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Study of Protein-Bacteriochlorophyll and Protein-Lipid Interactions of Natural and Model Light-Harvesting Complex 2 in Purple Bacterium Rhodobacter Sphaeroides

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Study of Protein-Bacteriochlorophyll and Protein-Lipid Interactions of Natural and Model Light-Harvesting Complex 2 in Purple Bacterium Rhodobacter Sphaeroides Study of protein-bacteriochlorophyll and protein-lipid interactions of natural and model light-harvesting complex 2 in purple bacterium Rhodobacter sphaeroides DISSERTATION DEPARTMENT OF BIOLOGY I BOTANIC LUDWIG-MAXIMILIANS-UNIVERSITÄT MÜNCHEN Submitted by Lee Gyan KWA March 2007 1. Referee: PD. Dr. P. Braun 2. Referee: Prof. Dr. H. Scheer Date of oral defence: 1 June 2007 Acknowledgements I am grateful to my supervisor PD. Dr. Paula Braun, who shared her experience in light-harvesting complex with me and for her advice throughout the years and during the preparation of this dissertation. I would like to express my gratitude to Brigitte Strohmann and Prof. Dr. Hugo Scheer for sharing their expertise with me. Many thanks to Prof. Dr. Gerhard Wanner (LMU, Biology Department I, Munich) for providing the EM facilities; Dr. Dominik Wegmann and PD. Dr. Britta Brügger (University of Heidelberg, Germany) for the ESI-MS measurements; Dr. Wolfgang Doster and Dr. Ronald Gerhardt (Technical University Munich, Germany) for light scattering and high pressure measurements; Ulrike Oster for her help with the TLC analyses; Silvia Dobler for the EM preparations and Dr. Alexander Pazur for his help with the PS statistical analyses. Sources of bacteria, Prof. Dr. Neil Hunter (University Sheffield, UK) is gratefully acknowledged. For fruitful and valuable discussions, I would like to thank my colleagues in the laboratory and institute; who have contributed to this work with their technical advices and their friendships. My special thanks are devoted to Kee Ping Wee, Han Ting How, Jörg Naydek, Ralf Kaiser, everyone in the small group and in MICC for their wonderful friendship, encouragement and for providing me with their time. I thank my family and all the people that have supported, encouraged and motivated me over the years. Thanks to Chong Yew for being the light of my days. Above all, I give praise to HIM! Summary The natural design of the photosystems of plants and photosynthetic bacteria using chlorophylls (Chls) or bacteriochlorophylls (BChls) as photoreceptors are robust. The basic principles of the biological system of light-harvesting complex 2 (LH2) are studied with the use of natural and model sequences expressed in vivo in modified Rhodobacter (Rb) sphaeroides strains. Three aspects have been explored in the thesis: (1) BChl’s macrocycle-protein interactions, (2) BChl’s phytol-protein interactions underlying the structural and functional assembly of the pigment-protein complexes, and (3) LH2-lipid interactions and the role of these interactions in photosynthetic membrane morphogenesis. BChls’ macrocycle-protein interactions: Residues at the immediate BChl- B850/protein interface are found to have little effect on specifying the BChl-B850 array, and their light-harvesting activity in LH2. Nevertheless, these residues are important for the structural thermal stability. With the use of ‘rescue’ mutagenesis of the model BChl binding site, the hydrogen-bond between αSer -4 and the C131 keto carbonyl group of βBChl-B850 is shown to be a crucial motif for driving the assembly of model LH2 complex. Possibilities for residue modifications are limited in the β-subunits as compared to the α-subunits, which suggests that the two polypeptides have distinct roles in complex assembly. In the β-subunits, there are residues detected adjacent to the BChl-B850 site which are critical for the assembly of LH2. BChls’ phytol-protein interactions: Mutagenesis of residues closely interacting with the BChl-B850 phytol moiety result in the pronounced loss of BChl-B800 from LH2. Dephytylation of bound BChls within assembled LH2 to BChlides also resulted in the loss of BChl-B800 and destabilisation of LH2 structural assembly. Thus, the phytol chains were shown to be important for optimal pigment binding, especially for BChl-B800; which appears to be highly sensitive to the proper packing of the phytols. The pattern of phytol interactions with their surrounding environments are significantly different for α- and β-ligated (B)Chls. The phytols of β-ligated (B)Chls, as opposed to α-ligated (B)Chls, have ample and specific interactions with residues of the binding helix which may contribute to the tertiary interactions of helices. LH2-lipids interactions: Phospholipid determination of LH2 only expressing strains of Rb sphaeroides shows that the nonbilayer-forming phospholipid, phosphatidylethanolamine (PE) is present in elevated amounts in the intracytoplasmic membranes and in the immediate vicinity of the LH2 complex. In combination with βGlu -20 residue and the