2 3 Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät Auf Antrag von Prof. Dr. Andreas Engel & Prof. Dr. Jean-Louis Rigaud Basel, den 19. Dezember 2000 Prof. Dr. Andreas D. Zuberbühler Dekan der Philosophisch Naturwissenschaftlichen Fakultät 4 To my parents, my brothers, and my friends. 5 6 Index 7 8 1. AFM and EM in structural biology........................................................................15 1.1. General introduction..................................................................................................15 1.1.1. The driving force to do science...........................................................................15 1.1.2. Membrane proteins............................................................................................15 1.1.3. 2D crystals allow the acquisition of structural information on membrane proteins in a native-like environment................................................................................16 1.1.4. Atomic force and electron microscopy cover large resolution ranges and together provide both surface and volume information.....................................................17 1.1.5. Results and Perspectives....................................................................................18 1.1.6. References..........................................................................................................19 1.2. Atomic force microscopy: A powerful tool to observe the assembly and function of native proteins........................................................................................................21 1.2.1. Abstract..............................................................................................................21 1.2.2. Introduction........................................................................................................21 1.2.3. Conditions for single molecule imaging.............................................................22 1.2.4. Imaging the ion-driven rotor of the ATP synthase..............................................23 1.2.5. Conformational flexibility of proteins................................................................25 1.2.6. The tongue-and-groove interaction of MIP tetramers.........................................26 1.2.7. Imaging the subcomplexes of the GroE chaperonin system: GroEL and GroES27 1.2.8. Observing the assembly of membrane proteins..................................................28 1.2.9. Outlook..............................................................................................................29 1.2.10.Acknowledgement..............................................................................................30 1.2.11.References..........................................................................................................30 1.3. Imaging streptavidin 2D-crystals on biotinylated lipid monolayers at high resolution with the atomic force microscope...........................................................35 1.3.1. Summary............................................................................................................35 1.3.2. Introduction........................................................................................................35 1.3.3. Materials and Methods.......................................................................................36 1.3.3.1. 1.3.3.2. 1.3.3.3. 1.3.3.4. 1.3.3.5. 1.3.4.1. 1.3.4.2. 1.3.4.3. 1.3.4.4. Materials................................................................................................36 Hydrophobicity measurement......................................................................36 Crystallization of streptavidin on biotin-lipid monolayer..................................36 Atomic force microscopy (AFM).................................................................37 Transmission electron microscopy (TEM).....................................................37 Hydrophobicity and topography of HOPG.....................................................37 Crystallization of streptavidin on biotin-lipid monolayer..................................38 AFM of streptavidin crystals......................................................................39 TEM of streptavidin crystals.......................................................................40 1.3.4. Results...............................................................................................................37 1.3.5. Discussion.........................................................................................................40 1.3.6. Acknowledgment................................................................................................42 1.3.7. References..........................................................................................................43 9 2. Application of high resolution AFM.......................................................................47 2.1. High resolution AFM topographs of the Escherichia coli waterchannel aquaporin Z................................................................................................................47 2.1.1. Abstract..............................................................................................................47 2.1.2. Introduction.......................................................................................................47 2.1.3. Results...............................................................................................................49 2.1.4. Discussion.........................................................................................................50 2.1.5. Materials and methods.......................................................................................54 2.1.5.1. 2.1.5.2. 2.1.5.3. 