of the rotational correlation time, b) Generation and Detection of a_ Nanosecond Light Pulse

A circuit diagram of the nanosecond lamp and gating system is described below:

R Lamp(L) Thyratron(T) 50 ft

236 When the voltage, V^ (about 4 kV) is first applied to the lamp circuit, the capacitances across L and T are slowly charged through

R. In the fully charged state T does not conduct so that V^ is divided between L and T. The lamp is fired by applying a gating pulse to the grid of the thyratron which increases the conductance of T, resulting in the rapid discharge of the capacitance across the thyratron. The voltage V^ is now placed entirely on the lamp which also discharges across the eletrode gap producing a short light pulse. The process begins again with the re-charging of the capacitances across L and T, and the rate of the gating pulses supplied to the thyratron controls the flash frequency.

The intensity, duration and spectral output of the lamp pulses are dependent a variety of factors which must be customized to the particular application required. To obtain very short pulses of duration about 1 ns, a narrow electrode gap, low flash frequency

(17 kHz), hydrogen or deuterium gas filling and a low applied voltage (3-4 kV) must be used. These pulses will be extremely weak

(barely visible), however with these gas fillings a fairly broad spectral output is obtained in the ultra-violet from 240 to 440 nm.

If the compound being studied has a long fluorescence lifetime, but rapid data collection from a weakly fluorescent sample is required then a broad electrode gap, high flash frequency (50 to

100 kHz), nitrogen gas filling and a high applied voltage (6 kV) must be used. With these conditions a broad pulse with a FWHM of about 6 ns is obtained, and with the nitrogen filling, only certain sharp lines centred around 310, 335 (maximum), 356 and 380nm can be used to provide more intense excitation. Even with these conditions, the light intensity reaching the cuvette is extremely low,

237 and can only be viewed in the dark, thus extreme sensitivity from

the detection system is needed.

A block diagram of the nanosecond spectrophotometer is shown in

Figure 47. The stop photomultiplier is protected from exposure to

high light intensities by an automatic shutter and monochromator,

and the start and stop photomultiplie rs are linked to the same

power supply. The start photomultiplier is small and relatively

insensitive but is selected for its extremely rapid rise time.

Earlier spectrophotometers used a bare wire close to the lamp which

detected the lamp discharge to provide the start signal in the

timing circuitry. The negative pulse from the start photomultiplier

is passed through a pulse-height discriminator and then through a nanosecond variable delay box (fine-tuning of the delay is obtained by using different lengths of co-axial cable). The start pulse is used to trigger the time-to-amplitude converter (TAC) which sets up a linear voltage ramp from 0 to 10 V which it reaches in a desig- nated time period. In 98% of the cases, no stop pulse is received and the TAC voltage re-sets to zero volts, in 2% of the cases a stop pulse is received from the fluorescence detection system, which stops the ramp in the TAC at a voltage which is used as the output to the multi-channel analyser. Thus, the output voltage is proportional to the time delay between the start and stop pulses.

The multi-channel analyser builds up a histogram of the number of counts collected against channel number (or voltage, or time).

A description of the computing techniques used to analyse the data is presented in Appendix 2.

238 Thyratron

FIGURE 47 Block diagram of the Applied Photophysics nanosecond fluorescence spectrophotometer, and the electronic components for the detection and analysis of the data.

239 Appendix 2_

Computing Procedures

The analysis of the fluorescence decay data was acheived with software written by Applied Photophysics Ltd. assuming single or double exponential decays, and by using a non-linear regression fitting procedure. These programs are not listed here.

The analysis of the decay of DPH fluorescence anisotropy was performed with software written on the Vector microcomputer

(software for this task was not available from Applied Photophysics

Ltd.). A summary of the steps involved is presented below:

1) Data collection: The fluorescence decays with the polarizing prisms parallel and perpendicular were collected on the multi- channel analyser and then transferred to permanent files on a floppy-disc via the computer. The lamp profiles and instrument response function were also collected for the perpendicular and parallel cases and also transferred to files.

