Table Of ContentUNIVERSITE D’AIX-MARSEILLE
Ecole Doctorale des Science de la Vie et de la Santé
Laboratoire d'Ingénierie des Systèmes Macromoléculaires
CNRS-LISM UMR7255
Thèse présentée pour obtenir le grade universitaire de docteur
Biologie-Biochimie Structurale
Dong WANG
Making dimers of light-harvesting complexes from purple
bacteria using copper–free click chemistry
Soutenue le 21/03/2017 devant le jury:
Maité PATERNOSTRE (CNRS-I2BC) Rapporteur
Laurent CATOIRE (CNRS-IBPC) Rapporteur
Sonia LONGHI (CNRS-AFMB) E x a m inateur
Hervé DARBON (CNRS-AFMB) Examinateur
James N. STURGIS (CNRS-LISM) D i r e cteur de thèse
Résumé
Les complexes collecteurs de lumière des bactéries photosynthétiques absorbent l'énergie
solaire, et transfert l'energie avec grande efficacité aux centres réactionnelles, siege ou elle est
captée pour l'utilisation par la cellule. Nous savons peu des détails du transfert d'énergie entre
les differents complexes collecteurs de lumière. Dans cette thèse, j'ai isolé différent complexes
collecteurs de lumière a partir de plusieurs souches de bactéries pourpres. J'ai construit de
modèles 3D par homologie et les structures possibles de dimères ont également été éxaminés.
Les sites de pontage dans ces protéines montre la possibilité de constuire des dimeres avec des
stuctures biologiquement pertients. J'ai développé un protocole pour construire de dimères de
protéines collectrices de lumière fortement oligomériques. Le protocole j'ai mis en place
contient trois grandes étapes: d'abord la réaction de lysines dans les complexes à un très faible
degré de réaction, et la purification des protéines marquées. Ensuite, les groupes réactives de
dibenzocyclooctyne (DBCO) ou de l'azoture sont introduits au complexes. Finalement, la
réaction sans cuivre de cycloaddition azoture-alcyne promue par distorsion a pour conséquence
la synthèse de dimères.
MOT-CLES: complexes collecteurs de lumière, modélisation par homologie, dimère, chimie-
click sans cuivre
2
Abstract
The light harvesting apparatus of photosynthetic bacteria absorb the energy from sunlight and
transfer the energy with high efficiency to the reaction center, where it is captured for use by
the cell. We know little about the details of energy transfer between different light-harvesting
complexes. In this thesis I isolated several different types of light-harvesting complex from
various stains of purple bacteria. 3D models were built, based on homology modeling, and
possible dimer structures were examined. The cross linking sites in these protein shown the
possiblity of forming biologically relevant dimer structures. I have developed a protocol to
make dimers, from highly oligomeric light harvesting proteins. The protocol developed
contains three main steps: first reaction of lysines in the complex at a very low degrees of
reaction and purifying the labelled protein. Then coupling the reactive groups of
dibenzylcyclootyne (DBCO) or of azide separately to the different complexes. Finally, the
copper free strain promoted azide-alkyne cycloaddition reaction occurred to synthesize the
dimer.
Key words: light harvesting apparatus, homology modeling, dimers, copper free click
chemistry
3
Acknowledgements
First and foremost, I would like to express my heartfelt gratitude to my supervisor, Prof. James
N. Sturgis both for his intellectual guidance and for his warm and constant encouragement
during the process of finish this thesis. Four years ago, he gave me a chance to join his group
and study with him. As a chemistry student, I am poor in biology but he taught me the
knowledge step by step with patience and prudence. With his help, I got a great advancement
in how to do a scientific research. In china, there is an ancient proverb“ He who teaches me
for one day is my father for life”. Thanks for my “father in science”, James, whose profound
knowledge of science triggers my love for this interesting work and whose earnest attitude tells
me how to study it. The profit that I gained from his profound knowledge, remarkable expertise
and intellectual ingenuity will be of everlasting significance to my future life and career.
Without his consistent and illuminating instruction, this thesis could not have reach its present
form.
Second, my cordial and sincere thanks go to to Dr. Jean-Pierre Duneau. I still remember he
picked me up at aix-marseille airport in 2012. It was our first meeting. During my Ph.D program,
he gave me a lot assistance especially in computer and 3D model building.
I am very grateful to Ms. Valérie Prima, my “mother in science”. Her interesting and
experiment skills have benefited me a lot during these years. Each time when I met a difficult
in experiment, she always gave me invaluable advices. In lab life, her generous assistance helps
me greatly in my thesis.