2.1.5.4. Reconstitution..........................................................................................54 Trypsin digestion......................................................................................54 Atomic force microscopy............................................................................54 Image processing......................................................................................55 2.1.6. Acknowledgment...............................................................................................55 2.1.7. References.........................................................................................................55 2.2. High resolution AFM topographs of Rubrivivax gelatinosus light-harvesting complex LH2...............................................................................................................59 2.2.1. Abstract..............................................................................................................59 2.2.2. Introduction.......................................................................................................59 2.2.3. Results...............................................................................................................61 2.2.4. Discussion.........................................................................................................65 2.2.5. Materials and methods.......................................................................................67 2.2.5.1. 2.2.5.2. 2.2.5.3. 2.2.5.4. 2.2.5.5. 2.2.5.6. Materials.................................................................................................67 Isolation, purification and proteolysis of LH2 complex....................................67 Biochemical and biophysical techniques.........................................................67 Reconstitution and 2D crystallization............................................................67 Atomic force microscopy............................................................................67 Image processing......................................................................................68 2.2.6. Acknowledgment...............................................................................................68 2.2.7. References.........................................................................................................68 3. Combining surface and projection techniques....................................................73 3.1. The aquaporin sidedness revisited...........................................................................73 3.1.1. Summary...........................................................................................................73 3.1.2. Introduction.......................................................................................................73 3.1.3. Results...............................................................................................................74 3.1.4. Discussion.........................................................................................................78 3.1.5. Materials and Methods......................................................................................80 3.1.5.1. 3.1.5.2. 3.1.5.3. 3.1.5.4. 3.1.5.5. 2D crystallization......................................................................................80 Trypsin digestion......................................................................................80 Atomic force microscopy............................................................................80 Freeze-drying & metal-shadowing.................................................................80 Cryo electron microscopy...........................................................................80 3.1.6. Acknowledgment...............................................................................................81 3.1.7. References.........................................................................................................81 10 4. Structural studies of a membrane transporter...................................................87 4.1. The functional Escherichia coli lactose permease LacY/Cytb562/6His forms trimers: A 2.8 nm 3D reconstruction and preliminary electron crystallographic data...............................................................................................................................87 4.1.1. Summary............................................................................................................87 4.1.2. Introduction........................................................................................................87 4.1.3. Results and discussion.......................................................................................89 4.1.3.1. 4.1.3.2. 4.1.3.3. Protein purification...................................................................................89 Single particle analysis..............................................................................90 Reconstitution and 2D crystallization...........................................................91 4.1.4. Perspectives........................................................................................................93 4.1.5. Material and Methods........................................................................................94 4.1.5.1. 4.1.5.2. 4.1.5.3. 4.1.5.4. Materials................................................................................................94 Protein Expression and Purification.............................................................94 Reconstitution.........................................................................................94 Electron microscopy and image processing....................................................95 4.1.6. References..........................................................................................................95 5. General discussion and conclusions.......................................................................101 6. Acknowledgment..........................................................................................................107 7. Curriculum vitae..........................................................................................................113 7.1. Education...................................................................................................................113 7.2. Teaching.....................................................................................................................113 7.3. Publications...............................................................................................................113 7.4. Meetings.....................................................................................................................114 11 12 1. AFM and EM in structural biology 13 14 1. AFM and EM in structural biology 1.1. General introduction 1.1.1. The driving force to do science What does it look like? What does it do? It is curiosity, the wish to know, that induces these questions. It is human nature - and it is the driving force to do fundamental research in any field ranging from astronomy to physics of smallest matter. The primary questions of molecular biologists are: What is the structure, what is the function, of a biomolecule? Once the structure and function of a biomolecule are known, its physiological role and interactive mechanisms can be brought into context within the framework of the cell and the whole organism. It is our ultimate goal to understand the molecular mechanisms of all processes in each cell of our body, ranging from neurons working in our brain, muscle cells allowing us to move, to the cells of our skin ultimately defining the borderlines of ourselves and our environment. 