2) The steady-state fluorescence anisotropy (rg) was determined for the samples as described in section 2.11.2.

3) The total number of counts in each file (over a specific channel range) was determined using a simple file-access and integration program written in Basic.

4) The total fluorescence, F,p(t), and total lamp, L^(t) data were calculated by the computer and then written to new files using the first part of the Basic program listed later.

FT(t) = Fi;L(t) + 2 Fj(t) .G

LT(t) = LXI(t) + 2 L^(t).G

G is calculated from:

G = W (1 - rs)/ VH (1 + 2rs)

240 Where VV is the total counts for the fluorescence decay with the

polarizers parallel ( ), VH is the total for the decay

measured with the prisms perpendicular ( F^(t) ) and rg is the

steady-state anisotropy. Usually G was found to be about 0.6 which

means that the excitation light was already slightly polarized

ccc perpendicular to the first polarizing prism. This is actually an advantage to the system since this results in the total counts VV and VH being approxiately equal and so the systematic errors due to background counts and noise are equal in both cases.

5) The total lamp and fluorescence data were then analysed to obtain the best-fit lifetime ( and pre-exponential parameters

(A^) assuming a double exponential fluorescence decay. This part of the procedure was performed with software written by Applied Photo- physics Ltd.

6) The best-fit parameters obtained from step 5 were then used in an analysis of the anisotropy decay:

A non-linear regression analysis (written in Basic) was again employed with a two-parameter fitting routines. The parameters r

(time-infinity anisotropy value), and the rotational correlation time, , were first estimated (by the operator) and then the estimates were constantly improved until the best-fit values were approached. This condition was judged to have been reached when:

reached a minimum value

Fp caic^t) was calculated from the following integral: t- =t- t

I Where Y = A1exp[-(t-ti)/TI] + A2exp[-(t-ti)/T2]

241 that is the decay of fluorescence between t^ and t after an infin-

itely short (delta-function) pulse of light.

Also Z = (rQ - rJ exp[-(t-tj.)/ ] + r^

(the decay of anisotropy between t^ and t after a delta-function

type pulse of light).

The convolution integral seems a complicated equation, but the

basic relationship used is that the difference fluorescence data is

equal to the total fluorescence data times the anisotropy:

FD(t) = FIX(t) - FI(t) = [FIX(t) + 2 Fx(t)] x r(t)

The main complication is that the computer must superimpose (or

convolute) the lamp profile with this relationship to obtain the

best fit curve to the actual difference decay data. In doing so it

used the best-fit motional parameters r^ and

succesively closer approximations (iterations), and this operation

led to the final results.

The convolution is performed as follows:

For each channel the computer calculates the total fluorescence,

the anisotropy and hence, the difference fluorescence:

a) The total fluorescence in a given channel (Fc) is given by

(i) +(ii) (as follows):

(i) The new value of the total fluorescence from the previous

channel which has decayed by an increment given by the the A^ and values previously obtained.

(ii) The total fluorescence in that channel resulting from the

total lamp counts in that channel times the Aj[ values.

b) The anisotropy in the channel is given by [(iii) + (iv)]/Fc

(as follows):

(iii) The new value of anisotropy which has decayed from the

242 previous channel (calculated using the estimated values for r^ and

<})), times the ""weighting' for that anisotropy value given by the

total fluorescence in (i).

(iv) The (maximum) anisotropy value in that channel as a result of

a delta-pulse excitation times the ^weighting' for the anisotropy

value given by the total fluorescence in (ii).

c) The calculated difference decay at that channel is given by the

calculated anisotopy times the calculated total fluorescence.

A listing of the Basic program used to perform the non-linear

regression procedure is presented in the following pages.

243 Non-linear Regression Program For Calculating the Best-Fit Difference

Decay

The program is divided into two sections, the first controls the

reading and writing of data from and to files on the floppy disc and

the second part performs the non-linear regression deconvolution

calculations. The non-linear regression analysis is based on the

method of Duggleby (1980), and is for a two-parameter fitting

routine.

