Circular Dichroism of the Laser–Induced Blue State Of

CIRCULAR DICHROISM OF THE LASER–INDUCED BLUE STATE OF

BACTERIORHODOPSIN, SPECTRAL ANALYSIS AND NEW INSIGHTS INTO THE

PURPLE→BLUE COLOR CHANGE

Thesis Submitted to The College of Arts and Sciences of the UNIVERSITY OF DAYTON

In Partial Fulfillment of the Requirements for The Degree of Master of Science in Chemistry

By Anusha Rudraraju Dayton, Ohio August, 2015

CIRCULAR DICHROISM OF THE LASER–INDUCED BLUE STATE OF

BACTERIORHODOPSIN, SPECTRAL ANALYSIS AND NEW INSIGHTS INTO THE

PURPLE→BLUE COLOR CHANGE

Name: Rudraraju, Anusha

APPROVED BY:

______Mark B. Masthay, Ph.D. Angela Mammana, Ph.D. Associate Professor Assistant Professor Committee Chairman Committee Member

______Matt Lopper, Ph.D. Shawn Swavey, Ph.D. Associate Professor Professor Committee Member Committee Member

ABSTRACT

CIRCULAR DICHROISM OF THE LASER–INDUCED BLUE STATE OF

BACTERIORHODOPSIN, SPECTRAL ANALYSIS AND NEW INSIGHTS INTO THE

PURPLE→BLUE COLOR CHANGE

Name: Rudraraju, Anusha University of Dayton

Advisor: Dr. Mark B. Masthay

The purple membrane (PM) of the salt–loving bacterium H. Salinarium owes both its color and physiological function to the protein bacteriorhodopsin (BR). The PM is comprised of BR trimers arranged in a crystalline hexagonal lattice. PM converts to an ultraviolet–induced colorless membrane (UVCM) upon exposure to diffuse ultraviolet

(UV) light through a 1–monomer 1–photon process and to a laser–induced blue membrane (LIBM) upon exposure to intense green laser pulses through a 1–monomer 2– photon process. The color changes which BR molecules undergo depend on the (1) BR aggregation state (e.g. crystalline trimeric PM and monomers) (2) wavelength of light (3) intensity of the light and (4) divalent cations, as earlier results indicate that calcium ions

(Ca2+) are removed from the PM surface during the PM  LIBM photoconversion. The origin of the unconventional biphasic band in CD spectrum has caused much debate.

iii

The principle purpose of the research is to further elucidate the mechanisms responsible for the well–known PMLIBM and PMUVCM photoconversions. Both of these processes have significant implications regarding potential photocooperative processes and exciton coupling between BR molecules within the PM. They also have important implications for the long–standing debate about the unusual “bisignate” circular dichroism (CD) spectrum of PM in the visible region of the spectrum, which has been attributed to both exciton coupling between the BR molecules and BR protein heterogeneity within the trimer. To accomplish this objective, I characterized the changes in the absorption and CD spectra of five separate BR species upon irradiation with intense 532 nm laser pulse and diffuse 254nm–UV–light: Native PM, Delipidated PM,

Monomeric BR, cation–free blue membrane and calcium saturated PM. Conclusions were drawn about the relative roles of inter–trimer and intra–trimer photocooperativity, exciton coupling and BR protein heterogeneity in the photochemistry and color changes of BR in

PM.

ACKNOWLEDGEMENTS

I would like to express my sincere gratitude towards Dr. Mark B. Masthay, for giving me the opportunity to work in his lab and for guiding me through my entire graduate course. Without his constant help and concern this thesis would not have been possible. Thank you for being so patient and kind to me. Further I would like to thank Dr.

Angela Mammana who has helped me patiently though all the experiments. Thank you for your great concern and compassion. Thanks to both of them for making my stay at

UD very memorable.

I would like to express my appreciation to my committee members Dr. Matt

Lopper and Dr. Shawn Swavey. I would also like to thank everybody in the chemistry department for being so nice and kind, which made my two years of study at the

University of Dayton unforgettable. Last but not least, I would like to thank my family and friends, for all their love and support.

TABLE OF CONTENTS

ABSTRACT……………………………………………………………………..……….iii

ACKNOWLEDGEMENTS……………………………………………………………….v

LIST OF FIGURES……………………………………………………………………….x

LIST OF SCHEMES…………………………………………………………………….xv

LIST OF GRAPHS……………………………………………………...... xvi

LIST OF TABLES…………………………………………………………………….xviii

LIST OF ABBREVIATIONS…………………………………………………………....xx

INTRODUCTION……………………………………………………………………..….1

MATERIALS AND METHODS………………………………………………………...11

A. BR Purchase……………………………………...... ………………………………11

B. Laser Studies and Spectroscopy………………………..…………………………..11

C. Laser and Hg–Lamp Description…………………………………………………...11

D. Sample Preparation…………………………………………………………………13

1. PM in Phosphate Buffer……..…………………...………………………....…13

(a) Laser–Induced Blue Membrane (LIBM).………..………………………….13

(b) Ultraviolet–Induced Colorless Membrane (UVCM).……….……….....…...13

2. Delipidated Purple Membrane (DLPM)………...…………………………...... 13

(a) Laser–Induced Delipidated Blue Membrane (LI–DLBM)………….………14

(b) Ultraviolet–Induced Delipidated Purple Membrane (UV–DLPM)………....14

3. Monomer (MON)...... ……………………...……………………………………14

(a) Laser Induced Colorless Monomer (LI–CMON).……………..…..………..15

(b) Ultraviolet–Induced Colorless Monomer (UV–CMON).…………….…….16

4. Cation Free Blue Membrane (CFBM) ………………………………..……..…16

(a) Laser Induced Cation Free Blue Membrane (LI–CFBM)……….………….16

(b) Ultraviolet–Induced Cation Free Blue Membrane (UV–CFBM)…….…….16

5. Calcium Saturated Purple Membrane (CaPM)…………………………….……17

(a) Laser–Induced Calcium Saturated Purple Membrane (LI–CaPM)…………17

(b) Ultraviolet–Induced Calcium Saturated Purple Membrane (UV–CaPM)..…17

E. ICP Experiment……………………………………………………………..………17

RESULTS………………….…………………………………………………………….20

(1) Laser Species…………………………………………………………………………20

(a) LIBM–Absorption spectra…………………….……………………………..……20

(b) LIBM–CD spectra……………………………………..………………………….21

(d) LI–DLBM–CD spectra...………………………………………………………….24

(e) LI–CMON–Absorption spectra…………...………………………….……...……25

(f) LI–CMON–CD spectra………...……………………………………………...... 26

(g) LI–CFBM–Absorption spectra…………………………………...…….……....…27

vii

(h) LI–CFBM–CD spectra……………………………………………...... …..………28

(i) LI–CaPM–Absorption spectra………………………………………………….....29

(j) LI–CaPM–CD spectra……………...……………………………………………...30

(2) Hg–Lamp Species………...……………………………………………………….…31

(a) UVCM–Absorption spectra………………………………...……………………..31

(b) UVCM–CD spectra……………………………………………...…...…………...32

(d) UV–DLPM–CD spectra………………………….………………..……...……….34

(e) UV–CMON–Absorption spectra………………………………………...... ……35

(f) UV–CMON–CD spectra…………………………...……………………………....36

(g) UV–CFBM–Absorption spectra……………………………...……..…………...... 37

(h) UV–CFBM–CD spectra………………………………..………………...………..38

(i) UV–CaPM–Absorption spectra…………………………………..………………...39

(j) UV–CaPM–CD spectra………………………………………………..……………40

(3) ICP Results……………………………………………….………………………..…41

(4) Spectral Analysis Tables………………………………………………..……………42

DISCUSSION……………………………………………………………………………47

(a) One Photon/ Two Photon Absorption–Spectral Analysis………………………...….47

(b) Proposed Mechanisms for Generation of LIBM and UVCM…………………...... 50

(d) Exciton Coupling/ Protein Heterogeneity…………………………………………....54

(e) Cation Binding……………………………………………………….………………58

viii

CONCLUSIONS………..……………………………………….………………………60

FUTURE STUDIES………………………...... …………………………………………61

REFERENCES………………………………….…………………….…………………63

APPENDIX…………………………………….………………………………………...72

LIST OF FIGURES

Figure 1. (A) ATRPSB chromophore is located roughly at the center of the channel formed by the 7 α– helices……………….………………………………..3

Figure 1. (B) Chemical structure of ATR, ATRPSB chromophore and reduced ATRPSB……………………...…………………………………………….…….3

Figure 2.Structure of PM lattice which shows the Purple BR monomers arranged in a heptamer of trimers………………………………..………………………..3

Figure 3. Upper – CD spectrum of the PM depicting the bisignate band.

Lower – Absorbance band of PM with absorbance maximum at 568 nm.

The zero point of the bisignate band would coincide with the absorbance maximum of the absorption spectra…………………...…………………………………..6

Figure 4. Top – Schematic representations of the native PM; Middle –

LIBM; Bottom – UVCM. The dark blue represents the BR570; lighter blue represents the P605 and white represent the P360……………...………………...…….8

Figure 5. Schematic of the sample irradiation with 532 nm pulses from a

Spectra Physics Model INDI–40 Pulsed Nd:YAG laser……………………………..…..12

Figure 6. Absorption Spectra of the photo–conversion of PM to LIBM induced by 6ns, 15 mJ pulse–1 (3.46 MWcm–2), 532 nm pulses for 230 min

(spectra taken after 0, 10, 30, 50, 70, 90, 110, 130, 150, 180, 230 minutes of irradiation)…...……………………………………………………………….……...... …20

Figure 7. CD Spectra of the photo–conversion of PM to LIBM induced by 6ns, 15 mJ pulse–1(3.6 MWcm–2), 532 nm pulses for 230 min (spectra taken after 0, 10, 30, 50, 70, 90, 110, 130, 150, 180, 230 minutes of irradiation)………………………………………………………………...……...………21

Figure 8. Absorption Spectra of the photo–conversion of DLPM to LI–DLBM induced by 6ns, 15 mJ pulse–1 (3.6 MWcm–2), 532 nm pulses for 120 min (spectra taken after 0, 3, 6, 9, 12, 18, 24, 30, 36, 48, 60, 84, 120 minutes)……….……………....23

Figure 9. CD Spectra of the photo–conversion of DLPM to LI–DLBM induced by 6ns, 15 mJ pulse–1 (3.6 MWcm–2), 532 nm pulses for 120 min (spectra taken after 0, 3, 6, 9, 12, 18, 24, 30, 36, 48, 60, 84, 120 minutes)………………...... …….24

Figure 10. Absorption Spectra of the photo–conversion of MON to LI–CMON induced by 6ns, 15 mJ pulse–1 (3.6 MWcm–2), 532 nm pulses for 60 min

(spectra taken after 0, 3, 6, 9, 12, 15, 20, 25, 30, 60 minutes of irradiation)………………………………………………………………………….…….25

Figure 11. CD Spectra of the photo–conversion of MON to LI–CMON induced by 6 ns, 15 mJ pulse–1(3.6 MWcm–2), 532 nm pulses for 60 min

(spectra taken after 0, 3, 6, 9, 12, 15, 20, 25, 30, 60 minutes of irradiation)…………………………...…………..…………………………………….…26

Figure 12. Absorption Spectra of the photo–conversion of CFBM to

LI–CFBM induced by 6ns, 15 mJ pulse–1 (3.6 MWcm–2),532 nm pulses for 90 min (spectra taken after 0, 3, 9, 15, 21, 27, 36, 45, 60 and

90 minutes of irradiation)………………………………………………………..…....….27

Figure 13. CD Spectra of the photo–conversion of CFBM to LI–CFBM induced by 6ns, 15 mJ pulse–1(3.6 MWcm–2), 532 nm pulses for 90 min

(spectra taken after 0, 3, 9, 15, 21, 27, 36, 45, 60 and 90 minutes of irradiation)…….....28

Figure 14. Absorption Spectra of the photo–conversion of CaPM to

LI–CaPM induced by 6 ns, 15 mJ pulse–1 (3.6 MWcm–2), 532 nm pulses for 42 min (spectra taken after 0, 6, 12, 18, 24, 30, 42 minutes)…………..………….…29

Figure 15. CD Spectra of the photo–conversion of CaPM to LI–CaPM induced by 6 ns, 15 mJ pulse–1(3.6 MWcm–2), 532 nm pulses for 42 min

(spectra taken after 0, 6, 12, 18, 24, 30, 42 minutes)……………………………...... …..30

Figure 16. Absorption Spectra of UVCM generated by irradiating PM suspensions with the 254 nm line of a 100 Watt Mercury lamp for 480 min

(spectra taken after 0, 10, 30, 60, 90, 120, 150, 180, 210, 240, 270, 300,

330, 360, 390, 420, 480 minutes of irradiation)………………………………………….31

Figure 17. CD Spectra of UVCM generated by irradiating PM suspensions with the 254 nm line of a 100 Watt Mercury lamp for 480 min (spectra taken after 0, 10, 30, 60, 90, 120, 150, 180, 210, 240, 270, 300, 330, 360, 390, 420,

480 minutes of irradiation)……………………………………………………………….32

Figure 18. Absorption Spectra of UV–DLPM generated by irradiating

DLPM suspensions with the 254 nm line of a 100 Watt Mercury lamp for

300 minutes (spectra taken after 0, 2, 10, 10, 30, 60, 90, 120, 120, 150,

180, 210 minutes of irradiation)………………………………………………………….33

Figure 19. CD Spectra of UV–DLPM generated by irradiating DLPM suspensions with the 254 nm line of a 100 Watt Mercury lamp for 300 minutes

xii

(spectra taken after 0, 2, 10, 10, 30, 60, 90, 120, 120, 150, 180, 210 minutes of irradiation)………………………………………………………………………….…….34

Figure 20. Absorption Spectra of UV–CMON generated by irradiating Mon suspensions with the 254 nm line of a 100 Watt Mercury lamp for 270 minutes

(spectra taken after0, 15, 30, 45, 60, 75, 90, 120, 150, 210, 270 minutes of irradiation)……………………………………………………………………………..…35

Figure 21. CD Spectra of UV–CMON generated by irradiating Mon suspensions with the 254 nm line of a 100 Watt Mercury lamp for 270 minutes

(spectra taken after 0, 15, 30, 45, 60, 75, 90, 120, 150, 210, 270 minutes of irradiation)………………………………………………………………………………..36

Figure 22. Absorption Spectra of UV–CFBM generated by irradiating CFBM suspensions with the 254 nm line of a 100 Watt Mercury lamp 240 minutes

(spectra taken after 0, 20, 40, 60, 60, 80, 110, 145, 180, 240 minutes of irradiation)……………………………………………………………………………..…37

Figure 23. CD Spectra of UV–CFBM generated by irradiating CFBM suspensions with the 254 nm line of a 100 Watt Mercury lamp 240 minutes

(spectra taken after 0, 20, 40, 60, 60, 80, 110, 145, 180, 240 minutes of irradiation)…………………………………………………………………………...... …38

Figure 24. Absorption Spectrum of UV–CaPM generated by irradiating

CaPM suspensions with the 254 nm line of a 100 Watt Mercury lamp for

250 minutes (spectra taken after 0, 40, 70, 100, 130, 130, 160, 190, 250, 250 minutes of irradiation)…………………………………………………………………....39