1.1.2. Membrane proteins Intrinsic or integral membrane proteins are defined as proteins that penetrate into and, most often, traverse the lipid bilayer of a biological membrane. Protein structures, which partition into lipid rather than remain in aqueous solution have specific chemical properties. They are rich in exposed hydrophobic amino acids and are restricted in their secondary structure. A consequence of these physico-chemical properties is that an integral membrane protein can only be brought into aqueous solution when solubilized in the presence of detergents. The challenge of understanding membrane proteins and transporters has attracted our interest. Figure 1 represents an interesting result of a genome study. Genomes of different organisms (E. coli, M. jannaschii, H. sapiens) were screened for their protein coding open reading frames and these open reading frames were translated into amino acid chains. The peptides were discriminated by their hydrophobicities. This resulted in two families of gene products: The hydrophilic cytoplasmic proteins, and the membrane proteins, which contain large hydrophobic stretches (e.g., representing transmembrane helices). By this relatively simple approach, it has been demonstrated that 20 - 30 % of genes (the higher the organism, the larger the percentage) code for strongly hydrophobic proteins which are most probably integrated into cell membranes. This intriguing result emphasizes the extreme importance of membrane proteins for living organisms. Membrane proteins connect the cytoplasm with the extracellular space of each living cell, form junctions between living cells or play an important role in the intracellular compartments. Hence, in bacteria, such molecules work in transport, secretion and bioenergetic processes. Multicellular organisms even require active communication between their cells. Consequently a large number of membrane proteins have evolved, working as receptors for intercellular trafficking or cellular adhesion and recognition. Evolution has also created highly specific channels and transporters, which are essential for the survival of biological systems; the deletion of many membrane proteins is lethal or leads to severe disease. The study of membrane protein structure is a difficult challenge: Membrane proteins remain only folded in their active state when 15 Figure 1. Number of genes of different organisms as a function of the hydrophobicity of their gene product. Integration over the two peaks corresponding to cytoplasmic and membrane proteins shows that 20 - 30 % of all genes code for membrane proteins. their hydrophobic transmembrane domains are embedded in a hydrophobic environment e.g., a lipid bilayer or a detergent micelle. This prerequisite makes the growth of 3D crystals for structure determination by X-ray diffraction difficult. Consequently, ~2000 atomic structures of water-soluble proteins are available but only ~20 atomic structures of integral membrane proteins. The structures so far determined, divide membrane proteins into two categories: αhelical and β-barrel membrane proteins. The majority of the structures were determined using 3D crystallization and X-ray diffraction. However, three structures have been solved by electron crystallography: Plant light-harvesting complex II, bacteriorhodopsin, and human aquaporin 1. The electron crystallography was carried out using 2D crystals of protein integrated in lipid bilayers. 1.1.3. 2D crystals allow the acquisition of structural information on membrane proteins in a nativelike environment In order to acquire biologically valid information, it is important to study the structure of membrane proteins under conditions where they remain functional. To this end, membrane proteins are reconstituted into 2D crystals in the presence of lipids which mimic their native membrane environment within a cell (chapters 2.1, 2.2, 3.1, 4.1). Although only 3 membrane protein structures have been solved to atomic resolution (below 4Å) using electron crystallography (Kühlbrandt et al., 1994; Henderson et al., 1990; Kimura et al., 1997; Murata et al., 2000), numerous proteins have been solved to medium resolution (4Å-10Å) in 2D projection or 3D density maps from electron micrographs. Such medium resolution maps revealed helix arrangements 16 or/and structural similarities within the aquaporin protein family (Stahlberg et al., 2001). 1.1.4. Atomic force and electron microscopy cover large resolution ranges and together provide both surface and volume information Structural biology encompasses a range of techniques, to elucidate structures and interactions of biomolecules. The table below summarizes some of the advantages and disadvantages of the various approaches. The atomic force and the electron microscope are our tools to investigate the fascinating microcosm of membrane proteins. As listed above, a combination of these two techniques covers a resolution range from micrometers to atomic scale, and yields both surface and volume information of proteins in the close to native environment of 2D crystals. Electron microscopy of negatively stained samples allows the efficient acquisition of structural information. Despite the limited resolution of ~15Å, the technique provides a high signal-to-noise ratio (chapters 1.3, 4.1). By these means, samples of 2D crystals of membrane proteins can be checked and a first Table 1. Advantages and disadvantages of techniques applied in structural biology technique X-ray crystallography advantages -atomic resolution disadvantages -well ordered 3D crystals are required -absence of phases -requires large amounts of protein -requires protein labeling -problems with large molecules (>40 kDa) -requires well ordered 2D crystals -technically difficult -resolution limit ~5Å -problems with small molecules (