I also want to thanks other people who work in the laboratory of “LISM”, Ms. Victoria Schmidt,
Dr. Prierre Hubert, Dr. Julie Viala and so on. They have given me a lot of help and courage
during my stay at the laboratory and throughout the process of writing this thesis. I also thank
for the Mr. Olivier Uderzo who prepared the mediums for the bacteria during 4 years.
The biggest thanks go to my family who has shared with me my worries, frustrations, and
hopefully my ultimate happiness in eventually finishing this thesis. Thanks for my father and
mother. Without their love, I cannot finish my study and this thesis cannot be finished.
Last but not least, I want to thanks for people who read and examine this thesis. Thanks very
much!
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Abbreviations and Acronyms
2-ME 2-mercaptoethanol
BChl Bacteriochlorophyll
CC Core Complex
CuAAC Copper(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition
DBCO Dibenzylcyclooctyne
DTT Dithiothreitol
EDC N-ethyl-3-N’,N’-dimethylaminopropylcarbodiimide
EPL Expressed Protein Ligation
ER Endoplasmic Reticulum
Fmoc 9-fluorenylmethyloxycarbonyl
HPLC High Performance Liquid Chromatograph
ICM IntraCytoplasmic Membranes
LH Light-harvesting
MS Mass Spectrum
NCL Native Chemical Ligation
NHS N-hydroxysuccinimide
NHS-Biotin Succinimidyl-2-(biotinamido)ethy-1,3-dithiopropionate
NHS-ester N-hydroxysuccinimide Easter
Phsp. Phaeospirillum
PSU Photosynthetic Unit
PTM Post-translational Modification
RC Reaction Center
Rsb. Roseobacter
Rb. Rhodobacter
SPAAC Strain-promoted Alkyne-Azide Cycloaddition
SPSS Solid-phase Peptide Synthesis
t-Boc Tert-butoxycarbonyl
TCEP Tris (2-carboxyethyl) phosphine
TEM Transmission Electron Microscope
5
Contents
Résumé ...................................................................................................................................... 2
Abstract ..................................................................................................................................... 3
Acknowledgements .................................................................................................................. 4
Abbreviations and Acronyms ................................................................................................. 5
Chapter 1 Chemical Modification of Proteins ....................................................................... 8
1.1 Chemical Modifications of Proteins ........................................................................................... 8
1.1.1 Post-Translational Modifications ....................................................................................... 8
1.1.1.1 Protein Phosphorylation ................................................................................................................. 9
1.1.1.2 Protein Glycosylation ................................................................................................................... 10
1.1.2 Chemical Engineering of Proteins .................................................................................... 12
1.2 How to Modify Proteins? .......................................................................................................... 13
1.2.1 Reactive Groups and Modifying Agents .......................................................................... 13
1.2.1.1 Physical Chemistry of Chemical Modification ............................................................................ 13
1.2.1.2 Organic Chemistry of Reactive Group ......................................................................................... 17
1.2.1.3 Comparison of Reactions ............................................................................................................. 29
1.2.2 Chemical Synthesis of Proteins ......................................................................................... 30
1.2.2.1 Chemical Synthesis of Peptides .................................................................................................... 30
1.2.2.2 Biological Incorporation of Non-standard Amino Acids ............................................................. 33
1.2.2.3 Peptide Ligation ............................................................................................................................ 34
1.3 Crosslinking Proteins ................................................................................................................ 37
1.3.1 Classic Chemical Crosslinkers .......................................................................................... 38
1.3.1.1 Homobifunctional Crosslinkers .................................................................................................... 38
1.3.1.2 Heterobifunctional Crosslinkers ................................................................................................... 39
1.3.1.3 Formaldehyde ............................................................................................................................... 40
1.3.2 Bioorthogonal Chemistry of Proteins ............................................................................... 40
1.3.2.1 Introduction of Bioorthogonal Chemistry .................................................................................... 41
1.3.2.2 Staudinger Ligation ...................................................................................................................... 41
1.3.2.3 Click Chemistry ............................................................................................................................ 42
1.3.2.4 Other Bioorthogonal Reactions .................................................................................................... 47
1.4 Bioorthogonal Strategy in My Thesis ...................................................................................... 48
Chapter 2 Photosynthesis in Purple Bacteria ...................................................................... 50
2.1 Introduction of Photosynthesis System ................................................................................... 50
2.1.1 Photosynthesis in Plants and Purple Bacteria ................................................................. 51
2.1.1.1 Photosythesis in Plants ................................................................................................................. 51
2.1.1.2 Photosynthesis in Purple Bacteria ................................................................................................ 54
2.2 The Structure and Function of Purple Bacterial Light-harvesting ...................................... 57
2.2.1 The Organization of Photosynthetic Units ....................................................................... 57
2.2.2 The Structure of Photosynthetic Proteins ........................................................................ 60
2.2.2.1 The Pigments in Photosynthetic Proteins ..................................................................................... 60
2.2.2.2 LH II ............................................................................................................................................. 63
2.2.2.3 Core Complex ............................................................................................................................... 67
2.2.3 The Function of PSU in Purple Bacteria .......................................................................... 71
2.2.3.1 Intra-protein Energy Transfer ....................................................................................................... 71
2.2.3.2 Inter-protein Energy Transfer ....................................................................................................... 75
2.3 Energy Transfer and My thesis Project .................................................................................. 76
Chapter 3 Materials and Methods ........................................................................................ 80
3.1 Protein Structural Modelling ................................................................................................... 80
3.1.1 Introduction to Homology Modelling ............................................................................... 80