The following arrays are used:

ARRAYS (Data stored in the computers short-term memory) vv(i) Fluorescence decay data, representing the number of counts in

the i th channel recorded with polarizers parallel. vh(i) As above but with polarizers perpendicular. pm(i) Number of counts in the i th channel recorded for the lamp profile. vt(i) Calculated total fluorescence decay data (F^,). vd(i) Calculated difference decay (Fp)« w(i) Statistical weighting parameter for the vd(i) value. b(i), q(i) Best-fit parameters required, and (q(i)) standard deviation from these values for the data.

244 100 DIM vd(580): DIM vc(580): DIM w(580): DIM x(580) 110 DIM vt(580): DIM pm(580): DIM vh(580): DIM w(580) 120 PRINT"*************************************************" 130 PRINT 140 PRINT 150 PRINT "ANISOTROPY DECAY, NON-LINEAR REGRESSION" 160 PRINT " AND DECONVOLUTION PROGRAM " 170 PRINT 180 PRINT 190 PRINT"*************************************************" 200 PRINT 210 PRINT 220 PRINT"W DECAY FILENAME AT LINE 260 " 230 PRINT"VH DECAY FILENAME AT LINE 320 " 240 PRINT"TOTAL FILENAME AT LINE 540 " 250 PRINT"PUMP FILENAME AT LINE 380 " 260 open"r",//l,"wdecay.fil" 270 gosub 3000 280 for i=32 to 544 290 vv(i)=x(i) 300 next i 310 close//1 320 open"r", //1,"vhdecay.fil" 330 gosub 3000 340 for i=32 to 544 350 vh(i)=x(i) 360 next i 370 close//1 380 open"r",//1, "lampdat. fil" 390 gosub 3000 400 for i=32 to 544 410 pm(i)=x(i) 420 print 430 print 440 input"What is the correction factor from rs values";cf 450 for i=32 to 544 460 vt(i)=vv(i)+vh(i)*cf*2 470 vd(i)=vv(i)-vh(i)*cf 480 next i 490 print"Do you wish to proceed to non-linear regression analysis" 500 print"or write the total decay data to file? type p or w" 510 input q$ 520 if q$="p" then 1000 530 close//l 540 open"r",//l,"totdeca.fil" 550 rf=l: gosub 3000 560 z=0 570 for j=l to 17 580 lset al$=mks$(vt(z+l)):lset a2$=mks$(vt(z+2)):lset a3$=mks$(vt(z+3)) 590 lset a4$=mks$(vt(z+4)):lset a5$=mks$(vt(z+5)):lset a6$=mks$(vt(z+6)) 600 lset a7$=mks$(vt(z+7)):lset a8$=mks$(vt(z+8)) 610 z=z+8 620 lset bl$=mks$(vt(z+l)):lset b2$=mks$(vt(z+2)):lset b3$=mks$(vt(z+3))

245 630 lset b4$=mks$(vt(z+4) ):lset b5$=mks$(vt(z+5) :lset b6$=mks$(vt(z+6)) 640 lset b7$=mks$(vt(z+7)):lset b8$=mks$(vt(z+8) 650 z=z+8 660 lset cl$=mks$(vt(z+l)):lset c2$=mks$(vt(z+2) :lset c3$=mks$(vt(z+3)) 670 lset c4$=mks$(vt(z+4)):lset c5$=mks$(vt(z+5) ilset c6$=mks$(vt(z+6)) 680 lset c7$=mks$(vt(z+7)):lset c8$=mks$(vt(z+8) 690 z=z+8 700 lset dl$=mks$(vt(z+l)):lset d2$=mks$(vt(z+2) :lset d3$=mks$(vt(z+3)) 710 lset d4$=mks$(vt(z+4)):lset d5$=mks$(vt(z+5) :lset d6$=mks$(vt(z+6)) 720 lset d7$=mks$(vt(z+7)):lset d8$=mks$(vt(z+8) 730 z=z+8 740 put//1 > j 750 next j 760 system 1000 rem calculating weighting parameters 1010 for i=32 to 544 1020 if vd(i)=0 then 1150 1030 if vd(i)10 then 2260