carotenoid headgroup at the N-terminus of the transmembrane β-helices is shown to influence the composition of lipids surrounding LH2. Specific local interactions between LH2 protein and lipids not only promote LH2 protein stability but appear to modulate the morphology of intracytoplasmic membranes. Based on these findings, the presence of LH2-lipid specificity is postulated. The approach of using model αβ-sequences with simplified pigment binding sites allows us to study the underlying factors involved in LH2 assembly and function. This gives rise to a better understanding of the interplay between BChl, apoproteins and membrane lipids in the assembly of a highly efficient light-harvesting complex in its native lipid-environment. TABLE OF CONTENTS TABLE OF CONTENTS Chapter 1: Introduction 1.1 The photosynthetic machinery of purple non-sulphur bacteria 1 1.1.1 Rhodobacter sphaeroides as a model organism for photosynthetic studies 3 1.1.1.1 The characteristic of LH complexes of Rb sphaeroides 4 1.1.1.2 Energy transfer in the photosystem of Rb sphaeroides 6 1.1.1.3 Biogenesis of the photosynthetic membrane of Rb sphaeroides 7 1.2 Purple non-sulphur bacterial light-harvesting complexes 9 1.2.1 Structures of light-harvesting complexes 9 1.2.1.1 The structure of light-harvesting complex 2 10 1.3 Photosynthetic pigments: bacteriochlorophyll and carotenoid 14 1.3.1 Bacteriochlorophylls: structures and functions 14 1.3.1.1 BChl-protein interactions in LH2 complex of Rb sphaeroides 17 1.3.2 Carotenoids: structures and functions 18 1.3.2.1 Carotenoid-protein interactions in LH2 complex of Rb sphaeroides 19 1.4 The photosynthetic membrane in Rb sphaeroides 23 1.4.1 Membrane morphology of Rb sphaeroides 23 1.4.2 Membrane phospholipid composition 24 1.5 Summary and approaches 27 1.5.1 Summary 27 1.5.2 Approaches 28 1.6 Thesis objectives and organisation of chapters 30 TABLE OF CONTENTS Chapter 2: Materials and methods 2.1 Materials 31 2.1.1 Bacterial strains 31 2.1.2 Plasmids and vectors 32 2.1.3 Buffers and solutions 32 2.1.4 Column materials and solutions 35 2.1.5 Enzymes, chemicals and kits 35 2.1.5.1 Antibiotics 35 2.1.5.2 Phospholipids 36 2.1.5.3 Markers 36 2.1.5.4 Others 36 2.1.6 Technical devices 37 2.1.6.1 Centrifugation 37 2.1.6.2 Spectrophotometers 37 2.1.6.3 Others 37 2.2 Methods 38 2.2.1 Media and growth conditions 38 2.2.2 General molecular biological methods 38 2.2.3 Mutagenesis 38 2.2.3.1 Primers design 39 2.2.3.2 Polymerase chain reaction (PCR) 39 2.2.4 Agarose gel electrophoresis 40 2.2.5 Conjugation 40 2.2.6 Bacterial cultivation 41 2.2.7 LH2 intracytoplasmic membrane isolation 41 2.2.8 LH2 isolation 42 2.2.9 Sodium dodecyl sulphate polyacrylamide (SDS-PAGE) gels 43 2.2.10 Chlorophyllase experiment 44 2.3 Analytical methods 45 2.3.1 Absorption spectroscopy 45 2.3.2 Electron microscopy (EM) analysis 45 TABLE OF CONTENTS 2.3.3 Electrospray ionization mass spectroscopy (ESI-MS) 45 2.3.4 Circular dichroism (CD) spectroscopy 47 2.3.5 Fluorescence spectroscopy 48 2.3.6 Protein quantification 48 2.3.7 Thin layer chromatography (TLC) 48 2.3.7.1 Phospholipids extraction 49 2.3.8 Statistic analysis of pigment-protein interactions in PSI 49 2.3.9 Structural modelling 49 2.3.10 Websites 50 RESULTS PART I: Protein-bacteriochlorophyll interactions in model and native light-harvesting complex 2 Chapter 3: Bacteriochlorophylls’ macrocycle-protein interactions underlying LH2 assembly and function 3.1 Introduction 53 3.2 Results and discussion 60 Simplification of the BChl-B850 binding site: model sequences in the TMH of the β-subunit 60 Design of the model LH2 αWT/βAL 60 The functional and structural characteristics of αWT/βAL 61 Study of interaction motifs at the model and native BChl-protein interface: H-bonding between the hydroxyl of serine and the keto carbonyl of BChl-B850 66 H-bond donor residue at position -4 of α-subunit 67 H-bond donor residue at position -4 of β-subunit 71 3.3 Conclusions 77 TABLE OF CONTENTS Chapter 4: Bacteriochlorophylls’ phytol chain-protein interactions underlying LH2 assembly and function 4.1 Introduction 79 4.2 Results and discussion 84 Study of the role of BChls phytol by mutating protein binding residues 84 Removal of the BChl phytol tails in assembled LH2 by enzymatic digestion 96 Statistical analyses of α- and β-ligated (B)Chls 103 Analysis of phytyl-protein interactions in PSI 106 Analysis of phytyl-amino acid contacts 108 PART II: Protein-lipid and pigment-lipid interactions in model and native light-harvesting complex 2 Chapter 5: Study of the relationship between Rb sphaeroides LH2 protein- phospholipids interactions and ICM morphology 5.1 Introduction 117 5.2 Results and discussion 128 Study of the relationship between LH2 and ICM morphology 128 Phospholipid composition of Rb sphaeroides expressing LH2 WT and model LH2 αALS-4/βAL 132 Phospholipid headgroup compositions 132 Phospholipid hydrocarbon chain compositions 134 Phospholipid composition of Rb sphaeroides LH2
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