3.2 Protein Purification ................................................................................................................... 84
3.2.1 Growth of Bacteria ............................................................................................................. 84
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3.2.1.1 Bacterial Strains ............................................................................................................................ 84
3.2.1.2 Growth Conditions ....................................................................................................................... 84
3.2.1.3 Harvesting Bacteria ...................................................................................................................... 86
3.2.2 Preparation of Membranes ............................................................................................... 86
3.2.3 Protein Purification ............................................................................................................ 87
3.2.3.1 Extraction ..................................................................................................................................... 87
3.2.3.2 Isolation of Proteins ...................................................................................................................... 88
3.2.3.3 Characterization of Proteins ......................................................................................................... 90
3.3 Chemical Labelling .................................................................................................................... 94
3.4 The Location of Lysine Modification ....................................................................................... 95
3.5 Formation of Dimers ................................................................................................................. 95
3.5.1 Overview of Synthesis ........................................................................................................ 95
3.5.2 Reaction with NHS-SS-Biotin ........................................................................................... 96
3.5.3 Maleimide Reaction ........................................................................................................... 97
3.5.4 Copper Free Click Chemistry Reaction ........................................................................... 97
3.6 Dimer Characterization ............................................................................................................ 97
3.6.1 Transmission Electron Microscope .................................................................................. 97
3.6.2 High Performance Liquid Chromatography ................................................................... 97
3.6.3 Dynamic Light Scattering .................................................................................................. 98
Chapter 4 3D Models of Photosynthetic Proteins ............................................................... 99
4.1 Monomeric Complexes .............................................................................................................. 99
4.1.1 LH II .................................................................................................................................... 99
4.1.1.1 LH II of Rb. sphaeroides .............................................................................................................. 99
4.1.1.2 LH II of Rsb. denitrificans .......................................................................................................... 102
4.1.1.3 LH II of Phsp. molischianum ..................................................................................................... 103
4.1.2 CC ...................................................................................................................................... 106
4.1.2.1 CC of Rsb. denitrificans ............................................................................................................. 106
4.1.2.2 CC of Phsp. molischianum ......................................................................................................... 107
4.1.3 Comparison of Models ..................................................................................................... 109
4.2 Models of Dimers ..................................................................................................................... 110
4.2.1 Biological Relevant Models ............................................................................................. 110
4.2.1.1 Dimers of LH II .......................................................................................................................... 110
4.2.1.2 Dimers of LH II and CC ............................................................................................................. 112
4.2.2 Non-Biological Relevant Models ..................................................................................... 113
Chapter 5 Protein Labelling and Dimer Formation ......................................................... 115
5.1 Protocol Outline ....................................................................................................................... 115
5.2 Understanding the Degree of Labeling .................................................................................. 116
5.2.1 Degree of Labeling ........................................................................................................... 116
5.2.2 Location of Lysine Modifications ................................................................................... 120
5.3 Crosslinking of Proteins .......................................................................................................... 129
5.3.1 Lysine Modification ......................................................................................................... 129
5.3.2 Binding to Avidin-Agarose .............................................................................................. 129
5.3.3 Disulfide Bond Reduction ................................................................................................ 131
5.3.4 Reaction with Maleimides ............................................................................................... 132
5.3.5 Click Chemistry ................................................................................................................ 133
5.3.6 Analysis of Reaction Products ......................................................................................... 134
5.3.6.1 Transmission Electron Microscope ............................................................................................ 134
5.3.6.2 HPLC .......................................................................................................................................... 135
5.4 Discussion ................................................................................................................................. 137
5.5 Conclusions .............................................................................................................................. 142
Chapter 6 Summary and Perspective ................................................................................ 144
References ............................................................................................................................. 149
Appendix ............................................................................................................................... 178
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Chapter 1 Chemical Modification of
Proteins
1.1 Chemical Modifications of Proteins
Biological protein modification is widespread, increasing the diversity of protein structure and
1
function greatly . However, our ability to synthetically mimic nature’s capacity to create such
modifications is limited by the chemistry that is available. Chemical modification of proteins
2
has emerged as an invaluable tool for the development of modified proteins, in the laboratory .