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Figure 25. CD Spectra of UV–CaPM generated by irradiating CaPM suspensions with the 254 nm line of a 100 Watt Mercury lamp for 250 minutes (spectra taken after 0, 40, 70, 100, 130, 130, 160, 190, 250, 250 minutes of irradiation) ……..…40

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LIST OF SCHEMES

Scheme 1. BR photocycle upon absorption of a single photon. BR570

* (light adapted BR) undergoes photoexcitation to I460 and then cycles through I460, J625, K590, L550, M410, N560 and O640 thermal intermediates……………..…...2

Scheme 2. The two photon absorption mechanism: The PM→LIBM

** photoconversion is mediated through the doubly excited intermediate I460 ………….…..4

Scheme 3. Diagram represents the proposed mechanism for the PM to

LIBM photoconversion…………………………………………………………….…….51

LIST OF GRAPHS

(a) Laser Species – Absorbance versus Time………….………………………….……..73

(b) Hg Lamp Species – Absorbance versus Time…………………………………….….74

(d) Hg Lamp Species – CD versus Time……………………………………..……….....76

(e) Laser Species – Absorbance spectra– Wavelength versus Time…………………...... 77

(f) Hg Lamp Species – Absorbance Spectra– Wavelength versus Time……………..…78

(g) Laser Species – Wavelength Shift in Absorption maximum and

CD + and CD – Peak……………………………………………………………….....….79

(h) Hg–Lamp Species – Wavelength Shift in Absorption maximum and

CD + and CD – Peak……………………………………………………………...….…..80

(i) Overlaid Band Intensity vs Time Graphs…………...... ………81

(j) Overlaid Wavelength vs Time Graphs………………………….…………………….82

(k) The rate of absorbance change in the absorbance spectra at the wavelength corresponding to the positive band in CD………………………...….83

(l) The rate of absorbance change in the absorbance spectra at the wavelength Maximum………………………………………………………..………84

(m) The rate of absorbance change in the absorbance spectra at the wavelength corresponding to the negative band in CD……………………...………85

xvi

(n) Absorbance of P340 P360 P380 plotted against time – Laser Species………....………..86

(o) Absorbance of P340 P360 P380 plotted against time – Hg–Lamp Species…...……..…..87

(p) CD vs T Plots in UV bands–Laser Species…………………………………….…….88

(q) CD vs T Plots in UV bands–Hg –Lamp Species……………………………..……….89

(r) ICP Calibration Curve……….………………………………………………………..90

xvii

LIST OF TABLES

Table 1. Summarizes the wavelength maximum, energy maximum, absorbance maximum, width of the half peak and extinction coefficient of different BR species for both laser and Hg lamp irradiations…………...... …...42

Table 2. Summarizes the wavelength maximum for CD+, energy maximum,

CD maximum for positive CD band and width of the half peak of different

BR species for both laser and Hg lamp irradiations…………………………...... 43

Table 3. Summarizes the wavelength maximum for CD–, energy maximum,

CD maximum for negative CD band and width of the half peak of different

BR species for both laser and Hg lamp irradiations………………………...... 44

Table 4. Summarizes the change in the magnitude of absorbance at 532 nm, change in the magnitude of CD+ band and change in the magnitude of CD– band.

Time of irradiation has also been displayed. All the values have been normalized……..45

Table 5. Summarizes the change in the rate of absorbance at 532 nm, rate of CD+ band drop and rate of CD– band rise. Time of irradiation has also been displayed. All the values have been normalized.…………………………….…….46

Table 6. Changes in circular dichroism and absorption spectra of various purple and blue bacteriorhodopsin species during various 2hν (532nm)– induced photoconversions………………………………………………………...……...91

xviii

Table 7. Rate of Intensity Changes (qualitative observations) Un-

Normalized………...... ……….92

Table 8. Magnitude of Intensity from the graphs (quantitative observations)

Un-Normalized………………………………………………………………………...... 93

Table 9. Magnitude of Intensity – ranking (quantitative observations) – Un-

Normalized………….……………………………………………………………………94

Table 10. Rate of change of Intensity (quantitative observations)–Un-

Normalized…………………...... ……95

Table 11. Rate of change of Intensity (ranking)-quantitative observations –

Un-Normalized……………………………………………………………………....…..96

Table 12. Rate of Wavelength Changes (qualitative observations) – Un-

Normalized………….………………………………………………………………..…..97

Table 13. Magnitude of wavelength changes –quantitative observations – Un-

Normalized………………………………………………………………………….……98

Table 14. Magnitude of wavelength changes (ranking)-quantitative observations

– Un-Normalized……………………………………………………………………...…99

Table 15. Rate of Wavelength changes (quantitative)– Un-Normalized………………100

Table 16. Rate of wavelength changes ranking (quantitative) – Un-Normalized……...101

Table 17. Magnitude changes (quantitative) ranking – Normalized…………...………102

Table 18. Rate changes (quantitative) ranking Normalized….…………………….…..103

xix

LIST OF ABBREVIATIONS

1. Amax: Absorbance maximum

2. Asp: Aspartic acid

3. ATP: Adenosine triphosphate

4. ATR: All–trans retinal

5. ATRPSB: All–trans retinal protonated Schiff base

6. BR: Bacteriorhodopsin

7. BR570: The light adapted BR without any irradiation

8. CaPM: Calcium saturated purple membrane

9. CD: Circular Dichroism

+ 10. CDmax: CD maximum for the positive band

– 11. CDmax: CD maximum for the negative band

12. CFBM: Cation free blue membrane

13. CHAPS : 3–[(3–cholamidopropyl) dimethylammonio]–1–propanesulfonate

14. DLBM: Delipidated blue membrane

15. DLPM: Delipidated purple membrane

16. EC: Exciton Coupling

17. ICP–OES: Inductively Coupled Plasma–Optical Emission Spectroscopy

18. LA : Light–Adapted

19. LIBM : Laser–induced blue membrane

20. LI–CaPM: Laser–induced calcium saturated purple membrane

21. LI–CFBM: Laser–induced cation free blue membrane

22. LI–CMON: Laser–induced colorless monomer

23. Lys: Lysine

24. MON: Monomer

25. MT: Multiple Transitions

26. Nd:YAG : Neodymium Yttrium Aluminum Garnet

27. P360: Photoproduct at Absorbance 360 nm for laser species

28. P360/UV: Photoproduct at Absorbance 360 nm for Hglamp species

29. P605: Blue photoproduct that is formed in PM→LIBM and DLPM→LI–DLBM photoconversions

30. P642: Photoproduct which is observed at absorbance 642 nm in some species

31. PH: Protein Heterogeneity

xxi

32. PM: Purple Membrane

33. Rh: Rhodopsin

34. TRITON–X–100: polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether

35. Trp: Tryptophan

36. UVCM: Ultraviolet induced colorless membrane

37. UV–CaPM: Ultraviolet induced calcium saturated purple membrane

38. UV–CFBM: Ultraviolet induced cation free blue membrane

39. UV–CMON: Ultraviolet induced colorless monomer

40. λmax: Wavelength maximum in the absorption spectra

– 41. λmax: Wavelength maximum for the negative CD band

+ 42. λmax: Wavelength maximum for the positive CD band

xxii

INTRODUCTION

Bacteriorhodopsin (BR) was discovered in 1971[1, 2], and since then has become one of the most studied of all proteins. BR is a trans–membrane protein responsible for the color of the purple membrane (PM) of the salt–loving bacterium Halobacterium

Salinarium [1, 2]. BR is structurally similar to the visual pigments rhodopsin (Rh) and iodopsin which are present in the light–sensitive rod and cone cells respectively, which mediate human vision. BR is thus a good model for human visual pigments, while at the same time offering two practical experimental advantages over them: (1) BR is more readily available and (2) more photostable under laboratory conditions than the visual pigments[3].

Structurally, BR consists of 7 α–helices arranged around a common center (Figure

1–A) which serves as a light–activated proton channel. The all–trans–retinyl protonated

Schiff’s base (ATRPSB) chromophore (Figure 1–B) of BR is bound covalently to the amino group of Lys–216 and is located roughly in the center of the channel. Upon absorption of a visible wavelength photon, the light–adapted (LA) form of BR known as

BR570 goes through a photocycle in which it is first photoexcited to its singlet excited

* state I460 and then sequentially cycles through several spectroscopically distinct thermal

species designated J625, K590, L550, M410, N560 and O640 which appear and then disappear, until finally the original BR570 is restored.

The subscripts designate the absorption wavelength maxima of the species in nanometers

(See Scheme 1).[3, 4]

 ,200 sfh * 500 fs 5 ps 2 s 50s BR 570  I 460  J625  K590  L550  M410

1ms 3ms 5ms  N560  O630  BR570.

Scheme 1: BR photocycle upon absorption of a single photon. BR570 (light adapted BR) undergoes photoexcitation to I and then cycles through I460, J625, K590, L550, M410, N560 and O640 thermal intermediates.

During this photocycle the ATRPSB chromophore isomerizes to the 13–cis configuration thereby inducing a series of conformational changes which facilitate pumping the proton initially on the Schiff base nitrogen from the inside to the outside of the cell (see Scheme 1). The resulting trans–membrane protein gradient is coupled to the generation of ATP which enables H. salinarium to survive under hypoxic conditions.[3]

BR is localized in a purple membrane (PM) which grows in halobacteria under low oxygen conditions. The PM is a rigid, tightly packed “crystalline” hexagonal lattice of BR trimers (Figure 2). Upon exposure to 532 nm laser pulses of intensity  0.4 MW cm–2,[5] PM converts to a Laser–Induced–Blue–Membrane (LIBM) via a sequential two– photon absorption process in which the ATRPSB chromophores of individual BR molecules nearly simultaneously absorb two 532 nm photons, inducing the reduction of

7 9 11 13 15 1 2 6 O 8 10 12 14 3 5 4

all-trans-retinal (ATR)

N BO O+ H

all-trans - retinyl protonated Schiff base (ATRPSB) (ATRPSB) (ATRPSB)

N H2 BO O+

Reduced-ATRPSB

N H 2 BO O+

retro- ATRPSB

Figure 1: (A) ATRPSB chromophore is located roughly at the center of the channel formed by the 7 α–helices; (B) Chemical structure of ATR, ATRPSB chromophore and reduced ATRPSB

the doubly–excited chromophores to an unprotonated Schiff base product which absorbs maximally near 360 nm.[3, 5-10] The generation of this colorless photoproduct, designated P360, is accompanied by the simultaneous or nearly simultaneous conversion of proximal BR monomers containing unreduced chromophores to a blue species designated P605 (Scheme 2).

Figure 2: Structure of PM lattice which shows the Purple BR monomers arranged in a heptamer of trimers.

In contrast, PM converts into an Ultraviolet–Induced–Colorless–Membrane

(UVCM) when the ATRPSB chromophore absorbs a single 254 nm photon.[3, 11] These singly–excited chromophores are reduced to an unprotonated Schiff base P360/UV photoproduct which absorbs in the same region as the P360 product in LIBM. The P360/UV photoproduct in UVCM manifests significantly less vibronic structure than the P360 product in LIBM, and is not accompanied by the generation of a blue species; UVCM gradually loses its intense purple color, turning first lavender and then colorless.

h 532 * PM = BR 570 BR 570 BR 570  I 460 BR BR a b c a

** I 460 BR BR  P 360 P 605 P 605 = LIBM a a b c

Scheme 2: The two photon absorption mechanism: The PM→LIBM photoconversion is mediated ** through the doubly excited intermediate I 460 .

One of the most interesting characteristics of PM is its circular dichroism (CD) spectrum. CD spectroscopy involves the differential absorption of left–and right–handed circularly polarized light. A CD signal is indicative of intrinsic or induced chromophore chirality. CD spectra provide detailed information about the structure and arrangement of chromophores in PM,[10, 12-47] visual pigments,[48-53] and related macromolecular structures.[54, 55] Although caution must be taken to avoid complications from the light scattering characteristic of PM and other membrane protein suspensions.[37, 56-58].

Monosignate CD spectra (i.e., spectra with a single positive or negative CD band) are characteristic of chiral chromophores. Bisignate CD bands in which the magnitude of

4 the positive band is equal to the magnitude of negative band is indicative of exciton coupling between two or more chromophores. The PM manifests a bisignate band in

+ – which the positive band (휆푚푎푥 = 535.2 nm) is twice as intense as the negative band (휆푚푎푥

= 599.8nm; see Figure 3). Although this bisignate CD spectrum is strongly suggestive of

EC between the ATRPSB chromophores of proximal BR monomers in PM the origin of the bisignate band has been of much debate and has still not been conclusively proved.

Three principle models have been proposed to explain the bisignate CD spectrum of PM: (1) an Exciton Coupling (EC) model, in which the bisignate CD spectrum results from the overlap of the intrinsic protein band and a bisignate band that arise from exciton coupling;[14-16, 21-24, 30, 47] (2) a Protein Heterogeneity (PH) model, in which the environment in which the three chromophores are present differ slightly resulting in

Cotton effects of different sign and/or size;[12, 13, 18-20, 25-29, 31-46] and (3) a now largely discredited[15, 29] Multiple Transitions (MT) model,[24] in which the CD spectrum is explained in terms of two close–lying transitions in BR[59] with opposite rotational strengths characteristic of polyenes[60] similar to the ATRPSB chromophore of BR. In this thesis I compare our data with the existing models and provide further insights into these models based on what our results suggest.

The principal purpose of my research was to utilize the PM→LIBM and

PM→UVCM photoconversions to provide new insights into the relative roles of EC and

PH in CD spectrum of PM. While a number of research groups have reported the generation of a “LIBM” upon irradiating the PM with 532 nm laser pulses,[3, 5-10].

Figure 3: Upper – CD spectrum of the PM depicting the bisignate band. Lower – Absorbance band of PM with absorbance maximum at 568 nm. The zero point of the bisignate band would coincide with the absorbance maximum of the absorption spectra.

Rhinow, et.al.[ 10] were the only group to report CD of LIBM to date. While these authors did not discuss the changes the morphology of the CD bands during the

PM→LIBM photoconversion, their spectra clearly indicate that the wavelength

+ maximum 휆푚푎푥 of the positive band shifts bathochromically whereas the wavelength

– maximum 휆푚푎푥 of the negative band remains constant. This observation plus the fact that

PM converts to a UVCM upon absorption of 254 nm UV light suggests that the purple → blue and purple → colorless photoconversions may provide novel insights into the relative roles of EC and PH in inducing the bisignate band in PM.

In my studies, various BR species consisting of various quantities of native membrane lipids and divalent cations were studied in order to characterize their effect on the structures of PM and BR. Delipidated PM was prepared by treating PM with

CHAPS[61] or Triton X-100[15, 62] detergents to remove ~75 % of the native lipids

(intertrimeric lipids), which keeps the trimers intact but reduces the unit cell dimensions to 59 Å instead of 62.4 Å, therefore contracting the lattice and bringing the chromophores closer. PM was treated with Triton X-100, which results in the loss of the hexagonal lattice of the PM and the generation of independent BR monomers solubilized in detergent micelles. By passing the PM through cation exchange column the cations were removed from the surface of PM to form cation free blue membrane (CFBM); yet and another species was prepared where the PM was saturated with excess amount of calcium to form calcium saturated purple membrane. The changes in the absorption and CD spectra of these various BR species were characterized as they were exposed to intense

532 nm laser pulses and the diffuse 254 nm line from a mercury lamp.