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2 3 Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät Auf Antrag von Prof. Dr. Andreas Engel & Prof. Dr. Jean-Louis Rigaud Basel, den 19. Dezember 2000 Prof. Dr. Andreas D. Zuberbühler Dekan der Philosophisch Naturwissenschaftlichen Fakultät 4 To my parents, my brothers, and my friends. 5 6 Index 7 8 1. AFM and EM in structural biology........................................................................15 1.1. General introduction..................................................................................................15 1.1.1. The driving force to do science...........................................................................15 1.1.2. Membrane proteins............................................................................................15 1.1.3. 2D crystals allow the acquisition of structural information on membrane proteins in a native-like environment................................................................................16 1.1.4. Atomic force and electron microscopy cover large resolution ranges and together provide both surface and volume information.....................................................17 1.1.5. Results and Perspectives....................................................................................18 1.1.6. References..........................................................................................................19 1.2. Atomic force microscopy: A powerful tool to observe the assembly and function of native proteins........................................................................................................21 1.2.1. Abstract..............................................................................................................21 1.2.2. Introduction........................................................................................................21 1.2.3. Conditions for single molecule imaging.............................................................22 1.2.4. Imaging the ion-driven rotor of the ATP synthase..............................................23 1.2.5. Conformational flexibility of proteins................................................................25 1.2.6. The tongue-and-groove interaction of MIP tetramers.........................................26 1.2.7. Imaging the subcomplexes of the GroE chaperonin system: GroEL and GroES27 1.2.8. Observing the assembly of membrane proteins..................................................28 1.2.9. Outlook..............................................................................................................29 1.2.10.Acknowledgement..............................................................................................30 1.2.11.References..........................................................................................................30 1.3. Imaging streptavidin 2D-crystals on biotinylated lipid monolayers at high resolution with the atomic force microscope...........................................................35 1.3.1. Summary............................................................................................................35 1.3.2. Introduction........................................................................................................35 1.3.3. Materials and Methods.......................................................................................36 1.3.3.1. 1.3.3.2. 1.3.3.3. 1.3.3.4. 1.3.3.5. 1.3.4.1. 1.3.4.2. 1.3.4.3. 1.3.4.4. Materials................................................................................................36 Hydrophobicity measurement......................................................................36 Crystallization of streptavidin on biotin-lipid monolayer..................................36 Atomic force microscopy (AFM).................................................................37 Transmission electron microscopy (TEM).....................................................37 Hydrophobicity and topography of HOPG.....................................................37 Crystallization of streptavidin on biotin-lipid monolayer..................................38 AFM of streptavidin crystals......................................................................39 TEM of streptavidin crystals.......................................................................40 1.3.4. Results...............................................................................................................37 1.3.5. Discussion.........................................................................................................40 1.3.6. Acknowledgment................................................................................................42 1.3.7. References..........................................................................................................43 9 2. Application of high resolution AFM.......................................................................47 2.1. High resolution AFM topographs of the Escherichia coli waterchannel aquaporin Z................................................................................................................47 2.1.1. Abstract..............................................................................................................47 2.1.2. Introduction.......................................................................................................47 2.1.3. Results...............................................................................................................49 2.1.4. Discussion.........................................................................................................50 2.1.5. Materials and methods.......................................................................................54 2.1.5.1. 2.1.5.2. 2.1.5.3. 