The complementary use of both chemical and genetic methods provides a large toolbox that
allows the preparation of almost unlimited protein constructs with either natural or synthetically
3
modified residues .
Some of the earliest attempts to use chemical modification procedures to identify particular
amino acid residues required for the biological activity of a protein were conducted in the group
4,5
of Heinz Fraenkel-Conrat . A few of those procedures are still used, with little change, to this
day. Over the past two decades, a number of newer methodologies have emerged for the
modification at both natural and non-standard amino acid residues, in vitro and in vivo, building
6
on a previously more limited toolkit for modification primarily at cysteine and lysine residues .
The applications of modified proteins are many. They are as varied as the in vivo tracking of
7
protein-fluorophore conjugates , the polyethylene glycolylation (PEGylation) of therapeutic
8 9
proteins to reduce immunogenicity , the production of new bio-materials with novel properties ,
10
and probing the mechanism of pathological enzymes . In general, chemical modification of
2
proteins covers post-translational modifications and chemical engineering of proteins. The first
based on the chemical reactions with amino acids residues, so it is effected by the chemical
reaction types and the activity of residues. While the chemical engineering of proteins can
introduce some non-nature amino acids into peptides and synthesis peptide as scientists
designed without the limits imposed by the genetic code. Despite the vast progress in the field
of modification of proteins, scientists still face many challenges, not only synthetically but also
11–13
from processing, manufacturing, safety, and stability perspective . I will investigate these in
this chapter…
1.1.1 Post-Translational Modifications
The chemical modification of proteins, to attain the chemodiversity usually achieved after
2
synthesis, and commonly referred to as post-translational modification (PTM) , is impressive.
It increases the functional diversity of the proteome by: the covalent addition of functional
groups or whole new proteins; the proteolytic cleavage of regulatory subunits. Another
common type of post-translational modification is formation or reductive cleavage of disulfide
bonds. This maturation occurs in the endoplasmic reticulum (ER), Golgi complex, post-Golgi
complex vesicles, and mitochondrial intermembrane space in eukaryotic cells and in the
8
14 15
periplasmic space in bacteria . Liu et al reported the disulfide bonds could affect the antibody
structure, stability and biological functions based on the investigation of lgG proteins. For
16 17
example, individual domains of CL domain , CH3 domain and single-chain variable
18
fragment without the complete intra-chain disulfide bond showed lower stability. These
modifications also include acylation, methylation, phosphorylation, sulfation, farnesylation,
ubiquitination, glycosylation, among others, and play a pivotal role in many important cellular
19
processes including trafficking, differentiation, migration and signaling . Phosphorylation and
glycosylation, as two of the most common modifications, are introduced as examples below.
1.1.1.1 Protein Phosphorylation
Phosphorylation of proteins is a post-translational modification (PTM) in which an amino acid
residue is phosphorylated by a protein kinase by the addition of a covalently bound phosphate
20
group . It was first proposed in 1906 by Phoebus Levene at the Rockefeller Institute for
21
Medical Research with the discovery of phosphorylated vtellin and was first detected in 1932
22
in casein . It is estimated that about 10% to 50% of proteins in any particular cell can be
23
phosphorylated . Usually, phosphorylation of proteins is considered as one of the more
24
extensively modification for a much fast response to environmental signals, as it often leads
25
to a shift between active and inactive forms . Considering the chemistry of the bond between
phosphate and an amino acid side chain, phosphoramino acids include phosphomonoesters
(serine, threonine and tyrosine), phosphoramidates (histidine, arginine and lysine),
20
acylphosphates (aspartate and glutamate), and thiophosphate (cysteine) . Reversible, multisite
phosphorylation of protein Ser, Thr and Tyr residues mediates numerous signal transduction
26 27
pathways in eukaryotic and prokaryotic cells. Histidine and cysteine phosphorylation are
well known critical processes involved in a bacterial phosphoenolpyruvate-dependent
28
carbohydrate transport system . Aspartate residue acts as a phosphate acceptor in P-type
29 30
ATPases and certain class of phosphotransferases . Histidine and aspartate phosphorylations
are engaged in two-component and multi-component phospho-relaying signalling systems in
31 32 33
bacteria , fungi and plants , involved in linking an extracellular stimulus, such as changing
osmolarity, oxygen, nitrogen, phosphorus or ethylene levels, to gene-regulating events. Such
systems have not been discovered in higher eukaryotes, nevertheless it is becoming increasingly
evident that histidine phosphorylation plays important regulatory roles also in mammalian
20
cellular signal transduction .