The following species were created by irradiating PM with intense 532 nm laser pulses

Purple membrane (PM) → Laser–Induced Blue Membrane (LIBM)

Delipidated Purple Membrane (DLPM) → Laser–Induced Delipidated Blue Membrane (LI–DLBM)

Monomer (MON) → Laser–Induced Colorless Monomer (LI–CMON)

Cation Free Blue Membrane (CFBM) → Laser–Induced Cation Free Blue Membrane (LI–CFBM)

Calcium Saturated Purple Membrane (CaPM) → Laser–Induced Calcium Saturated Purple Membrane (LI–CaPM)

The following species were created by exposing PM to 254 nm ultraviolet light from a

100 watt mercury lamp.

PM → Ultraviolet–Induced Colorless Membrane (UVCM)

DLPM→ Ultraviolet –Induced Delipidated Blue Membrane (UV– DLPM)

MON → Ultraviolet –Induced Colorless Monomer (UV–LI–CMON)

CFBM → Ultraviolet –Induced Cation Free Blue Membrane (UV–CFBM)

CaPM → Ultraviolet –Induced Calcium Saturated Purple Membrane (UV–CaPM)

The resulting color changes and absorption and CD spectral trends for all the species were characterized in order to specify whether the positive and the negative bands bear any direct relationship to the blue (P605) or colorless (P360) species present in LIBM

(P360 + P605) and UVCM (P360/UV only).

Figure 4: Top – Schematic representations of the native PM; Middle – LIBM; Bottom – UVCM. The dark blue represents the BR570; lighter blue represents the P605 and white represent the P360.

The mechanism involved in the color change of the PM when irradiated with the

532 nm laser pulses and diffuse 254 nm light have not been yet determined. Because divalent cations (Ca2+ and Mg2+) and native PM lipids regulate the color of PM, PM was treated to remove the cations (CFBM) and lipids (DLPM, MON), or saturated with

8 excess of calcium to make (CaPM). The resulting spectra were studied in order to determine the effect of cations and lipids on the color change observed during the PM →

LIBM and PM → UVCM photoconversions. Because PM converts to a CFBM upon removal of Ca2+ and Mg2+. Dr Masthay proposed a model for the color change as the PM is converted to LIBM (Figure 5) which involves the light–induced removal of cations from the purple membrane leading to a blue P605 species similar to that in CFBM. The absorption and CD spectral trends were studied in order to test this model. This mechanism displays the involvement of the divalent cations (M2+ = Ca2+, Mg2+) in the regulation of the formation of the colored product during the two–photon induced photoreduction process.

The PM binds ~ 4 moles of Ca2+ and Mg2+ per one mole of BR[63]. Dr Masthay’s

Model: During the photoreduction of trimer by 532 nm laser pulses, the two photons are absorbed by a single chromophore in the trimer. This chromophore is reduced to P360 and changes its conformation thereby inducing a conformational change in the adjacent two chromophores in the trimer. This conformational change induces the loss of cations from the two unreduced chromophores, resulting in a purple → blue color change in those two monomers.[3] This model was tested via an ICP experiment in which PM was converted to LIBM and UVCM. The centrifuged supernatant was tested for the presence of Ca2+ and Mg2+ using an ICP before and after the photoconversions.

Significantly, when PM is reduced with borohydride, a broad, nearly structure less

PUV band (i.e., similar to the P360 product in LIBM) results, concomitant with the loss of the absorbance at 570 nm but with no color change.[12, 64, 65] The PM simply turns progressively lighter purple, then lavender, then colorless with increasing percentage of

9 chromophore reduction. Even so, the bisignate CD bands undergo a bathochromic shift similar to that observed in LIBM and LI–DLBM.[12] Interestingly, then, the absorption spectra are like that of the CaPMLI–CaPM transition, but the CD bands behave like those of the PMLIBM and DLPMLI–DLBM photoconversions, suggesting that the loss of bisignate band intensity is due to reduction of the chromophore but is not intrinsically connected to the color of the non–reduced BR570 or P605 species.[12]

MATERIALS AND METHODS

A. BR Purchase

PM was purchased from Dr. Robert Birge’s research lab at the University of

Connecticut in Storrs, CT at a cost of $40 mg–1, and prepared according to the procedures typically used in the Birge group.[66, 67]

B. Laser Studies and Spectroscopy

During the course of each photoconversion overlaid UV–visible absorption spectra were obtained using a Jasco V–630 spectrophotometer and CD spectra was obtained using a Jasco J–815 spectrophotometer to provide insights into the changes in the chromophore conjugation pathway and binding site chirality, respectively.

C. Laser and Hg–Lamp Description

Solutions of different BR species (see below) were irradiated with 532 nm pulses from a Spectra Physics Model INDI–40 Pulsed Nd:YAG laser (0.9 cm beam diameter;

~6.5 ns pulse width) operating in TEM00 mode at 10 pulse sec–1 with actinic intensities of ~ 2.4 – 3.6 MW cm–2 (10 –15 mJ pulse–1). Beam intensities above the two–photon saturation threshold of ~ 0.4 MW cm–2[3, 5] were generated by attenuating the beam to

10% of its full intensity with a 10%–reflective beam sampler (Newport Model

10B20NC.1) which was antireflection coated on the back surface to eliminate ghosting

11 and wedged at an angle of 30±15 arc min to eliminate internal fringes. The intensities were characterized by inserting a Newport Model 818P–020–12 Power Detector coupled to a Newport Model 1918–C Power Meter between the polarizer and sample cuvette holder prior to irradiation sessions. A complete schematic diagram of the intensity– controlled laser irradiation system is shown in Figure 5.

During irradiation samples were stirred with a magnetic Starna “Spinette” stirrer, and sample temperatures were maintained at ambient temperatures of 20–22º C.

Figure 5: Schematic of the sample irradiation with 532 nm pulses from a Spectra Physics Model INDI–40 Pulsed Nd:YAG laser.

254 nm UV products were generated using output from a 100 W mercury arc lamp (Newport Model 6281) in combination with a 254 nm line filter (Oriel Model

#564000). All the irradiation experiments were performed in the dark, although the ambient room light did not interfere with the photochemistry of BR.

D. Sample Preparation

1. PM in Phosphate Buffer

Aqueous PM suspensions were prepared at BR concentrations of 3.8×10–4 –

–4 –6 –5 6.1×10 M and diluted to concentrations of 10 M < [BR]0 < 3×10 M in pH 6.9 –7.4 phosphate buffer prior to all experiments unless otherwise specified. PM suspensions were placed in standard 1 cm path length quartz cuvettes (Starna Model G14–5) and light–adapted using light from a slide projector. Concentrations and molar extinction

퐿퐴 coefficients for all species are specified assuming a molar excitation coefficient 휀570 =

63,000 M–1 cm–1 for light adapted (LA) PM. The concentration of BR in PM was found to be

1.94 ×10–5 M.

(a) Laser–Induced Blue Membrane (LIBM) was generated by irradiating PM suspensions prepared as described above with 12 mJ (2.9 MWcm–2) pulses until the absorbance maximum of the chromophore band dropped to 28% of its initial value and the samples appeared blue, with 579 < λmax < 590 nm.

(b) Ultraviolet–Induced Colorless Membrane (UVCM) was generated by irradiating PM suspensions prepared as described above with the 254 nm line of a 100 Watt Mercury lamp placed in a homemade Al bloch cuvette holder with a 1 cm diameter aperture; all the Hg lamp output was focused into the aperture. The 254 nm line was selected using an

Oriel # 56400 line filter until the absorbance dropped to 31 % its initial value.

2. Delipidated Purple Membrane (DLPM)

PM (500 µL) was added to 5 mL of 20 mM 3–[(3–cholamidopropyl) dimethylammonio]–1–propanesulfonate (CHAPS) detergent in 5.6 mM acetate

13 buffer.[61] The solution was transferred into a polycarbonate centrifuge tube of 10.4 mL capacity (# 355603). A balance tube was prepared with water. The two tubes were mounted in a Beckman L 70 Ultra centrifuge with type 70 Ti rotor. The solutions were spun at an acceleration of 45,000×g for 20 min at 4 °C. After centrifugation the purple supernatant was harvested. 0.15µL of the supernatant was added to 2.85 mL of Potassium

Phosphate buffer (25 mM, pH=7).

(a) Laser–Induced Delipidated Blue Membrane (LI–DLBM) was prepared by irradiating

DLPM suspensions prepared as described above with 10 mJ (2.4 MWcm–2) laser pulses until the absorbance maximum of the chromophore band dropped to 25 % of its initial value and the samples appear blue, with 579 < λmax < 590 nm.

(b) Ultraviolet–Induced Delipidated Purple Membrane (UV–DLPM) was generated by irradiating DLPM suspensions prepared as described above with the 254 nm line of 100

Watt Mercury lamp until the absorbance dropped to 33 % its initial value.

3. Monomer (Mon)

Mon was prepared by solubilizing PM in reduced Triton X–100 detergent

(reduced–TX; Sigma–Aldrich, used as purchased). Reduced–TX was used instead of normal TX to facilitate spectroscopic analysis in the UV region. Both the 532 nm pulsed laser and 254 nm UV (Hg lamp) irradiation experiments were performed on the same day simultaneously with identical solutions to minimize potential errors caused by differences in samples.

In the initial PM solubilization step we added 2.25 mL of a 1% v:v (1% w:w) solution of reduced–TX in pH 6.9 phosphate buffer to 600 µL of a 9.6 mg BR/mL

14 aqueous suspension of PM. The resulting suspensions consisted of BR monomers incorporated into reduced–TX micelles (critical micelle concentration = 0.015% v:v with a 4:1 w:w reduced–TX:BR ratio and a 0.5% v:v detergent:buffer ratio). The solution was prepared in a 10 ml volumetric flask and was sonicated in a Fisher Scientific model

FS20H sonicator for 1 minute with the flask to touching the bottom of the sonicator to maximize the sonicator efficiency. The solution was incubated in the dark for 36 hours to allow for the completion of the monomerization and the solution was transferred into a centrifuge tube. A balance tube was prepared with water. The two tubes were mounted in a Beckman L 70 Ultra centrifuge with type 70 Ti rotor. The solutions were spun at an acceleration of 200,000×g (44,000 rpm) for 45 min at 4 °C. After centrifugation 4.5 ml of the purple supernatant was harvested. The pellet comprised of the PM lipids was purple in color indicating some loss of BR. The sonication time was increased to one minute rather than the conventional sonication time of 20 seconds. This increase in time of sonication reduced the amount of BR lost in the pellet by 33 %. These concentrated Mon suspensions were diluted with buffer (0.5 % v:v detergent:buffer) making a total of 6 ml solution. The 6 ml solution was separated into two 3 ml solutions and filled in two separate cuvettes. The cuvettes have been marked as “Laser” and “HgLamp” to be distinguished from one another.

(a) Laser–Induced Colorless BR Monomer (LI–CMON) was prepared by irradiating suspensions of Mon prepared as described above with 10 mJ (2.4 MWcm–2) pulses until the absorbance maximum of the chromophore band dropped to 5 % of its initial value and the samples were faint purple or colorless to the eye.

(b) Ultraviolet–Induced Colorless Monomer (UV–CMON) was generated by irradiating

Mon suspensions with the 254 nm line of the 100 watt mercury lamp until the absorbance dropped to 12 % its initial value.

4. Cation–Free Blue Membrane (CFBM)

CFBM was prepared by passing PM suspensions (un–buffered to avoid adding

Na+ and K+ ions to the suspensions) through a 15 mm, 20 cm gravity column packed with

9 – 10 g of Bio–Rad AG50W–X8 biotechnological grade 100 – 200 mesh hydrogen form cation–exchange resin at pH = 5 according to the technique of Kimura, et. al.[68] The column slurry was rinsed with milliQ water until the pH = 5.9. Concentrated PM suspensions with [BR] = 3.8×10–4 M and volume of 200 µL were placed at the top of the columns; ~1 mL aliquots of CFBM were collected at the bottom of the columns. ~2.5 mL of BR was collected and it appeared to be blue with pH of 4.0.

(a) Laser–Induced Cation–Free Blue Membrane (LI–CFBM) was prepared by irradiating suspensions of CFBM prepared as described above with 2.4 MWcm–2, 10 mJ pulses until the absorbance maximum of the chromophore band dropped to 30 % of its initial value and the samples were faint blue or colorless to the eye.

(b) Ultraviolet–Induced Cation–Free Blue Membrane (UV–CFBM) was generated by irradiating CFBM suspensions prepared as described above with the 254 nm line of a 100

Watt Mercury lamp until the absorbance dropped to 46 % its initial value.

5. Calcium Saturated Purple Membrane (CaPM)

CaCl2 solution of 3.0 M was prepared and was centrifuged and the clear supernatant was collected. 85 µL of PM was added to 2.915 mL of CaCl2 solution. The solution appeared full of dispersed aggregates. Hence, to prevent the dispersed aggregated from settling, the solution was stirred for the whole run.

(a) Laser–Induced Calcium Saturated Purple Membrane (LI–CaPM) was prepared by irradiating suspensions of CaPM prepared as described above with 2.4 MWcm–2, 10 mJ pulses until the absorbance maximum of the chromophore band dropped to 29 % of its initial value and the samples were faint purple or colorless to the eye.

(b) Ultraviolet–Induced Calcium Saturated Purple Membrane (UV–CaPM) was generated by irradiating CaPM suspensions prepared as described above with the 254 nm line of a

100 Watt Mercury lamp until its absorbance dropped to 40 % is initial value.

E. ICP Experiment

The rich media that was used to grow BR contained the following per 1L prep:

250 g NaCl, 9.77 g MgSO4, 3.00 g tri–sodium citrate, 2.00 g KCl and 10 g peptone. The media was prepared at pH 7.2 using purified milliQ water (essentially ddH20) is used.

Although Ca2+ was never directly added to the medium but minute quantities of Ca2+ ions were found to be present. The experiments were performed on an Agilent 700 Series

Model 68467A ICP Optical Emission Spectrometer. Since we were looking for very small concentrations of Ca2+ and Mg2+ ions, we had to soak and wash all the glassware used to prepare standards with 16 M HNO 3, to replace the minute quantities of cations bound to the surface of the glassware with H+. The glassware was washed multiple times

17 with 18.0 MΩ·cm milliQ water after the acid wash. The glassware was washed in batches and the same acid solution was reused for all the batches. Ca2+ standards of 0.02, 0.05,

0.10, 0.25, 0.50, 0.60 and 0.70 ppb and Mg2+ standards of 5, 10, 25, 50, 70, 75 and 80 ppb were prepared from 1000 ppm stock ICP standard solutions [Agilent 700 Series Model

68467A ICP OES] for Ca2+ and Mg2+ ions. Each standard was run 5 times with 5 water runs between each run of the standard. The standardization process itself lasted for 15 hours. The standardization was performed on the same day of the irradiation step.