246 1650 for i=n0 to nl 1670 gosub 2280 1680 z=vd(i)-g 1690 r5=sqr(w(i))*z 1700 rl=rl+abs(r5) 1710 rem calculating the partial derivatives 1720 b(l)=b(l)*l.02 1730 gosub 2340 1740 u=gw 1750 b(l)=b(l)*.98/1.02 1760 gosub 2380 1770 b(l)=b(l)/.98 1780 p(l)=(u-gx)/(.04*b(1)) 1790 b(2)=b(2)*l.02 1800 gosub 2420 1810 u=gy 1820 b(2)=b(2)*.98/1.02 1830 gosub 2460 1840 b(2)=b(2)/.98 1850 p(2)=(u-gz)/(.04*b(2)) 1860 rem form the various sums 1870 sl=sl4w(i)*p(1)~2 1880 s2=s2+w(i)*p(l)*p(2) 1890 s3=s3+w(i)*p(2)~2 1900 s4=s4+w(i)*p(l)*z 1910 s5=s5+w(i)*p(2)*z 1920 s6=s6+w(i)*z~2 1930 next i 1940 rem correct the parameters and check for convergence 1950 r2=6*rl/(nl-nO) 1960 d=sl*s3-s2~2 1970 q(I)=(s3*s4-s2*s5)/d 1980 q(2)=(sl*s5-s2*s4)/d 1990 c=abs(q(l)/b(l))+abs(q(2)/b(2)) 2000 b(l)=b(l)+q(l) 2010 b(2)=b(2)+q(2) 2020 print" 11 2030 print"estimates for iteration";il 2035 print"rinf=";b(1)" rot. correl. time=";b(2)" sum sqrs=";s6 2040 if c>.0001 then 1580 2050 rem final values for fitting 2060 v=s6/((nl-n0)-2) 2070 q(l)=sqr(v*s3/d) 2080 q(2)=sqr(v*sl/d) 2090 print 2100 print"FINAL VALUES " 2105 print 2110 print"rinf=";b(1),"+/-";q(1) 2120 print"rot. correl. time=",b(2),"+/-";q(2) 2130 print 2140 input"do you wish to see the final best-fit and real values";q$ 2145 if q$="y" then 2150 2147 end 2150 print"channel Fd Fd(calc) Diff" 2160 print