Another effect of the addition of a phosphate molecule to a hydrophobic amino acid residue is
that it can convert a portion of protein from hydrophobic to hydrophilic. This can induce a
34
conformational change in the structure of the protein . The conformational change of enzymes
23
introduced by phosphate molecule make them become actived or deactived . A classic example
is Protein kinase B/Akt, which plays crucial roles in promoting cell survival and mediating
insulin responses. When the Ser 474 is phosphorylated, the order transition of the αC helix is
disordered and reconfiguration of the kinase structure. These conformational changes active the
35 36
function of the enzyme . Another example is the phosphorylation of src tyrosine kinase . It
9
leads to the conformational change in the enzyme, resulting in a fold in the structure, which
masks its kinase domain, and is thus shut “off”.
Protein phosphorylation is a reversible post-translational protein modification that is the result
of kinases and phosphatases, which phosphorylate and dephosphorylate substrates,
23,37
respectively . Indeed, the size of the phosphoproteome in a given cell is dependent upon the
temporal and spatial balance of kinase and phosphatase concentrations in the cell and the
38
catalytic efficiency of a particular phosphorylation site . Over the past two decades, scientists
have used chemical biology methods to install the phosphate moiety in proteins in order to
39,40
understand more deeply the function and mechanism of phosphorylation . Both enzymatic
41
and chemical reactions have been used in these studies. Volkman et al reported that the
structure of nitrogen regulatory protein C (NtrC) can be changed when phosphate group was
introduced by kinases in vitro at Asp86. This phosphorylation turns NtrC into active state for
42,43 44
signal transduction . An example of chemical modification in vitro is the Cohen group
β-TrCP *
investigated the regulation of phosphorylation of IkB Kinase in SCF dependent
degradation. Protein Phosphorylation also can have biotechnological applications. For example,
45
in food science, Lei used the chemical reaction to add the sodium tripolyphosphorylate to the
soybean protein and found that the creaming index and particle size distribution of
phosphorylated soybean protein concentrate (SPC) were increased and the emulsifying property
of SPC improved by phosphorylation.
1.1.1.2 Protein Glycosylation
Another example of PTM is glycosylation, it involves the attachment of sugar moieties to
46
proteins . Formation of the sugar–amino acid linkage is a crucial event in glycosylation and
the biosynthesis of the carbohydrate units of glycoproteins. Glycosylation usually involves the
coordinated effort of a complex array of enzymes, in specific sub-cellular organelles to
47
successfully generate complex carbohydrate-associated PTM . These reactions have been
48–50
founded in all the three domain (eukaryotes, eubacteria and archaea) of life . At least 9 of
51
20 amino acids can be modified by a variety of carbohydrates , ranging from a single
52
monosaccharide to glycan chains containing hundreds of monosaccharides . For instance, in
mammals the major glycans contains 10 different monosaccharide units: glucose (Glc),
galactose (Gal), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), fucose
(Fuc), mannose (Man), xylose (Xyl), glucuronic acid (GlcA), iduronic acid (IdoA), and 5-N-
51
acetylneuraminic acid (Neu5Ac, or sialic acid)—all derivable from glucose in every cell .
They set in motion a complex series of post-translational enzymatic steps that lead to the
47
formation of a host of protein-bound oligosaccharides with diverse biological functions .
Glycopeptide bonds can be categorized into specific groups based on the nature of the sugar-
peptide bond and the oligosaccharide attached, including N-, O- and C-linked glycosylation,
* β-TrCP
The SCF complex is the ubiquitin ligase responsible for mediating phosphorylation dependent
579
ubiquitination of I kappa B alpha .
10