Concentrated BR suspension of volume 800 µL was added to 6.3 mL of milliQ which gave an absorbance of 1.27 at 휆푚푎푥= 563.4 nm. The 7.100 mL of BR was transferred into a polycarbonate centrifuge tube of 10.4 mL capacity (Beckman #

355603). A balancing tube was prepared with DI water. The sample and balance tubes were placed in a Beckman Coulter Type 70.1 Ti Rotor (one exactly opposite to the other) and mounted into the Beckman Coulter Preparative Ultra Centrifuge L – 70.

Centrifugation was performed at a speed of 33,000 rpm at 4 °C for 35 minutes. 5 mL of the supernatant was collected and run through the ICP. The BR pellet was re–suspended in milliQ water and the process was repeated two more times. A total of 3 supernatants were tested and the final pellet was re–suspended in milliQ water. The resulting PM which was virtually devoid of all the calcium and magnesium ions was divided into two cuvettes. One cuvette was irradiated with 15 mJ (3.6 MWcm–2), 532 nm laser pulses at 10

Hz for 240 minutes. The other cuvette was irradiated with the 254 nm line of our 100

Watt Mercury lamp for 545 minutes. Both the absorption and CD spectra were obtained for the solutions at the beginning and end of the irradiation; in addition, absorption spectra were obtained at regular intervals for both the species throughout the irradiation.

The supernatants from the resultant LIBM and UVCM were then tested for Ca2+ and

Mg2+ ions using the ICP.

RESULTS

(1) Laser Species

(a) LIBM – Absorption Spectra

Figure 6. Absorption Spectra of the photo–conversion of PM to LIBM induced by 6ns, 15 mJ pulse–1 (3.46 MWcm–2), 532 nm pulses for 230 min (spectra taken after 0, 10, 30, 50, 70, 90, 110, 130, 150, 180, 230 minutes of irradiation).

The PM sample was irradiated for a total of 230 min, with spectra taken after regular intervals. The absorption maximum for our light adapted PM samples was 568 nm consistent with literature reports. A568 drops from 1.27 to 0.354 after 230 minutes of irradiation which is a 72 % drop in the absorption maximum; the λmax shifted from 568 nm to 594 nm (776 cm–1). A three peaked photoproduct is observed with peaks at 340 nm, 360 nm and 380 nm. This product is called the P360 photoproduct. The shape of the

P360 is not very structured and appears to be broad. It becomes more structured with the

Formation of the blue P605 photoproduct. The absorbance at 360 nm increased by 59 % from 0.584 to 0.931. An isosbestic region is present between 434 nm and 447 nm. An isosbestic point is initially present at 610 nm, but disappears after 50 minutes of irradiation. The absorbance of the P642 photoproduct starts at 0.233 and increases up to 80 minutes (0.379) and then starts to fall, with its final absorbance being 0.256. The absorbance of the 255 nm dip is stable throughout the run. The 280 nm Tryptophan band was also observed to have no significant change in its absorbance.

(b) LIBM – CD Spectra

+ The wavelength maximum for the positive CD band (휆푚푎푥) of BR in pure PM

– was at 535 nm; The wavelength maximum for the negative CD band ( 휆푚푎푥 ) of BR in

+ pure PM was at 600 nm. The magnitude of the positive band (퐶퐷푚푎푥 ) was 10.6 mdeg

– + and the magnitude of negative band (퐶퐷푚푎푥) was –5.67 mdeg. 휆푚푎푥 shifted from 535 nm

21 to 560 nm (a 821.1 cm–1 bathochromic shift) during the course of the irradiation and

– + 휆푚푎푥 did not show a significant shift in its wavelength. 퐶퐷푚푎푥 fell from 10.6 to 0.551 (95

– % drop) and 퐶퐷푚푎푥 rises from –5.67 to 0.491 (108 % rise). An isosbestic point was observed between the positive and the negative band which initially is at 569 nm. The isosbestic point shifts with the irradiation time. The isosbestic point also differs from the zero point (the point where the positive and the negative band intersect with the horizontal axis). The zero point shifts from 576 nm to 600.4 nm (705.6 cm–1 bathochromic shift) after 70 minutes of irradiation. The CD spectra became monosignate after 70 minutes of irradiation. The initial magnitude of the positive CD band was almost twice that of the negative CD band. A band is found at 263 nm which corresponds to the tryptophan in the absorption spectra. This band became less positive shifted slightly to longer wavelengths with time. Another band was found at 318 nm which becomes less negative and rises. This band did not shift to longer or shorter wavelengths. There was also what appeared to be a two peaked band with the peaks at ~361 and ~ 379 nm which disappears as the time of irradiation increases.

The delipidated PM sample was irradiated for a total of 120 min, with spectra taken after regular intervals. The absorption maximum for BR in pure DLPM was at 563 nm. Amax drops from 1.30 to 0.324 (75 % drop) after 120 minutes of irradiation. λmax

–1 shifts from 563 nm to 596 nm (996.1 cm bathochromic shift). The P360 3 peaked product is more pronounced and sharp. The absorbance of the P360 product rose by 73 % from 0.513 to 0.887 [The appearance of the three peaked product is sudden and is nearly simultaneous with the purple–to–blue color change]. As the three peaked product gets more significant after 18 min irradiation, the isosbestic point which was a range (427.4 nm – 450 nm) becomes a single point (456 nm). A second isosbestic point is observed at

606 nm and disappears after 18 minutes of irradiation. The absorbance P642 nm photoproduct goes from 0.239 to 0.263 (9.1 % rise). This band increases for the first 48 minutes of irradiation and then starts to fall. The ~ 255 nm dip shows no significant decrease in its absorbance. The small decrease in its absorbance might be attributed to the

23 decrease in the absorbance of the adjacent tryptophan band, as A280 decreases from 2.23 to 2.05 (8 %).

(d) LI–DLBM – CD Spectra

+ – The 휆푚푎푥 for BR in pure DLPM was at 527 nm. The 휆푚푎푥 for BR in pure DLPM

+ – + was at 619 nm. While the 퐶퐷푚푎푥 was 6.31 mdeg and 퐶퐷푚푎푥 was –6.43 mdeg. 휆푚푎푥 shifts from 526.6 to 547.8 nm (735.0 cm–1 bathochromic shift) during the course of the

– –1 irradiation and 휆푚푎푥 shifts from 589 nm to 619 nm (823 cm bathochromic shift).

+ – 퐶퐷푚푎푥 fell from 6.31 to 0.00 (100 % drop) and 퐶퐷푚푎푥 rises from –2.53 to 0.432 (83 % rise). An isosbestic point was observed between the positive and the negative band which initially is at 563 nm. The isosbestic point shifted with the irradiation time to 576 nm.

The isosbestic point also differs from the zero point. The zero point is present at 574.2 nm which also shifts with time. As with PM the initial magnitude of the positive CD band is almost twice the negative CD band. The 263 nm band became less positive with time and fell, but did not shift to longer wavelengths with time. The 318 nm band becomes

24 less negative and rises, but did not shift to longer or shorter wavelengths. In contrast to

LIBM the ~361 and ~ 379 nm peaks were very intense, sharp and structured.

(e) LI–CMON – Absorption Spectra

Figure 10. Absorption Spectra of the photo–conversion of MON to LI–CMON induced by 6ns, 15 mJ pulse–1 (3.6 MWcm–2), 532 nm pulses for 60 min (spectra taken after 0, 3, 6, 9, 12, 15, 20, 25, 30, 60 minutes of irradiation).

The Monomer sample was irradiated for a total of 60 min, with spectra taken after regular intervals. The absorption maximum for BR in pure MON was at 555.6 nm.

Amax drops from 0.813 to 0.044 (95% drop) after 60 minutes of irradiation. λmax shifts

–1 from 556 nm to 560 nm (141.4 cm bathochromic shift). The absorbance of the P360 band increases with time but no 3 peaked product is seen, instead a broad band is seen. The

P360 absorbance increases from 0.219 to 0.445 (50.8 % increase). A very stable and significant isosbestic point is seen at 421.8 nm. 255 nm dip migrates to 250 nm, which is stable throughout the entire run. The 280 nm band is also very stable with time. The P642 product is absent in the monomer.

(f) LI–CMON – CD Spectra

Figure 11. CD Spectra of the photo–conversion of MON to LI–CMON induced by 6 ns, 15 mJ pulse– 1(3.6 MWcm–2), 532 nm pulses for 60 min (spectra taken after 0, 3, 6, 9, 12, 15, 20, 25, 30, 60 minutes of irradiation).

+ – The 휆푚푎푥 for BR in pure MON was at 526 nm. The 휆푚푎푥 for BR in pure MON was at

+ – + 598 nm. While the 퐶퐷푚푎푥 was 3.38 mdeg and 퐶퐷푚푎푥 was –1.45 mdeg. 휆푚푎푥 shifts from

–1 – 526 to 538 nm (437.9 cm bathochromic shift) and 휆푚푎푥 does not shift and remains

+ constant at 598 nm during the course of the irradiation. 퐶퐷푚푎푥 falls from 3.38 to – 0.11

(103 % drop) and the negative CD band is completely absent in MON. The zero point was present at 574 nm. The 258 nm band becomes less positive and 318 nm becomes less negative with time. There is not even a slight manifestation of the ~361 and ~ 379 nm band.

(g) LI–CFBM – Absorption Spectra

Figure 12. Absorption Spectra of the photo–conversion of CFBM to LI–CFBM induced by 6ns, 15 mJ pulse–1 (3.6 MWcm–2),532 nm pulses for 90 min (spectra taken after 0, 3, 9, 15, 21, 27, 36, 45, 60 and 90 minutes of irradiation).

The CFBM sample was irradiated for a total of 90 min, with spectra taken after regular intervals. The absorption maximum for BR in pure CFBM is found to be 602 nm.

Amax goes from 0.972 to 0.288 (70% drop) after 90 minutes of irradiation. λmax shifts from

–1 602 nm to 576 nm (761.88 cm hypsochromic shift). No P360 product is formed, not even a broad band is observed. Although there is no manifestation of the P360 product there is an isosbestic point at 493 nm. The P642 product was not present in the CFBM. There is a slight decrease in the absorbance of the ~ 255 nm dip from 1.85 to 1.78 (3.78 % drop).

There is a decrease in the absorbance of the 280 nm band. The absorbance goes from 2.22 to 2.11 (4.95 % drop).

(h) LI–CFBM – CD Spectra

Figure 13. CD Spectra of the photo–conversion of CFBM to LI–CFBM induced by 6ns, 15 mJ pulse– 1(3.6 MWcm–2), 532 nm pulses for 90 min (spectra taken after 0, 3, 9, 15, 21, 27, 36, 45, 60 and 90 minutes of irradiation).

+ – The 휆푚푎푥 for BR in pure CFBM was at 556 nm. The 휆푚푎푥 for BR in pure CFBM was at

+ – + 648.6 nm. While the 퐶퐷푚푎푥 was 5.81 mdeg and 퐶퐷푚푎푥 was –3.25 mdeg. 휆푚푎푥 remained

– + constant at 556 nm as did 휆푚푎푥 at 648 nm. 퐶퐷푚푎푥 falls from 5.81 to 1.18 (80 % drop)

– and 퐶퐷푚푎푥 rises from –3.24 to 0.33 (110 % rise). An isosbestic point is observed at

612nm. The zero point is present at 613 nm. The magnitude negative CD band is almost two thirds the positive CD band. The 263 nm band becomes less positive with time. 318 nm is at 333 nm in CFBM. This band becomes less negative and rises. Two peaked band does not appear with irradiation.

(i) LI–CaPM – Absorption Spectra

Figure 14. Absorption Spectra of the photo–conversion of CaPM to LI–CaPM induced by 6 ns, 15 mJ pulse–1 (3.6 MWcm–2), 532 nm pulses for 42 min (spectra taken after 0, 6, 12, 18, 24, 30, 42 minutes).

The CaPM sample was irradiated for a total of 42 min, with spectra taken after regular intervals. The P360 3 peaked product was present with the peaks slightly structured.

Isosbestic Point was present at 456 nm. The P642 product was absent. 256 nm dip rises little with its absorbance going from 1.27 to 1.36 (6.61% rise). 280 nm band was very stable. The absorption maximum for BR in pure CaPM was 563 nm. Amax goes from 1.01 to 0.29 (71 % rise) after 42 minutes of irradiation. λmax shifts from 563 nm to 566 nm

(81.59 cm–1 bathochromic shift).

(j) LI–CaPM – CD Spectra

Figure 15. CD Spectra of the photo–conversion of CaPM to LI–CaPM induced by 6 ns, 15 mJ pulse– 1(3.6 MWcm–2), 532 nm pulses for 42 min (spectra taken after 0, 6, 12, 18, 24, 30, 42 minutes).

+ – The 휆푚푎푥 for BR in pure CaPM was at 530.8 nm. The 휆푚푎푥 for BR in pure CaPM was at

+ – + 599.8 nm. While the 퐶퐷푚푎푥 was 10.3 mdeg and 퐶퐷푚푎푥 was –3.41 mdeg. 휆푚푎푥 shifts from 531 nm to 534 nm (98.8 cm–1 bathochromic shift) during the course of the

– –1 irradiation and 휆푚푎푥 shifts from 600 nm to 604 nm (127 cm bathochromic shift).

+ – 퐶퐷푚푎푥 falls from 10.3 to 1.88 (81 % rise) and 퐶퐷푚푎푥 rises from –3.41 to 1.53 (144 % drop). An isosbestic point was observed at 571.2 nm. The zero point was present at 575.6 nm. The magnitude negative CD band was almost two thirds the positive CD band [

2×퐶퐷+ CD – ]. The 263 nm band becomes less positive with time and 318 nm 3 ~ becomes less negative with time. ~361 and ~ 379 nm peaks appear with time. It has structure but not very pronounced.

(2) Hg – Lamp Species

(a) UVCM – Absorption Spectra

Figure 16. Absorption Spectra of UVCM generated by irradiating PM suspensions with the 254 nm line of a 100 Watt Mercury lamp for 480 min (spectra taken after 0, 10, 30, 60, 90, 120, 150, 180, 210, 240, 270, 300, 330, 360, 390, 420, 480 minutes of irradiation).

The PM sample was irradiated for a total of 480 min, with spectra taken after regular intervals. The absorption maximum for BR in pure PM was at 569 nm which is consistent with literature reports. Amax drops from 1.27 to 0.39 (70 % drop) after 480 minutes of irradiation

–1 λmax shifts from 569 nm to 564 nm (143.33 cm hypsochromic shift). The three peaked product is absent, instead a broad band is present which rises as the time of irradiation increases. The Absorbance at P360 increases from 0.576 to 0.873 (51.6 % rise). The isosbestic point disappears after 270 minutes of irradiation. The isosbestic point ranges from 392 nm to 458 nm up to 270 min irradiation. There is an isosbestic point at 608 nm which disappears after 90 minutes of irradiation. The absorbance of the P646 photoproduct starts at 0.23 and increases up to 80 minutes to 0.34 and then starts to fall, with its final absorbance being 0.24. The absorbance of the 255 nm dip increases with time where the

31 absorbance goes from 2.00 to 2.53. The 280 nm band also rises as the time of the irradiation increases. The absorbance goes from 2.42 to 2.62 (7.63 % rise).