247 2170 for i=n0 to nl 2180 gosub 2570 2190 print i,vd(i),gf,vd(i)-gf 2200 next i 2210 print"final values were " 2220 print"rinf=";b(l),"+/-,,;q(l),,,rot. correl=,,;b(2),,ns",,,+/-,,;q(2) 2230 close//l 2235 rem opening file of best-fit data 2240 open"r",//l,"bestfit.dat" 2250 goto 550 2260 print"terminated after 10 iterations" 2270 goto 2060 2280 rem ansiotropy calculations here 2290 rem assuming .163 ns/channel 2295 rem fluor. lifetime calc. also assuming .163 ns/channel 2296 rem convert input Ti values and final rot. correl. vales 2297 rem if time-scale is not .163 ns/channel 2300 u2=(u2*ul*exp(-.163/b(2))+(.39-b(l))*pm(i)*cf)/(vc(i-l)+pm(i)*cf) 2310 ul=vc(i) 2320 g=(u2+b(l))*vc(i) 2330 return 2340 o2=(o2*ol*exp(-.163/b(2))+(.39-b(l))*pm(i)*cf)/(vc(i-l)+pm(i)*cf) 2350 ol=vc(i) 2360 gw=(o2+b(1))*vc(i) 2370 return 2380 z2=(z2*zl*exp(-.163/b(2))+(.39-b(l))*pm(i)*cf)/(vc(i-l)+pm(i)*cf) 2390 zl=vc(i) 2400 gx=(z2+b(1))*vc(i) 2410 return 2420 f2=(f2*fl*exp(-.163/b(2))+(.39-b(l))*pm(i)*cf)/(vc(i-l)+pm(i)*cf) 2430 fl=vc(i) 2440 gy=(f2+b(l))*vc(i) 2450 return 2460 e2=(e2*el*exp(-.163/b(2))+(.39-b(l))*pm(i)*cf)/(vc(i-l)+pm(i)*cf) 2470 el=vc(i) 2480 gz=(e2+b(1))*vc(i) 2490 return 2500 gl=pm(i)*al/(al+a2)+gl*exp(-.163/tl) 2510 g2=pm(i)*a2/(al+a2)+g2*exp(-.163/t2) 2520 vc(i)=(gl+g2)/cf 2530 yy=yy+vc(i) 2540 print i,vt(i),vc(i),vd(i),vd(i)/vt(i) 2550 zz=zz+vt(i) 2560 return 2570 h2=(h2*hl*exp(-.163/b(2))+(.39-b(l))*pm(i)*cf)/(vc(i-l)+pm(i)*cf) 2580 hl=vc(i) 2590 gf=(h2+b(1))*vc(i) 2600 vt(i)=gf 2610 return 3000 FIELD//1,4 AS al$,4 AS a2$,4 AS a3$,4 AS a4$,4 AS a5$, 3010 FIELD//1,20 AS X$,4 AS a6$,4 AS a7$,4 AS a8$ 3020 FIELD//1,32 AS X$,4 AS bl$,4 AS b2$,4 AS b3$,4 AS b4$,4 AS b5$ 3020 FIELD//1,32 AS X$,20 AS X$,4 AS b6$,4 AS b7$,4 AS b8$ 3030 FIELD//1,32 AS X$,32 AS X$,4 AS cl$,4 AS c2$,4 AS c3$,4 AS c4$ 3040 FIELD//1,32 AS X$,32 AS X$,16 AS X$,4 AS c5$,4 AS c6$,4 AS c7$,4 AS

248 3050 FIELD//1,32 AS X$,32 AS X$,32 AS X$,4 AS dl$,4 AS d2$,4 AS d3$,4 AS d4$ 3060 FIELD//1,32 AS X$,32 AS X$,32 AS X$,16 AS X$,4 AS d5$,4 AS d6$ 3070 FIELD//1,32 AS X$,32 AS X$,32 AS X$,24 AS X$,4 AS d7$,4 AS d8$ 3080 IF rf=l THEN return 4010 for i=l to 17 4020 get//l,i 4030 z=32*(i-32) 4040 x(z+l)=cvs(al$):x(z+2)=cvs(a2$):x(z+3)=cvs(a3$):x(z+4)=cvs(a4$) 4050 x(z+5)=cvs(a5$):x(z+6)=cvs(a6$):x(z+7)=cvs(a7$):x(z+8)=cvs(a8$) 4060 z=z+8 4070 x(z+l)=cvs(bl$):x(z+2)=cvs(b2$):x(z+3)=cvs(b3$):x(z+4)=cvs(b4$) 4080 x(z+5)=cvs(b5$):x(z+6)=cvs(b6$):x(z+7)=cvs(b7$):x(z+8)=cvs(b8$) 4090 z=z+8 5000 x(z+l)=cvs(cl$):x(z+2)=cvs(c2$):x(z+3)=cvs(c3$):x(z+4)=cvs(c4$) 5010 x(z+5)=cvs(c5$):x(z+6)=cvs(c6$):x(z+7)=cvs(c7$):x(z+8)=cvs(c8$) 5020 z=z+8 5030 x(z+l)=cvs(dl$):x(z+2)=cvs(d2$):x(z+3)=cvs(d3$):x(z+4)=cvs(d4$) 5040 x(z+5)=cvs(d5$):x(z+6)=cvs(d6$):x(z+7)=cvs(d7$):x(z+8)=cvs(d8$) 5050 next i 5060 return

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