2.1.5.4. Reconstitution..........................................................................................54 Trypsin digestion......................................................................................54 Atomic force microscopy............................................................................54 Image processing......................................................................................55 2.1.6. Acknowledgment...............................................................................................55 2.1.7. References.........................................................................................................55 2.2. High resolution AFM topographs of Rubrivivax gelatinosus light-harvesting complex LH2...............................................................................................................59 2.2.1. Abstract..............................................................................................................59 2.2.2. Introduction.......................................................................................................59 2.2.3. Results...............................................................................................................61 2.2.4. Discussion.........................................................................................................65 2.2.5. Materials and methods.......................................................................................67 2.2.5.1. 2.2.5.2. 2.2.5.3. 2.2.5.4. 2.2.5.5. 2.2.5.6. Materials.................................................................................................67 Isolation, purification and proteolysis of LH2 complex....................................67 Biochemical and biophysical techniques.........................................................67 Reconstitution and 2D crystallization............................................................67 Atomic force microscopy............................................................................67 Image processing......................................................................................68 2.2.6. Acknowledgment...............................................................................................68 2.2.7. References.........................................................................................................68 3. Combining surface and projection techniques....................................................73 3.1. The aquaporin sidedness revisited...........................................................................73 3.1.1. Summary...........................................................................................................73 3.1.2. Introduction.......................................................................................................73 3.1.3. Results...............................................................................................................74 3.1.4. Discussion.........................................................................................................78 3.1.5. Materials and Methods......................................................................................80 3.1.5.1. 3.1.5.2. 3.1.5.3. 3.1.5.4. 3.1.5.5. 2D crystallization......................................................................................80 Trypsin digestion......................................................................................80 Atomic force microscopy............................................................................80 Freeze-drying & metal-shadowing.................................................................80 Cryo electron microscopy...........................................................................80 3.1.6. Acknowledgment...............................................................................................81 3.1.7. References.........................................................................................................81 10 4. Structural studies of a membrane transporter...................................................87 4.1. The functional Escherichia coli lactose permease LacY/Cytb562/6His forms trimers: A 2.8 nm 3D reconstruction and preliminary electron crystallographic data...............................................................................................................................87 4.1.1. Summary............................................................................................................87 4.1.2. Introduction........................................................................................................87 4.1.3. Results and discussion.......................................................................................89 4.1.3.1. 4.1.3.2. 4.1.3.3. Protein purification...................................................................................89 Single particle analysis..............................................................................90 Reconstitution and 2D crystallization...........................................................91 4.1.4. Perspectives........................................................................................................93 4.1.5. Material and Methods........................................................................................94 4.1.5.1. 4.1.5.2. 4.1.5.3. 4.1.5.4. Materials................................................................................................94 Protein Expression and Purification.............................................................94 Reconstitution.........................................................................................94 Electron microscopy and image processing....................................................95 4.1.6. References..........................................................................................................95 5. General discussion and conclusions.......................................................................101 6. Acknowledgment..........................................................................................................107 7. Curriculum vitae..........................................................................................................113 7.1. Education...................................................................................................................113 7.2. Teaching.....................................................................................................................113 7.3. Publications...............................................................................................................113 7.4. Meetings.....................................................................................................................114 11 12 1. AFM and EM in structural biology 13 14 1. AFM and EM in structural biology 1.1. General introduction 1.1.1. The driving force to do science What does it look like? What does it do? It is curiosity, the wish to know, that induces these questions. It is human nature - and it is the driving force to do fundamental research in any field ranging from astronomy to physics of smallest matter. The primary questions of molecular biologists are: What is the structure, what is the function, of a biomolecule? Once the structure and function of a biomolecule are known, its physiological role and interactive mechanisms can be brought into context within the framework of the cell and the whole organism. It is our ultimate goal to understand the molecular mechanisms of all processes in each cell of our body, ranging from neurons working in our brain, muscle cells allowing us to move, to the cells of our skin ultimately defining the borderlines of ourselves and our environment. 