(b) UVCM – CD Spectra

+ – 휆푚푎푥 of BR in pure PM was at 537 nm. 휆푚푎푥 of BR in pure PM was at 600 nm.

+ – + While the 퐶퐷푚푎푥 was 11.2 mdeg and 퐶퐷푚푎푥 was –4.9 mdeg. 휆푚푎푥 shifts from 537 to

–1 – 540 nm (110.5 cm bathochromic shift) during the course of the irradiation and 휆푚푎푥

–1 + shifts from 600 nm to 615 nm (406.2 cm bathochromic shift). 퐶퐷푚푎푥 falls from 11.30

– to 1.08 (90 % drop) and 퐶퐷푚푎푥 rises from – 4.95 to 1.08 (121 % rise). An isosbestic point was observed between the positive and the negative band which initially is at 573 nm. The isosbestic point does not shift significantly with the irradiation time. The zero point is present at 577 nm which shifts with time. It goes from 577 nm to 614 nm after

240 minutes of irradiation. The band becomes monosignate after that. The magnitude positive CD band is almost twice the negative CD band. The 263 nm band becomes less positive and the 318 nm band becomes less negative with time. The ~361 nm and ~ 379

32 nm which is present in the beginning starts to disappears as the time of irradiation increases

Figure 18. Absorption Spectra of UV–DLPM generated by irradiating DLPM suspensions with the 254 nm line of a 100 Watt Mercury lamp for 300 minutes (spectra taken after 0, 2, 10, 10, 30, 60, 90, 120, 120, 150, 180, 210 minutes of irradiation).

The delipidated PM sample was irradiated for a total of 300 min, with spectra taken after regular intervals. The absorption maximum for BR in pure DLPM was at 564 nm. Amax drops from 1.23 to 0.40 (67 % rise) after 300 minutes of irradiation. λmax shifts

–1 from 564 nm to 555 nm (288 cm hypsochromic shift). Peaks of the P360 three peaked product are not as sharp as the laser species and the absorbance goes from 0.498 to 0.589

(18.27 % rise). An initial isosbestic point ranges from 341 nm to 459 nm up to 90 minutes of irradiation and then the range shifts to 433 nm to 454 nm. The absorbance increases from 0.24 to 0.33 for 90 minutes and then starts to decrease finally to 0.203. A second isosbestic point is present up until 90 minutes of irradiation at 613 nm and then disappears. 255 nm dip shows very slight to almost no change. The absorbance changes from 1.70 to 1.75 (2.94% rise). There is a slight decrease in the absorbance of the 280 nm

33 band. The absorbance goes from 2.15 to 1.96 (8.83 % drop). There is an isosbestic point at 263 nm.

(d) UV–DLPM – CD Spectra

Figure 19. CD Spectra of UV–DLPM generated by irradiating DLPM suspensions with the 254 nm line of a 100 Watt Mercury lamp for 300 minutes (spectra taken after 0, 2, 10, 10, 30, 60, 90, 120, 120, 150, 180, 210 minutes of irradiation).

+ – The 휆푚푎푥 for BR in pure DLPM was at 528 nm. The 휆푚푎푥 for BR in pure DLPM

+ – + was at 597 nm. While the 퐶퐷푚푎푥 was 8.12 mdeg and 퐶퐷푚푎푥 was – 4.23 mdeg. 휆푚푎푥

– does not shift and stays at 528 nm during the course of the irradiation and 휆푚푎푥 shifts

–1 + from 597 nm to 599 nm (55.92 cm bathochromic shift). 퐶퐷푚푎푥 falls from 8.12 to 0.23

– (97 % rise) and 퐶퐷푚푎푥 rises from –4.23 to 0.89 (79 % drop). An isosbestic point is observed between the positive and the negative band which initially is at 573 nm. The zero point is present at 576 nm which shifts with time. The magnitude positive CD band is almost twice the negative CD band. The 263 nm band becomes less positive with time and falls. There is a small shift to longer wavelengths with time. 318 nm becomes less negative and rises. ~361 and ~ 379 nm peaks appear with time. They peaks are structured but not very sharp.

(e) UV–CMON – Absorption Spectra

Figure 20. Absorption Spectra of UV–CMON generated by irradiating Mon suspensions with the 254 nm line of a 100 Watt Mercury lamp for 270 minutes (spectra taken after0, 15, 30, 45, 60, 75, 90, 120, 150, 210, 270 minutes of irradiation).

The Monomer sample was irradiated for a total of 270 min, with spectra taken after regular intervals. The Absorption maximum for BR in pure MON was at 555 nm.

Amax goes from 0.817 to 0.101 (88 % drop) after 270 minutes of irradiation. λmax does not shift and remains stable at 555 nm. The absorbance of the P360 band increases with time but no 3 peaked product is seen, instead a broad band is seen. The P360 absorbance increases from 0.234 to 0.338 in 150 minute irradiation. It then starts to decrease to a final absorbance of 0.316 (after 270 min irradiation). After 150 minute irradiation the product formed also starts to degrade hence the drop in the absorbance. The isosbestic point at 422 nm is stable up to 150 nm and then disappears as the product starts to degrade. The length of the band between 448 nm to 493 nm seems to show some small peaks in its structure, these small peaks (disturbances) seem to be absent in other species.

(don’t know what these peaks mean). The far UV product is absent in the monomer. 255 nm dip migrates to 250 nm. This dip slowly rises but there is not a huge increase in absorbance. The 280 nm band loses absorbance with time

(f) UV–CMON – CD Spectra

Figure 21. CD Spectra of UV–CMON generated by irradiating Mon suspensions with the 254 nm line of a 100 Watt Mercury lamp for 270 minutes (spectra taken after 0, 15, 30, 45, 60, 75, 90, 120, 150, 210, 270 minutes of irradiation).

+ – The 휆푚푎푥 for BR in pure MON was at 533 nm. The 휆푚푎푥 for BR in pure MON

+ – + was at 611 nm. While the 퐶퐷푚푎푥 was 3.00 mdeg and 퐶퐷푚푎푥 was – 0.595 mdeg. 휆푚푎푥 does not shift with time and remains stable at 533 nm during the course of the irradiation

– + and 휆푚푎푥 is present at 611 nm (no negative band). 퐶퐷푚푎푥 falls from 3.00 to 0.302 (89 %

– drop ) and 퐶퐷푚푎푥 starts out at –0.595; considered to be completely absent. An isosbestic point was present at 569 nm (although it is a monosignate band so this value is not accurate). The zero point is present at 567 nm. The negative band in the CD is completely absent. The 260 nm band becomes less positive with time and 318 nm becomes less negative. There is not even a slight manifestation of the ~ 361 and ~ 379 nm band.

(g) UV–CFBM – Absorption Spectra

Figure 22. Absorption Spectra of UV–CFBM generated by irradiating CFBM suspensions with the 254 nm line of a 100 Watt Mercury lamp 240 minutes (spectra taken after 0, 20, 40, 60, 60, 80, 110, 145, 180, 240 minutes of irradiation).

The CFBM sample was irradiated for a total of 240 min, with spectra taken after regular intervals. The absorption maximum for BR in pure CFBM was at 603 nm. Amax drops from 1.05 to 0.484 (54 % drop) after 240 minutes of irradiation. λmax shifts from

–1 603 nm to 569 nm (997.1 cm hypsochromic shift). No P360 product is formed, not even a broad band is observed. Although there is no manifestation of the P360 product there is an isosbestic point at 506 nm. The P642 is not present in the CFBM. The absorbance at the

255 nm band does not significantly change. There is a decrease in the absorbance of the

280 nm band. The absorbance goes from 2.31 to 2.15 (6.92 % drop).

(h) UV– CFBM – CD Spectra

Figure 23. CD Spectra of UV–CFBM generated by irradiating CFBM suspensions with the 254 nm line of a 100 Watt Mercury lamp 240 minutes (spectra taken after 0, 20, 40, 60, 60, 80, 110, 145, 180, 240 minutes of irradiation).

+ – The 휆푚푎푥 for BR in pure CFBM was at 567 nm. The 휆푚푎푥 for BR in pure CFBM

+ – + was at 645 nm. While the 퐶퐷푚푎푥 was 6.91 mdeg and 퐶퐷푚푎푥 was – 4.31 mdeg. 휆푚푎푥 shifts from 567 to 546 nm (690.3 cm–1 hypsochromic shift) during the course of the

– –1 irradiation and 휆푚푎푥 shifts from 645 nm to 647 nm (38.4 cm bathochromic shift).

+ – 퐶퐷푚푎푥 falls from 6.91 to 1.90 (72 % drop) and 퐶퐷푚푎푥 rises from – 4.31 to – 0.29 (93 % rise). An isosbestic point is observed at 613 nm. The zero point is present at 616 nm. The magnitude negative CD band is almost two thirds the positive CD band. The 263 nm band becomes less positive with time. The 318 nm band is at 333 nm in CFBM. This band becomes less negative and rises. Two Peaked band does not appear to be present and does not appear with irradiation.

(i) UV–CaPM – Absorption Spectra

Figure 24. Absorption Spectra of UV–CaPM generated by irradiating CaPM suspensions with the 254 nm line of a 100 Watt Mercury lamp for 250 minutes (spectra taken after 0, 40, 70, 100, 130, 130, 160, 190, 250, 250 minutes of irradiation) .

The CaPM sample was irradiated for a total of 250 minutes, with spectra taken after regular intervals. The absorption maximum for BR in pure CaPM was at 563.2nm.

Amax drops from 1.00 to 0.40 (60 % drop) after 250 minutes of irradiation. λmax shifts from 563 nm to 546 nm (527.0 cm–1 hypsochromic shift). Absorbance at 360 nm increases from 0.37 to 0.64 (72.9 % rise), but there is no manifestation of the three peaked product. Instead a broad band is seen which rises as the time of irradiation increases. An isosbestic point is present at 467 nm. The P642 product seems to be absent.

255 nm dip rises with its absorbance going from 1.24 to 1.56 (25.8 % rise). The 280 nm band also increases with time with its absorbance going from 1.61 to 1.70 (5.5 % rise).

(j) UV–CaPM – CD Spectra

+ – The 휆푚푎푥 for BR in pure CaPM was at 526 nm. The 휆푚푎푥 for BR in pure CaPM was at

+ – + 598 nm. While the 퐶퐷푚푎푥 was 7.82 mdeg and 퐶퐷푚푎푥 was – 6.75 mdeg. 휆푚푎푥 does not

– shift and remains stable at 526 during the course of the irradiation, also 휆푚푎푥 does not

+ – shift and remain stable at 598. 퐶퐷푚푎푥 falls from 7.82 to 0.54 (93 % drop) and 퐶퐷푚푎푥 rises from –6.76 to 0.05 (101 % rise). An isosbestic point was observed at 571 nm. The zero point was present at 570 nm. The magnitude negative CD band was almost two thirds the positive CD band. The 263 nm band becomes less positive with time and falls.

The band does not shift to longer wavelengths with time. 318 nm becomes less negative with time. ~361 and ~ 379 nm peaks do not seem to be present. Rather a broad band is seen which rises as the time of irradiation increases.

(3) ICP Results

The concentration of our stock BR was 1.28 mg/mL and we used 0.8 mL to prepare 7.1 mL of BR with A570 = 1.24. The number of moles of BR in 3.5 mL of this solution

(solution in one cuvette) was calculated to be 1.94×10–8. If we assume the literature value of ~ 4 moles of Ca2+ and Mg2+ per a mole of BR, the calculated number of moles of cations would come to 7.77×10–8.

The ICP would give the results in the form of intensities of the solutions injected.

Therefore the intensities of all the standard solutions were recorded and calibration curves were constructed. From the calibration curves the amount of calcium and magnesium in the supernatant of LIBM were determined to be 0.11 ppb and 13.27 ppb respectively. Also the amount calcium and magnesium in the supernatant of the UVCM were determined to be 1.23 ppb and 84.18ppb. Although the amount of Ca2+ and Mg2+ in the UVCM supernatant were comparatively higher the amount of ions released was still considered to be small.

The calculated number of moles of calcium and magnesium in the LIBM supernatant were 0.00919×10–9 and 1.91×10–9 which come to a combined total of 1.92×10–9 moles of cations (0.1 cations per BR molecule). The calculated number of moles of calcium and magnesium in the UVCM supernatant were 0.11×10–9 and 12.12×10–9, respectively, which come to a combined total of 1.2×10–8 moles of cations (0.6 cations per BR molecule). These two values when compared with the theoretical value of 4 cations per

BR are very small and therefore it can be safely concluded that our experimental results do not show any release of Ca2+ and Mg2+ in to the supernatant during the PM→LIBM of the photochemical reaction.

(4) Spectral Analysis Tables

Table 1: Summarizes the wavelength maximum, energy maximum, absorbance maximum, width of the half peak and extinction coefficient of different BR species for both laser and Hg lamp irradiations.

LASER λmax –1 –1 –1 Emax(cm ) Amax –1 Ƹmax (cm M ) SPECIES (nm) W1/2(cm )

PM → LIBM 568 17606 1.2 3615 63000

DLPM → LI– 5623 17775 1.3 3283 – DLBM

MON → LI– 556 17999 0.8 3667 42000 CMON

CFBM → LI– 602 16611 1.0 4407 – CFBM

CaPM → LI– 563 17756 1.0 3671 – CaPM

HGLAMP λmax –1 –1 –1 –1 Emax(cm ) Amax W1/2(cm ) Ƹmax (cm M ) SPECIES (nm)

PM → UVCM 569 17581 1.2 3570 63000

DLPM → UV– 564 17737 1.2 3285 – DLPM

MON → UV– 555 18012 0.8 3692 42000 CMON

CFBM → UV– 603 16584 1.0 4344 – CFBM

CaPM → UV– 563 17775 1.0 3757 – CaPM

Table 2: Summarizes the wavelength maximum for CD+, energy maximum, CD maximum for positive CD band and width of the half peak of different BR species for both laser and Hg lamp irradiations. – W1/2(cm CD+ λmax –1 1 LASER SPECIES Emax(cm ) CDmax ) (nm)

PM → LIBM 535 18685 10.6 2564

DLPM → LI– 527 18990 6.3 2215 DLBM

MON → LI– 526 19012 3.0 2922 CMON

CFBM → LI– 556 17980 5.8 2813 CFBM

CaPM → LI–CaPM 531 18840 10.3 2909

– HGLAMP CD+ λmax –1 W1/2(cm Emax(cm ) CDmax SPECIES (nm) 1)

PM → UVCM 537 18636 11.3 2482

DLPM → UVDCM 528 18954 4.6 3296

MON → UV– 533 18776 3.0 2717 CMON

CFBM → UV– 567 17624 7.0 2327 CFBM

CaPM → UV– 526 19026 7.8 2351 CaPM

Table 3: Summarizes the wavelength maximum for CD–, energy maximum, CD maximum for negative CD band and width of the half peak of different BR species for both laser and Hg lamp irradiations. – W1/2(cm –1 1 LASER SPECIES CD–λmax (nm) Emax(cm ) CDmax )

PM → LIBM 600 16672 –5.67 783 DLPM → LI– 589 16978 –6.43 1136 DLBM

MON → LI– 598 16722 –1.00 916 CMON

CFBM → LI– 649 15418 –3.25 1176 CFBM

CaPM → LI–CaPM 600 16672 –3.41 877

– W1/2(cm 1 HGLAMP –1 ) CD–λmax (nm) Emax(cm ) CDmax SPECIES

PM → UVCM 600 16661 –5.0 931

DLPM → UVDCM 597 16750 –4.2 393

MON → UV– 611 16377 –0.6 1320 CMON

CFBM → UV– 645 15509 –4.3 1114 CFBM

CaPM → UV– 598 16734 –6.8 1187 CaPM

Table 4: Summarizes the change in the magnitude of absorbance at 532 nm, change in the magnitude of CD+ band and change in the magnitude of CD– band. Time of irradiation has also been displayed. All the values have been normalized.

LASER SPECIES ΔA ΔCD+ ΔCD– TIME (minutes)

PM → LIBM 118 180.2 237.9 230

DLPM → LI–DLBM 113 199.8 228.8 120

MON → LI–CMON 180 261.7 3.1 60

CFBM → LI–CFBM 57 132.1 244.8 90

CaPM → LI–CaPM 100.0 138.1 524.6 42

TIME HGLAMP SPECIES ΔA ΔCD+ ΔCD– (minutes)

PM → UVCM 101 165.0 312.6 480

DLPM → UVDLPM 87 180.5 307.0 300

MON → UV–CMON 155 163.4 206.3 270

CFBM → UV–CFBM 23 113.9 174.7 240

CaPM → UV–CaPM 68 174.3 203.2 250

Table 5: Summarizes the change in the rate of absorbance at 532 nm, rate of CD+ band drop and rate of CD– band rise. Time of irradiation has also been displayed. All the values have been normalized

PM → LIBM 0.51 0.78 1.03

DLPM → LI– 0.94 1.67 1.91 DLBM

MON → LI– 3.0 4.37 0.05 CMON

CFBM → LI– 0.63 1.47 2.72 CFBM

CaPM → LI– 2.38 3.30 12.50 CaPM

HGLAMP ΔA ΔCD+ ΔCD– SPECIES

PM → UVCM 0.21 0.34 0.65

DLPM → UV– 0.30 0.60 1.02 DLPM

MON → UV– 0.58 0.61 0.77 CMON

CFBM → UV– 0.10 0.47 0.73 CFBM

CaPM → UV– 0.27 0.70 0.81 CaPM

DISCUSSION

(a) One Photon/ Two Photon Absorption–Spectral Analysis

PM→LIBM and PM→UVCM Photoconversions

There are a number of similarities and differences in one–photon and two–photon photoproducts of the PM. In LIBM a bathochromic shift in the absorption λmax is observed as the solutions turn blue and a broad and unstructured P605 product is generated. The hypsochromically–shifted P360 product (which is formed concomitantly with P605 photoproduct) is vibronically structured with peaks at 340, 360 and 380 nm; these peaks become more pronounced as the time of irradiation increases. The CD peaks mimic the bathochromic absorption shift as both the positive and the negative CD peaks shift to longer wavelengths (See Tables 2 and 3). In contrast, no shift in the absorption

λmax is observed during the generation of UVCM; the absorption drops as the time of irradiation increases as the PM becomes first lavender and then colorless. A P360/UV product is present as a broad unstructured band which increases in intensity with decreasing wavelength. It is unclear if the increase A360 is due to the formation of a genuine P360/UV species or is a result of the increase in the absorbance of the 280 nm tryptophan band. Such a rise in absorbance could be due to (1) photoproducts formed from the apoprotein backbone, (2) increases in light scattering, or (3) a combination of these two effects. Changes in the CD spectrum during the generation of UVCM mimic

47 those of the absorption spectrum; both the positive and the negative CD bands lose intensity without shifting to longer wavelengths. The increase in the A640 in the

PM→LIBM and PM→UVCM photoconversions could be indicative of a bathochromically shifted “P640” photoproduct is similar for both the species. The tryptophan band is reasonably stable in the laser species but in the Hg lamp species its absorbance increases. Since tryptophan would be expected to photodegrade upon exposure to UV light, the increase in A280 could originate from (1) PM lipid photochemistry, (2) increases in light scattering due to aggregation of PM fragments, or

(3) a combination of these two factors.

DLPM → LI–DLBM and DLPM → UV–DLPM Photoconversions

Upon LI–DLBM formation the P360 product becomes very structured and prominent. This prominence in vibronic structure suggests that the DLPM–which is more compact and packed than native PM,[62, 69] places additional conformational constraints upon the P360 chromophore. In contrast to PM, the 280 nm band in LI–DLBM is clearly degrading in both the Laser and Hg lamp species, suggesting that the PM lipids might stabilize the PM apoprotein against photodegradation. UV–DLPM also shows a slight manifestation of the three peaked product which after a while starts to deteriorate.

CFBM→LI–CFBM and CFBM→UV–CFBM Photoconversions

A360 steadily increases in all the laser photoproducts except the LI–CFBM while in the Hg lamp species the absorbance at 360 nm increases early and then starts to drop.

For CFBM both the Laser and Hg lamp species the visible absorption band shifts to

48 shorter wavelengths; it is likely that this hypsochromic shift is an artifact caused by a combination of photodegradation of blue chromophore and increase in light scattering.

The P360 and the P360/UV products are completely absent in both LI–CFBM and UV–

CFBM. Significantly, the visible band due to the blue chromophore still loses its absorbance suggesting that its blue, P605 like chromophore undergoes irreversible photochemistry as does P605 in LIBM and LI–DLBM. This result is consistent with those reported by Rhinow et. al[10].

CaPM→LI–CaPM and CaPM→UV–CaPM Photoconversions

P360 is vibronically structured in LI–CaPM, while a broad unstructured UV band is seen in UV–CaPM. It was evident from the observations with LIBM and LI–DLBM that the P360 was more prominent in tightly packed lattices and during laser irradiation.

This might suggest that the UV irradiation causes a loss in the crystallinity of the structure which lessens the conformational constraints on the chromophores thereby lessening the vibronic structure of the three peaked product. This conclusion is reasonable since ∈532= 0 for the P360 and P360/UV products; they can completely degrade only via very weak virtual two–photon excitation process.

For all the laser species the absorbance of the P360 product steadily increases; in contrast this absorbance starts to drop after certain period of irradiation for all the Hg lamp species. I believe that this is occurring because P360/UV photoproducts absorb the

254 nm photon and degrade via a UV induced one–photon process, but do not absorb the

532 nm pulses and hence do not degrade via a two photon process in the laser species.

(b) Proposed Mechanisms for Generation of LIBM and UVCM

Previous work done by Dr. Masthay’s group[3] proved that the two–photon cross sections δ532 of PM and Mon (determined from the quantum yields for the generation of

LIBM and LI–CMON) were both very large and similar in magnitude, thereby showing that the processes involved sequential two–photon rather than virtual state–mediated two–photon excitation of the ATRPSB chromophore. More importantly, the similarities in the values of δ532 (PM→LIBM) and δ532 (Mon→LI–CMON) almost definitively prove that the PM→LIBM photoconversion is mediated by a 1–monomer–2–photon process rather than a 2–monomer–2–photon process. We believe this to be the case because δ532

(PM→LIBM) and δ532 (MON→LI–CMON) would differ significantly if the PM→LIBM photoconversion was mediated by a 2–monomer–2–photon process, as a 2–monomer–2– photon process is not possible for MON→LI–CMON). In this 1 monomer two–photon mechanism, a single monomer absorbs two–photons and the chromophore C=NH+ linkage is reduced to a C–N single bond, thus yielding P360. The colorless P360 product

(white in Figure 4) would most likely differ from BR570 (purple in Figure 4) conformationally; monomers which have been converted to P360 may thus induce additional conformational changes in nearby (unreduced) BR570 molecules,[3, 8, 10] which convert to the blue P605 photoproduct as a result.

hv532 * hv532 ** 570 570 BRBRBR 570  I 460 570 BRBR 570  I 460 570 BRBR 570 (1) a b c a b c a b c

** I 460 570 BRBR 570  P360 570 BRBR 570 (2) a b c a b c

ConformationalChanges LIBM LIBM LIBM 2+ P360 570 BRBR 570    P360 605 BRBR 605 = LIBM + M (3) a b c a b c

Scheme 3: Diagram represents the proposed mechanism for the PM to LIBM photoconversion

The P605 photoproduct which resembles the blue chromophore in CFBM forms only in the case of LIBM and LI–DLBM upon two photon irradiation and is absent in all the other BR species. Hence intense laser pulses are required to generate the P605 species.

Significantly the product formed by the reaction between the reducing agent NaBH4 and

PM contains a P360–like product but lacks the P605 like product; i.e., the borohydride reduction product —which should be similar to the reduction by laser pulses—does not show a shift in the λ [12, 64, 65] like that in the LIBM. Since NaBH4 is an exogenous reducing agent, the endogenous amino acids and water molecules in the chromophore binding site of BR (which serve as sources of e–, H. or H–) are inert during the laser and

Hg lamp induced photoreduction. While the generation of UVCM is believed to be occurring through a 1–monomer one–photon process. Individual BR570 (purple in figure

4–bottom) molecules which absorb one 254 nm photon are reduced into a colorless monomer(white in figure 4–bottom); i.e., the UVCM is generated via a 1–monomer–1– photon process. The generation of the UVCM is believed to follow a Poisson distribution of BR monomers throughout the membrane i.e., BR monomers are reduced randomly and independently of the each other throughout the membrane. This hypothesis was

51 consistent with our results, since we see a clear degradation of the tryptophan band and the P360/UV photoproduct in UVCM.

Also for the UVCM the monomer is believed to undergo an indirect reduction, rather than a direct reduction of chromophore as in the case of LIBM. In LIBM single monomers undergo the two photon excitation and are photo–reduced; both the 532 nm photons because the tryptophan undergoes two–photon excitation 4 orders of magnitude less efficiently at this wavelength. In the case of UVCM, both the tryptophan band and the P360 product are susceptible to photodegradation by the 254 nm photons. Therefore, the reduction of the ATRPSB chromophore in UVCM occurs via a Trp–to–ATRPSB energy transfer process. This hypothesis is further explored in the cation binding section.

Changes in different absorption and CD peaks were plotted against the time of irradiations to make quantitative judgments about the spectra for various BR species (See

Appendix – graphs k, l and m). Photocooperativity is present in the absorbance drop

ΔA532 of the chromophore during the generation of LIBM and LI–DLBM; when the same calculations were performed on the P360 photoproduct this cooperativity was absent. Two possibilities exist for the ΔA532 photocooperativity: (1) The sigmoidal curve for ΔA532 could be an artifact that is a result of the shift of the λmax in case of both LIBM and LI–

DLBM or (2) might be a genuine effect which suggests a cooperativity in the destruction of the chromophore. Assuming option (2), when initially the membrane is purple and rigidly crystalline, the generation of P360 is difficult. When the blue species starts to form and the crystallinity of the membrane is lost.[10]

Significantly, the ΔA532 cooperativity is absent in the MON. This result is not surprising for MON, since it lacks a membrane lattice: MON is inherently incapable of photocooperativity. It is somewhat surprising that the CFBM and CaPM do not manifest

ΔA532 photocooperativity, since they presumably maintain trimeric, hexagonal crystallinity in their membrane structures. However, these species do not undergo bathochromic shifts; the only means of loss of A532 is through loss of intensity; bathochromic shift does not contribute. Hence, this result suggests that the photocooperativity is partially the consequence of the bathochromic shift observed is during the generation of LIBM and LI–LI–DLBM. CaPM, CFBM do not change color during the course of the reaction therefore a conformational change is assumed to not be necessary for their generation; since the cooperativity models depends on the conformational change occurring in a trimer that increases the susceptibility of the surrounding trimers, this absence of cooperativity can be explained through the absence of conformational change. This scenario would also hold if the crystallinity of CaPM and

CFBM were less pronounced than that in PM and DLPM. It is highly significant that the

UVCM and UV–DLPM species show no cooperativity, thereby supporting our hypothesis of indirect reduction of the chromophore through energy transfer from the tryptophan. Due to a one–photon reduction process and an indirect reduction of ATRPSB chromophore via an endogenous reducing surrounding amino acids the UVCM undergoes no conformational changes during its reduction therefore would not account for the loss of cations and a blue color change.

(d) Exciton Coupling/ Protein Heterogeneity

There has been a long standing debate about the origin of the biphasic CD band which has still not been conclusively resolved. An exciton coupling hypothesis was argued by

Dencher and Heyn, [58, 59] who found that the bisignate band of PM disappeared when

PM was treated with Triton X–100 detergent. They proposed increased mobility of BR in membrane disrupted the exciton coupling. Ebrey and Becher, [36] who bleached the PM with hydroxylamine and then reconstituted it with all–trans–retinal; they demonstrated that the shape of the CD was dependent on the number of retinal binding sites occupied.

In addition, they showed that the distance between chromophores was 26–36 Å allows the chromophores to exhibit exciton coupling. While Dencher, Heyn, Ebrey and Becher assumed the exciton coupling within the trimers Cassim later argued for the presence of exciton coupling between the monomers by proposing a heptameric unit cell for exciton coupling within PM. Another model explaining the bisignate band is multiple transitions model where the combination of CD bands with opposite rotational strengths due to a retinal–apoprotein heterogeneity of the BR molecules or due to two possible close–lying long–wavelength transitions of the retinal of the BR with opposite rotational strengths. A number of other authors proposed a protein heterogeneity model, ElSayed and

Karnaukhova [24] where the three BR subunits in the trimer are not conformationally equal, and therefore, the bisignate CD spectrum of BR in the purple membrane occurs due to a superposition of the CD spectra from variously distorted BR subunits with opposite rotational strengths, in the trimer than interchromophoric exciton–coupling interactions. They also stated that this heterogeneous environment is created due to the conformational restraints of the lattice. Therefore if the membrane lattice is subjected to

54 any perturbation there would be a loss of the conformational restraint there by the monomer loses its heterogeneity (as clearly occurs when PM is converted to MON via solubilization in TX100 detergent).

The CD spectrum of LIBM and LI–DLBM show bisignate band where the positive band is almost twice the negative band while in case of the monomer it forms a monosignate band. The CD has almost equal magnitude of cotton effects in case of CFBM and CaPM, for both these cases the negative band is equal to 2/3rds the positive band. This would argue for PH because the loss to cations and lipids would cause changes in the structural constraints of the membrane thereby changing the internal environment and chirality of various ATRPSB chromophores within the PM. The positive CD band shifts bathochromically in case of LIBM and LI–DLBM, which is similar to the spectra by

Rhinow, et. al.[10] and also similar to NaBH4[12, 64, 65] reduction. (During the NaBH4 reduction the absorption spectrum does not shift while the CD spectrum shifts to longer wavelengths). We see a prominent increase of the 360 nm band in CD spectra. This very significant rise in the band is absent in all our species. We would rather see a small rise in this band with a manifestation of two peaks at ~ 360 nm and ~ 380 nm respectively.