1.1.2. Membrane proteins Intrinsic or integral membrane proteins are defined as proteins that penetrate into and, most often, traverse the lipid bilayer of a biological membrane. Protein structures, which partition into lipid rather than remain in aqueous solution have specific chemical properties. They are rich in exposed hydrophobic amino acids and are restricted in their secondary structure. A consequence of these physico-chemical properties is that an integral membrane protein can only be brought into aqueous solution when solubilized in the presence of detergents. The challenge of understanding membrane proteins and transporters has attracted our interest. Figure 1 represents an interesting result of a genome study. Genomes of different organisms (E. coli, M. jannaschii, H. sapiens) were screened for their protein coding open reading frames and these open reading frames were translated into amino acid chains. The peptides were discriminated by their hydrophobicities. This resulted in two families of gene products: The hydrophilic cytoplasmic proteins, and the membrane proteins, which contain large hydrophobic stretches (e.g., representing transmembrane helices). By this relatively simple approach, it has been demonstrated that 20 - 30 % of genes (the higher the organism, the larger the percentage) code for strongly hydrophobic proteins which are most probably integrated into cell membranes. This intriguing result emphasizes the extreme importance of membrane proteins for living organisms. Membrane proteins connect the cytoplasm with the extracellular space of each living cell, form junctions between living cells or play an important role in the intracellular compartments. Hence, in bacteria, such molecules work in transport, secretion and bioenergetic processes. Multicellular organisms even require active communication between their cells. Consequently a large number of membrane proteins have evolved, working as receptors for intercellular trafficking or cellular adhesion and recognition. Evolution has also created highly specific channels and transporters, which are essential for the survival of biological systems; the deletion of many membrane proteins is lethal or leads to severe disease. The study of membrane protein structure is a difficult challenge: Membrane proteins remain only folded in their active state when 15 Figure 1. Number of genes of different organisms as a function of the hydrophobicity of their gene product. Integration over the two peaks corresponding to cytoplasmic and membrane proteins shows that 20 - 30 % of all genes code for membrane proteins. their hydrophobic transmembrane domains are embedded in a hydrophobic environment e.g., a lipid bilayer or a detergent micelle. This prerequisite makes the growth of 3D crystals for structure determination by X-ray diffraction difficult. Consequently, ~2000 atomic structures of water-soluble proteins are available but only ~20 atomic structures of integral membrane proteins. The structures so far determined, divide membrane proteins into two categories: αhelical and β-barrel membrane proteins. The majority of the structures were determined using 3D crystallization and X-ray diffraction. However, three structures have been solved by electron crystallography: Plant light-harvesting complex II, bacteriorhodopsin, and human aquaporin 1. The electron crystallography was carried out using 2D crystals of protein integrated in lipid bilayers. 1.1.3. 2D crystals allow the acquisition of structural information on membrane proteins in a nativelike environment In order to acquire biologically valid information, it is important to study the structure of membrane proteins under conditions where they remain functional. To this end, membrane proteins are reconstituted into 2D crystals in the presence of lipids which mimic their native membrane environment within a cell (chapters 2.1, 2.2, 3.1, 4.1). Although only 3 membrane protein structures have been solved to atomic resolution (below 4Å) using electron crystallography (Kühlbrandt et al., 1994; Henderson et al., 1990; Kimura et al., 1997; Murata et al., 2000), numerous proteins have been solved to medium resolution (4Å-10Å) in 2D projection or 3D density maps from electron micrographs. Such medium resolution maps revealed helix arrangements 16 or/and structural similarities within the aquaporin protein family (Stahlberg et al., 2001). 1.1.4. Atomic force and electron microscopy cover large resolution ranges and together provide both surface and volume information Structural biology encompasses a range of techniques, to elucidate structures and interactions of biomolecules. The table below summarizes some of the advantages and disadvantages of the various approaches. The atomic force and the electron microscope are our tools to investigate the fascinating microcosm of membrane proteins. As listed above, a combination of these two techniques covers a resolution range from micrometers to atomic scale, and yields both surface and volume information of proteins in the close to native environment of 2D crystals. Electron microscopy of negatively stained samples allows the efficient acquisition of structural information. Despite the limited resolution of ~15Å, the technique provides a high signal-to-noise ratio (chapters 1.3, 4.1). By these means, samples of 2D crystals of membrane proteins can be checked and a first Table 1. Advantages and disadvantages of techniques applied in structural biology technique X-ray crystallography advantages -atomic resolution disadvantages -well ordered 3D crystals are required -absence of phases -requires large amounts of protein -requires protein labeling -problems with large molecules (>40 kDa) -requires well ordered 2D crystals -technically difficult -resolution limit ~5Å -problems with small molecules (
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