Evidence from Our Research Which Favors Protein Heterogeneity Model –

The work done in the past by Dr Masthay’s group[3] suggested the existence of PH but did not conclusively prove the PH hypothesis for two reasons. First, their earlier work proved that the two–photon cross sections δ532 of PM and Mon (determined from the quantum yields for the generation of LIBM and LI–CMON) were both very large and similar in magnitude, thereby showing that the processes involved sequential two–photon

55 rather than virtual state–mediated two–photon mechanisms. Second, and more importantly, the similarities in the values of δ532 (PM→LIBM) and δ532 (Mon→LI–

CMON) almost definitively prove that the PM→LIBM photoconversion is mediated by a

1–monomer–2–photon process rather than a 2–monomer, 2–photon process. We believe this to be the case because δ532 (PM→LIBM) and δ532 (Mon→LI–CMON) would differ significantly if the PM→LIBM photoconversion was mediated by a 2–monomer, 2– photon process, as a 2–monomer, 2–photon process is not possible for Mon→LI–

CMON). b. Exciton Delocalization Lifetimes. The exciton delocalization lifetimes of 1.3 fs (us) and 25 fs (El–Sayed) calculated using the time–energy uncertainty relationship ΔEΔt

≥h/4∏ are so short that they make a 1–monomer–2–photon process unlikely— particularly with nanosecond pulses like those we use. That said, 1–monomer–2–photon process could in principal occur in PM even with these short lifetimes, but the efficiency would be small due to depolarization of the transition moment vectors as the energy would rapidly migrate from one monomer to another. Our large cross sections help to argue against this possibility, making EC unlikely.

Khorana and coworkers[62] were able to successfully model both the absorption and CD spectra of PM by assuming that the broad (W1/2 ~3,800 cm–1), unstructured 570 nm absorption band of BR could be comprised of a sum of three narrower (W1/2 ~2,500 cm–

1) bands centered at 519, 555, and 591 nm. While this result supports the PH Model in its own right, it has strong implications for the PM→LIBM, Mon→LI–CMON, and

PM→UVCM mechanisms. In particular, Khorana’s model would allow for PM to turn blue upon 532 nm irradiation and colorless upon 254 nm irradiation, as follows.

In Fig. 3 of Khorana and coworkers, it is apparent that the extinction coefficient ∈532 is quite large for the 519 and 555 nm bands, but much smaller for the 591 nm band. Hence,

532 nm laser irradiation would be expected to selectively photodegrade the 519 and 555 nm bands via a sequential two–photon process; such degradation would be much less efficient for the 591 nm band. This scenario would result in (1) a bathochromic shift of the absorption band with a concomitant purple→blue color change, to a final maximum near 591 nm, and (2) a bathochromic shift of the positive CD band with little shift in the negative CD band. While this expected result using the Khorana model does not perfectly match our experimental results, it is similar enough to suggest that it has significant merit.

As additional support, note that the 591 nm band extends very far to the red. It could be that the apparently higher absorbance out near 640 nm toward the end of the PM–to–

LIBM photoconversion could be due to this band after the 519 and 555 nm bands are essentially eliminated. (This would not explain the high absorbance in this region late in the generation of the UVCM however, as it is likely that the 519, 555, and 591 bands would degrade at nearly equal rates upon 254 nm irradiation; see arguments below).

As additional support, note that the PH model would explain the lack of a blue photoproduct during the Mon→LI–CMON photoconversion. This agrees with our results because the 519, 555, and 591 bands are due to different chromophore environments for different members of a trimer in the PM. These would coalesce into a single band in the monomer centered near 555 nm. This single band would not shift in wavelength during the photodegradation, but would simply lose intensity, as is observed with Mon.

As yet another additional point of support, we have noted that P360 becomes more vibronically structured toward the end of the PM→LIBM and DLPM→LI–DLBM photoconversions. Since the vibronic structure in P360 is likely due to rigid conformational constraints on the reduced–ATRPSB chromophore, the vibronic structure of P360 would be consistent with the PH model if the 519 and 555 nm monomers—which photodegrade early—had more relaxed conformational constraints on the chromophore than the 591 monomers, which photodegrade late.

The Khorana model would also result in the generation of colorless photoproducts upon

254 nm irradiation, since 254 nm photons are principally absorbed by the apoprotein backbone, which then transfers energy or electrons to the chromophores in all three heterogeneous proteins (519, 555, and 591 absorbing) with roughly equal efficiency, so that the 591 band would be destroyed at the same rate as the 519 and 555 nm bands.

Hence, neither the 570 nm absorption band nor either of the CD bands would undergo shifts in wavelength; they would simply lose intensity during the PM→UVCM photoconversion.

It is important to note that one argument against the PH model of Khorana is that the 519,

555, and 591 nm heterogeneous absorption bands are somewhat narrow for retinyl protonated Schiff base chromophores.[70]

(e) Cation Binding

The number of divalent cations (M2+ = Mg2+ and Ca2+) released during the Hg lamp irradiation (0.6 M2+ per BR molecule) was greater than the number of cations released during the Laser irradiation (0.1 M2+ per BR molecule). In both cases the number of

58 cations lost is significantly less than the theoretical value (4 M2+ per BR for complete removal of cations). These results are consistent with earlier studies from Dr Masthay’s laboratory in which the M2+ binding constants of divalent cations as well as the selectivity of the membrane surface for divalent cations decreased significantly during the generation of LIBM. [3] These new results suggests that while some M2+ leaves the

PM surface during the generation of LIBM and UVCM, it is likely that the majority relocalizes on the surface as a result of conformational changes, induced by laser or Hg lamp. Most importantly, these results suggest that the purple→blue color change induced by the laser during the generation of LIBM is different than the non–photochemical removal of M2+ which occurs during the formation of CFBM.

CONCLUSIONS

Intensity of the visible band and photoreduction of Schiff base terminus of protonated retinyl Shiff base chromophore occurs regardless of whether color change occurs. P360 UV photoproduct becomes more vibronically structured with increasing packing of the membrane lattice. Negative CD bands are more photolabile than the positive CD bands in visible range. Lattice must be present for the negative CD band to be prominent because when the lattice structure was perturbed using TX100 we saw a loss of the bisignate band. This phenomenon is further supported by the loss of the bisignate band upon the conversion of PM→LIBM when the crystallinity of the membrane is slowly lost. In species that do not change color the bisignate bands are symmetric with a node between positive and negative band centered at the wavelength of the absorbance maximum. It is likely that lipid–cation interactions are important in this regard. The 280 nm “tryptophan” band is photostable and plays little to no role in the 532 nm laser induced BR photochemistry however, amino acids in the apoprotein backbone likely act as “endogenous photoreducing agents” in the UV–induced species. We believe that the endogeneous reducing amino acids is less proximal to the protonated Shiff base chromophore terminus in absence of divalent cations hence we don’t see any P360 product for the LI–CFBM.

FUTURE STUDIES

The following studies would provide additional insights in the current set of data and will add to the present understanding of the structure of the PM.

(1) The accumulation of the positive charges one side of the membrane could cause the

membrane to bend slightly due to the repulsion of the positive charges. This

phenomenon is known as “flapping” of the membrane. The origin of the bisignate

band could be further studied in regard to the flapping of the membrane. Thin

membrane studies can be made in order to study this phenomenon. Flapping of

fragments being associated with bisignate band could be eliminated as a possibility if

PM placed in a thin film does not manifest the bisignate band.[8, 10]. The stirring

effects on the bisignate band could be further studied to shed light on this matter.

(2) The studies based on the isolated trimers have not fully been explored. Hence the

future study might include a study of the CD and absorption of isolated trimers. This

would be particularly useful since Rhinow, et. al.[10] concluded that LIBM consists

of loosely associated trimers. We could expect anti–cooperativity with in a trimer if it

is the unreduced monomer with in a trimer (2 blue and 1 white) which are reduced

푏푙푢푒 푝푢푟푝푙푒 next, because the 휀532 < 휀532 due to the bathoshift (looking at the quantum yield

(ɸ) values would help resolve this. Cooperativity might still be apparent in the trimer

푏푙푢푒 푝푢푟푝푙푒 푏푙푢푒→푐표푙표푟푙푒푠푠 푝푢푟푝푙푒→푏푙푢푒 (3) if 훿532 > 훿532 or ∅532 > ∅532 . Cooperativity could also be

푝푢푟푝푙푒→푏푙푢푒 푏푙푢푒→푐표푙표푟푙푒푠푠 ascertained if we know ∅532 and ∅532 .

(4) We do not expect cooperativity to be apparent in UVCM because it is 254 nm light

which is being absorbed which excited the apoprotein. We should look to see how

푝푢푟푝푙푒 푏푙푢푒 푏푙푢푒 푝푢푟푝푙푒 휀254 and 휀254 compare because we would expect cooperativity if 휀254 > 휀254 .

The 254 nm light could be absorbed by ATRPSB or by the apo–protein. A good way

to test our model is to generate UVCM with ~1/3 monomers destroyed, irradiate with

laser and see if it turn blue. Likewise to irradiate BoroCM with laser. It would be

interesting to see if LIBM and UVCM form in immobilized membranes in thin films.

(5) Studies of BR mutants and the PM–to–LIBM transition in those can be studied.

(6) Two–photon polarization ratio experiments, as the PM–to–LIBM photoconversion

should be more efficient with circularly rather than linearly polarized light if EC is

present. We used linearly polarized laser pulses in our studies.

(7) The PH model allows for the shift to longer wavelengths during the PM–to –LIBM

process without requiring the loss of Ca2+ or Mg2+, and hence is for the most part

consistent with your ICP results. It does not preclude the loss of cations, but it also

does not require the loss of cations.

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APPENDIX

Parameters during the Laser Irradiation: All the laser–induced photoconversions were generated using the 532 nm pulsed laser output from a Spectra Physics INDI–40 Pulsed

Dye Laser (0.9 cm beam diameter) operating at 10 Hz at intensities of 10–15 mJ pulse–1

(2.4–3.6 MW cm–2). All the samples were kept at ambient temperatures throughout the experiment and were stirred during irradiation using a STARNA “SPINETTE” electronic cell stirrer (Model SCS 1.11)

Parameters during the HgLamp Irradiation: UV–induced photoconversions were generated using the filtered 254 nm (Newport Model 564000) output from a (Newport

Model 6281) 100 W Hg Lamp focused into a 1×4 cm Quartz cuvettes containing 2.4 –

3.5 mL of BR–containing solutions focused into an ~ 0.9 cm diameter spot size. All the samples were kept at ambient temperatures throughout the experiment and were stirred during irradiation using a STARNA “SPINETTE” electronic cell stirrer (Model SCS

1.11)

(A) GRAPHS (a) Laser Species – Absorbance versus Time

LIBM LI–DLBM

LI–CMON LI–CFBM

LI–CaPM

(b) Hg Lamp Species – Absorbance versus Time

UVCM UV–DLPM

UV–CMON UV–CFBM

UV–CaPM

LIBM LI–DLBM

LI–CMON LI–CFBM

LI–CaPM

(d) Hg Lamp Species – CD versus Time

UVCM UV–DLPM

UV–CMON UV–CFBM

UV–CaPM

(e) Laser Species – Absorbance spectra– Wavelength versus Time

LIBM LI–DLBM

LI–CMON LI–CFBM

LI–CaPM

(f) Hg Lamp Species – Absorbance Spectra– Wavelength versus Time

UVCM UV–DLPM

UV–CMON UV–CFBM

UV–CaPM

(g) Laser Species – Wavelength Shift in Absorption maximum and CD + and CD – Peak

LIBM (λmax vs Time) LIBM (λ vs T)–CD+ LIBM (λ vs T)–CD– m) 620 620m) 620 WAVELENTH (max) WAVELENTH WAVELENGTH(n 520 520WAVELENGTH(n 520 TIME(min) 0 TIME(min)100 200 0 100 200 0 TIME(min)100 200

DLBM (λ vs T)–CD+ DLBM (λmax vs Time) DLBM (λ vs T)–CD- 620 620 620 ) (max) WAVELENGTH

520 520 520WAVELENGTH(nm)

0 TIME(min)100 200 WAVELENGTH(nm 0 TIME(min)100 200 0 TIME(min)100 200

LI–CMON (λmax vs Time) LI–CMON (λ vs T)–CD+ LI–CMON (λ vs T)–CD- 620 620 620 WAVELNGTH(max) 520 520WAVELENGTH(nm) 520WAVELENGTH(nm) 0 TIME(min)100 200 0 TIME(min)100 200 0 TIME(min)100 200

LI–CFBM (λ vs T)–CD+ LI–CFBM (λmax vs Time) 620 620 620 LI–CFBM (λ vs T)–CD- WAVELENGTH(nm) 520 520WAVELENGTH(nm) 520 WAVELENTH(max) 0 TIME(min)100 200 0 TIME(min)100 200 0 TIME(min)100 200

LI–CaPM (λmax vs Time) LI–CaPM (λ vs T)–CD+ LI–CaPM (λ vs T)–CD- 620 620 620

570 570 570 WAVELENGTH(max) WAVELENGTH(nm) WAVELENGTH(nm) 520 520 520 0 TIME(min)100 200 0 TIME(min)100 200 0 TIME(min)100 200

(h) Hg–Lamp Species – Wavelength Shift in Absorption maximum and CD + and CD – Peak

570 UVCM (λmax vs T) 540 UVCM (λ vs T)–CD+ 620 UVCM (λ vs T)–CD-

600 Wavelength(nm)

560 535 580WAVELENGTH(nm) WAVELENGTH(nm) 0 200TIME(min)400 600 0 200TIME(min)400 600 0 200TIME(min)400 600

580 1000 600 UVPDLBM (λ vs T)–CD- UVPDLBM (λmax vs T) UVPDLBM (λ vs T)–CD+

560 m) m) WAVELENGTH(n WAVELENGTH(n

540Wavelength(nm) 0 595 0 TIME(min)200 400 0 TIME(min)200 400 0 100 TIME(min)200 300 400

555.5 1000 1000 UVLI–CMON (λ vs T)–CD- UVLI–CMON (λmax vs T) UVLI–CMON (λ vs T)–CD+

555 500 500 Wavelength(nm)

554.5 WAVELENGTH(nm) 0 WAVELENGTH(nm) 0 0 100TIME(min)200 300 0 100TIME(min)200 300 0 100TIME(min)200 300

650 600 650 UVLI–CFBM (λmax vs T) UVLI–CFBM (λ vs T)–CD+ UVLI–CFBM (λ vs T)–CD-

600 550 645 Wavelength(nm) WAVELENGTH(nm) 550 500WAVELENGTH(nm) 640 0 100TIME(min)200 300 0 100TIME(min)200 300 0 100TIME(min)200 300

570 1000 1000 UVLI–CaPM (λmax vs T) UVLI–CaPM (λ vs T)–CD+ UVLI–CaPM (λ vs T)–CD- 560 500 500 550 Wavelength(nm) WAVELENGTH(nm) 540 WAVELENGTH(nm) 0 0 0 100TIME(min)200 300 0 100TIME(min)200 300 0 100TIME(min)200 300

(i) Overlaid Band Intensity vs Time Graphs Overlaid Absorbance vs Time (Laser Species) Overlaid Absorbance vs Time (Hglamp Species)

Overlaid CD+ vs Time (Laser Species) Overlaid CD+ vs Time (Hglamp Species)

Overlaid CD– vs Time (Laser Species) Overlaid CD– vs Time (Hglamp Species)

(j) Overlaid Wavelength vs Time Graphs Overlaid λABS vs Time (Laser Species) Overlaid λABS vs Time (Hglamp Species)

Overlaid CD+ vs Time (Laser Species) Overlaid CD+ vs Time (Hglamp Species)

Overlaid CD– vs Time (Laser Species) Overlaid CD– vs Time (Hglamp Species)

(k) The rate of absorbance change in the absorbance spectra at the wavelength corresponding to the positive band in CD LASER SPECIES HGLAMP SPECIES

400 4.00E+02 UVCM – 536.2 nm bump LIBM – 534.4 nm bump

2.00E+02 200 (A0/At)*100 (A0/At)*100 0.00E+00 0 0 50 100Time 150(min) 200 250 0 200Time (min)400 600 5.00E+02 400 DLBM– 530 nm bump UV–DLBM– 531 nm bump

200 (A0/At)*100 0.00E+00 0

(A0/At)*100 0 50Time (min)100 150 0 100 200Time (min)300 400

2.00E+03 1000 LI–CMON– 530 nm bump UV–CMON– 530 nm bump

1.00E+03 (A0/At)*100 (A0/At)*100

0.00E+00 0 0 20 Time40 (min) 60 80 0 50 100Time150 (min)200 250 300

4.00E+02 200 LI–CFBM – 560 nm bump UV–CFBM – 567 nm bump

2.00E+02 100 (A0/At)*100 (A0/At)*100

0.00E+00 0 0 20 Time40 (min)60 80 100 0 50 100 Time150 (min) 200 250 300 4.00E+02 400 LI–CaPM– 531 nm bump UV–CaPM– 533.6 nm bump

2.00E+02 200 (A0/At)*100 (A0/At)*100

0.00E+00 0 0 10 Time20 (min)30 40 50 0 50 100 Time150 (min) 200 250 300

(l) The rate of absorbance change in the absorbance spectra at the wavelength Maximum LASER SPECIES HGLAMP SPECIES

4.00E+02 400 LIBM - 534.4 nm max UVCM - 568.8 nm max

2.00E+02 200 (A0/At)*100 (A0/At)*100 (A0/At)*100

0.00E+00 0 0 50 100TIME (min)150 200 250 0 100 200 Time300 (min)400 500 600

6.00E+02 400 UVDLPM - 563.8 nm DLBM - 562.6nm 200 4.00E+02 0 2.00E+02 0 100 200 300 400 (A0/At)*100 -200 (A0/At)*100 0.00E+00 0 50TIME (min)100 150 -400 Time (min)

2.00E+03 1000 LI–CMON - 555.6 nm UV–CMON - 555.2 nm 1.50E+03

1.00E+03 500 (A0/At)*100

5.00E+02 (A0/At)*100

0.00E+00 0 0 20 TIME40 (min) 60 80 0 50 100 Time150 (min)200 250 300

4.00E+02 400 LI–CFBM - 602 nm UV–CFBM - 603 nm

2.00E+02 200 (A0/At)*100 (A0/At)*100 (A0/At)*100 0.00E+00 0 0 20 TIME40 (min)60 80 100 0 50 100Time150 (min)200 250 300 60 300 LI–CaPM – 563.2 nm UV–CaPM - UV - 562.6 nm 40 200

20 100 (A0/At)*100 (A0/At)*100

0 0 0 2 TIME4 (min) 6 8 0 100Time (min)200 300

(m) The rate of absorbance change in the absorbance spectra at the wavelength corresponding to the negative band in CD LASER SPECIES HGLAMP SPECIES

3.00E+02 LIBM - 599.8 nm 300 UVCM - 536 .2 nm

2.00E+02 200

1.00E+02 100 (A0/At)*100 (A0/At)*100 0.00E+00 0 0 50 100Time (min)150 200 250 0 200Time (min)400 600

4.00E+02 300 DLBM - 589 nm UVDLPM - 597 nm 200 2.00E+02 100 (A0/At)*100 (A0/At)*100

0.00E+00 0 Time (min) 0 50 100 150 0 100 Time200 (min) 300 400

1.50E+03 600 LI–CMON- 598 nm UV–CMON- 610.6 nm 1.00E+03 400

5.00E+02 200 (A0/At)*100

0.00E+00 0 (A0/At)*100 0 20 Time40 (min) 60 80 0 100Time (min)200 300

4.00E+02 300 LI–CFBM - 648.6 nm UV–CFBM - 644.8 nm 200 2.00E+02 100 (A0/At)*100 (A0/At)*100

0.00E+00 0 0 20 Time40 (min)60 80 100 0 50 100 150Time 200(min) 250 300

4.00E+02 300 LI–CaPM - 599.8 nm UV–CaPM- 597.6 nm 200 2.00E+02 100 (A0/At)*100 (A0/At)*100

0.00E+00 0 0 10 Time20 (min)30 40 50 0 50 100 150Time (min)200 250 300

(n) Absorbance of P340 P360 P380 plotted against time – Laser Species LIBM LI–DLBM

LI–CMON LI–CFBM

LI–CaPM

(o) Absorbance of P340 P360 P380 plotted against time – HgLamp Species

UVCM UV–DLPM

UV–CMON UV–CFBM

UV–CaPM

(p) CD vs T Plots in UV bands–Laser Species

LIBM LI–DLBM

LI–CMON LI–CFBM

LI–CaPM

(q) CD vs T Plots in UV bands–HgLamp Species UVCM UV–DLPM

UV–CMON UV–CFBM

UV–CaPM

(r) ICP Calibration Curves ICP Calibration for Calcium

ICP Calibration for Magnesium

(B) Tables

Table 6. Changes in Circular Dichroic and Absorption Spectra of Various Purple and Blue Bacteriorhodopsin Species during Various 2hν (532nm)–Induced Photoconversions Circular Dichroism Spectrum

init,— init, init,— init, max CDmax CDmax P360 max pulse 2h532–Induced pulse — Photoconversion (PC) Absorption Spectrum

init A280 A532  max pulse pulse

Mod –2.9  10–5 1.2  10–4 543 609 Mod —  PM LIBM Strong –2.4  –2.8  10–5 568 Mod 10–6 Strong –1.1  10–5 1.6  10–4 527 589 Sharp — DLPMLI–LI– Strong –4.6  DLBM –2.6  10–5 563 Sharp –6 10 None –2.1  10–4 3.6  10–3 Mon 534 580 None — Strong –2.4  –6.8  10–5 556 Broad 10–6 None –8.6  10–6 6.3  10–5 556 649 None —  CFBM None –4.3  602 –6.7  10–6 None 10–7

Weak –7.4  10–5 2.9  10–4 CaPM 531 600 Broad — Strong –4.1  –5.2  10–5 565 Mod 10–6

Table 7. Rate of Intensity Changes (qualitative observations) Un-Normalized

LASER SPECIES A CD+ CD-

PM → LIBM 4 5 4

DLPM → LI–DLBM 3 2 2

MON → LI–CMON 1 1 5 (distant)

CFBM → LI–CFBM 5 4 2

CaPM → LI–CaPM 2 3 1

HGLAMP SPECIES A CD+ CD-

PM → UVCM 4 4 4

DLPM → UV– 3 3 3 DLPM

MON → UV– 1 1 5 (distant) CMON

CFBM → UV– 5 5 1 CFBM

CaPM → 2 2 2 UV0CaPM

Table 8. Magnitude of Intensity from the graphs (quantitative observations) Un- Normalized

LASER SPECIES A CD+ CD- 

230 PM → LIBM 0.704 -10.02 +6.16

DLPM → LI– 0.778 -6.301 +6.86 120 DLBM

MON → LI– 0.682 -3.42 +0.97 60 CMON

CFBM → LI– 0.271 -4.62 +3.5 90 CFBM

CaPM → LI– 0.825 -8.43 +4.94 42 CaPM

HGLAMP A CD+ CD-  SPECIES

PM → UVCM 0.634 -10.2 +6.03 480

DLPM → 0.621 -4.34 +5.11 300 UVDLPM

MON → UV– 0.632 -2.69 +0.60 270 CMON

CFBM → 0.133 -5.01 +4.02 240 UVCFBM

CaPM → 0.415 -7.286 +6.81 250 UVCaPM

Table 9. Magnitude of Intensity – ranking (quantitative observations) – Un-Normalized

LASER A CD+ CD-  SPECIES

230 PM → LIBM 3 1 2

DLPM → LI– 2 3 1 120 DLBM

MON → LI– 4 5 5 60 CMON

CFBM → LI– 5 4 4 90 CFBM

CaPM → LI– 1 2 3 42 CaPM

HGLAMP A CD+ CD-  SPECIES

PM → UVCM 1 1 2 480

DLPM → 3 4 3 300 UVDLPM

MON → UV– 2 5 5 270 CMON

CFBM → 5 3 4 240 UVCFBM

CaPM → 4 2 1 250 UVCaPM

Table 10. Rate of change of Intensity (quantitative observations)–Un -Normalized

LASER SPECIES A CD+ CD-

PM → LIBM 0.0030 -0.043 +0.026

DLPM → LI–DLBM 0.0064 -0.052 +0.057

MON → LI–CMON 0.0113 -0.057 +0.016

CFBM → LI–CFBM 0.0030 -0.051 +0.039

CaPM → LI–CaPM 0.013 -0.200 +0.12

HGLAMP SPECIES A CD+ CD-

PM → UVCM 0.0013 -0.021 +0.01

DLPM → UVDLPM 0.0020 -0.014 +0.01

MON → UV– 0.0023 -0.009 +0.00 CMON

CFBM → UVCFBM 0.0005 -0.020 +0.01

CaPM → UVCaPM 0.0016 -0.029 +0.02

Table 11. Rate of change of Intensity (ranking)-quantitative observations – Un- Normalized

LASER SPECIES A CD+ CD-

PM → LIBM 4 5 4

DLPM → LI–DLBM 3 3 2

MON → LI–CMON 2 2 5

CFBM → LI–CFBM 5 4 3

CaPM → LI–CaPM 1 1 1

HGLAMP SPECIES A CD+ CD-

PM → UVCM 4 2 4

DLPM → UVDLPM 2 4 2

MON → UV–CMON 1 5 5

CFBM → UVCFBM 5 3 3

CaPM → UVCaPM 3 1 1

Table 12. Rate of Wavelength Changes (qualitative observations) – Un-Normalized

LASER SPECIES A CD+ CD-

PM → LIBM 2 – Batho 3 – Batho 2 – Batho

DLPM → LI–DLBM 1 – Batho 1 – Batho 1 – Batho

MON → LI–CMON 3 – No Shift 2 – Batho 3 – No Shift

CFBM → LI–CFBM 5 – Hypso 4 – No Shift 3 – No Shift

CaPM → LI–CaPM 3 – No Shift 4 – No Shift 3 – No Shift

HGLAMP SPECIES A CD+ CD-

PM → UVCM 3 – Hypso 2 – No Shift 1 – Batho

DLPM → UVDLPM 3 – Hypso 2 – No Shift 2 – No Shift

MON → UV–CMON 5 – No Shift 2 – No Shift 2 – No Shift

CFBM → UVCFBM 1 – Hypso 1 – Hypso 2 – No Shift

2 – No CaPM → UVCaPM 2 – Hypso 2 – No Shift Shift

Table 13. Magnitude of wavelength changes –quantitative observations – Un-Normalized

LASER SPECIES A CD+ CD-

PM → LIBM +21 +24.6 +9

DLPM → LI–DLBM +33.4 +21.2 +30

MON → LI–CMON +4.4 +12.4 +0.2

CFBM → LI–CFBM -26.4 +0.2 -1.2

CaPM → LI–CaPM +2.6 +2.8 +4.6

HGLAMP SPECIES A CD+ CD-

PM → UVCM -4.6 +2.6 +15

DLPM → UVDLPM -9 0 +2

MON → UV–CMON 0 0 0

CFBM → UVCFBM -34.2 -21.4 +1.6

CaPM → UVCaPM -16.2 0 0

Table 14. Magnitude of wavelength changes (ranking)-quantitative observations – Un- Normalized

LASER SPECIES A CD+ CD-

PM → LIBM 3 1 2

DLPM → LI–DLBM 1 2 1

MON → LI–CMON 4 3 5

CFBM → LI–CFBM 1 5 4

CaPM → LI–CaPM 5 4 3

HGLAMP SPECIES A CD+ CD-

PM → UVCM 4 2 1

DLPM → UVDLPM 3 3 2

MON → UV– 5 3 4 CMON

CFBM → UVCFBM 1 1 3

CaPM → UVCaPM 2 3 4

Table 15. Rate of Wavelength changes (quantitative) – Un-Normalized

LASER SPECIES A CD+ CD-

PM → LIBM +0.09 +0.10 +0.039

DLPM → LI–DLBM +0.27 +0.17 +0.25

MON → LI–CMON +0.073 +0.20 +0.0033

CFBM → LI–CFBM -0.293 +0.0022 -0.013

CaPM → LI–CaPM +0.061 +0.066 +0.109

HGLAMP SPECIES A CD+ CD-

PM → UVCM +0.009 +0.005 +0.031

DLPM → UVDLPM -0.03 0 +0.006

MON → UV–CMON 0 0 0

CFBM → UVCFBM -0.14 -0.089 +0.006

CaPM → UVCaPM -0.064 0 0

100

Table 16. Rate of wavelength changes ranking (quantitative) – Un-Normalized

LASER SPECIES A CD+ CD-

PM → LIBM 3 3 3

DLPM → LI–DLBM 2 2 1

MON → LI–CMON 4 1 5

CFBM → LI–CFBM 1 5 4

CaPM → LI–CaPM 5 4 2

HGLAMP SPECIES A CD+ CD-

PM → UVCM 4 2 1

DLPM → UVDLPM 3 3 2

MON → UV– 5 3 3 CMON

CFBM → UVCFBM 1 1 2

CaPM → UVCaPM 2 3 3

101

Table 17. Magnitude changes (quantitative) ranking – Normalized

 LASER SPECIES A CD+ CD- 

PM → LIBM 2 3 3 230

DLPM → LI– 3 2 4 120 DLBM

MON → LI– 1 1 5 60 CMON

CFBM → LI– 5 5 2 90 CFBM

CaPM → LI– 4 4 1 42 CaPM

 HGLAMP A CD+ CD- SPECIES

PM → UVCM 2 3 1 480

DLPM → 3 1 2 300 UVDLPM

MON → UV– 1 4 3 270 CMON

CFBM → 5 5 5 240 UVCFBM

CaPM → 4 2 4 250 UVCaPM

102

Table 18. Rate changes (quantitative) ranking – Normalized

LASER SPECIES A CD+ CD-

PM → LIBM 5 5 4

DLPM → LI–DLBM 3 3 3

MON → LI–CMON 1 1 5

CFBM → LI–CFBM 4 4 2

CaPM → LI–CaPM 2 2 1

HGLAMP SPECIES A CD+ CD-

PM → UVCM 4 5 5

DLPM → UVDLPM 2 3 1

MON → UV–CMON 1 2 3

CFBM → UVCFBM 5 4 4

CaPM → UVCaPM 3 1 2

103