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Electron microscopy (EM) demarcates itself from other structural biology techniques by its applicability to a large range of biological objects that spans from whole cells to individual macromolecules. In single-particle cryo-EM, frozen-hydrated samples, prepared by vitrification with liquid ethane, retain macromolecules in a medium that approximates their natural aqueous environment and that, in this way, preserves high-resolution structural information. Nonetheless, the sensitivity of biological specimens to the high-energy electron beam introduces restrictions on the total dose that can be used during imaging while avoiding significant radiation damage. Consequently, the signal-to-noise ratio attained in each individual image is very low, and structures with high-resolution detail must be recovered by averaging thousands of projections in random orientations. This is achieved through the use of image processing algorithms capable of aligning and classifying particle images through the evaluation of cross-correlation functions between each particle and a reference.
In recent years, several innovations took place in the field of single-particle cryo-EM, among which the development of direct electron detectors must be highlighted. Direct electron detectors have a better detective quantum efficiency (DQE) than both photographic film and CCD cameras, and offer a fast readout, compatible with the acquisition of movie stacks. Additionally, new image processing software has become available, with more sophisticated algorithms and designed to take advantage of the specific characteristics of the movies produced with direct electron detectors. These technological advances in both hardware and software catalyzed a revolution in single-particle cryo-EM, which is now routinely used for the determination of near-atomic structures. As a result, the range of macromolecules accessible to cryo-EM has increased drastically, as targets that were unsuitable before for imaging due to their small dimensions can now be adequately visualized and refined to high-resolution.
During my doctoral work, I have used single-particle cryo-EM to structurally characterize challenging membrane proteins, with a strong emphasis on protein complexes from aerobic respiratory chains. In chapter I of this thesis, I present my results on the bovine respirasome, a mitochondrial supercomplex composed of complexes I, III and IV. Chapter II is dedicated to the analysis of the structure of alternative complex III (ACIII) from Rhodothermus marinus, a bacterial quinol:cytochrome c/HiPIP oxidoreductase unrelated to the canonical cytochrome bc1 complex (complex III). In addition, in chapter III I describe the structure of KimA, a high-affinity potassium transporter that drives the transport of its substrate by using the energy stored in the form of a proton gradient. These three membrane proteins, with molecular weights ranging from 140 kDa to 1.7 MDa, illustrate the possibilities and limitations faced in single-particle cryo-EM.
The aerobic respiratory chain is responsible for the generation of a transmembrane difference of electrochemical potential that is then used by ATP synthase for the production of ATP or for driving solute transport over the membrane. They catalyze the transfer of electrons from a substrate, such as NADH or succinate, to molecular oxygen and use the chemical energy released in these redox reactions to drive the translocation of protons, or in some cases sodium ions, to the intermembrane space in mitochondria or the periplasm in bacteria.
In mitochondria, the respiratory chain is composed of four complexes: complex I (NADH:ubiquinone oxidoreductase), complex II (succinate dehydrogenase), complex III (cytochrome bc1 complex) and complex IV (cytochrome c oxidase). While it was for a long time believed that these complexes existed as single entities in the membrane, the use of milder procedures for protein purification and analysis revealed that respiratory complexes associate into well-ordered structures, known as supercomplexes. These have been proposed to offer different structural and functional advantages that are still controversial, including substrate channeling, stabilization of individual complexes and reduction of reactive oxygen species (ROS) production. The most thoroughly studied respiratory supercomplex has been the respirasome, conserved in higher eukaryotes and composed of one copy of complex I, a complex III dimer and one complex IV. By single-particle cryo-EM analysis, I retrieved a 9 Å map of the respirasome from Bos taurus, which allowed the accurate docking of atomic models of the three component complexes. The structure shows that complex III associates to the concave side of the membrane arm of complex I, while complex IV is located between the end of the complex I hydrophobic arm and complex III. Several defined protein-protein contacts are observed between the component complexes, which are mediated predominantly by supernumerary subunits and close to the membrane surfaces. The interactions established between complex I and complex III are extensive and may support the argument that the association of complex I into supercomplexes is required for the stabilization or even the biogenesis of this complex.
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TeaABC from the halophilic bacterium Halomonas elongata belongs to the family of tripartite ATP-independent periplasmic (TRAP) transporters. It facilitates the uptake of the compatible solutes ectoine and hydroxyectoine which protect the cell from dehydration by accumulating in the cytoplasm during hyperosmotic stress. It is the only known TRAP transporter activated by osmotic stress. Ectoine and hydroxyectoine accumulation in H. elongata is regulated by the cytoplasmic universal stress protein TeaD. The gene encoding TeaD is located in the same operon as the TeaABC gene. TeaD regulates the cellular homeostasis of ectoine possibly by interacting directly or indirectly with TeaABC. All subunits of TeaABC and TeaD were expressed in E. coli and purified. With TeaD and the solute binding protein (SBP) TeaA high levels of expression suitable for crystallization could be obtained and their 3D structures solved. The small transmembrane protein TeaB and the transporter TeaC showed only moderate and low levels of expression respectively. Functional analysis on TeaA was performed using Isothermal Titration Calorimetry. The measurements demonstrate that TeaA is a high affinity ectoine-binding protein (Kd = 0.19 _M) that also has a significant affinity for hydroxyectoine (Kd = 3.8 _M). The structure of TeaA was solved using ab initio phase determination by MAD (multiple anomalous dispersion). TeaA structures were determined in three conformations: TeaA alone, TeaA in complex with ectoine and TeaA in complex with hydroxyectoine. The resolutions of the structures were 2.2, 1.55 and 1.80 Å, respectively. These represent the first structures of an osmolyte SBP associated to a TRAP transporter. The structures reveal similar ligand binding compared to osmolyte SBPs of ABC transporter pointing to coevolution of the ligand binding modes. Moreover, unique features such as the solvent-mediated specific binding of the ligands ectoine and hydroxyectoine could be observed for TeaA. The structure of TeaD in complex with its cofactor ATP was solved by molecular replacement at a resolution of 1.9 Å. Comparison with other structures of universal stress proteins shows striking oligomerization and ATP binding in TeaD. In conclusion, this work presents the first detailed analysis of the molecular mechanisms underlying ligand recognition of an osmoregulated transporter from the TRAP-transporter family.
Transport of proteins into or across cellular membranes is mediated by the conserved and ubiquitous Sec-machinery. The Sec-homologue in the inner membrane of Escherichia coli is SecYEG. Sec-mediated insertion of numerous membrane proteins is aided by YidC, another protein integral to the inner membrane of Escherichia coli. YidC fulfils in addition the integration of a variety of membrane proteins Sec-independently. It belongs to a conserved but structurally uncharacterised family of proteins important for membrane protein biogenesis and comprises homologues in mitochondria and chloroplasts. By modification of a former crystallisation protocol two-dimensional crystals of SecYEG were grown in presence of the signal sequence peptide of LamB. Recording of structural data by electron cryo-microscopy and calculation of a difference structure comparing a former SecYEG projection structure with the one of SecYEG crystallised in presence of the substrate revealed several new and vacant densities. These hint to signal peptide binding close to the translocation pore and to significant rearrangements in proximity to the lateral exit site for transmembrane domains in SecYEG. The difference structure suggests that dimeric SecYEG is an asymmetric molecule consisting of one active and one inactive SecYEG monomer. Detergent removal from a mixture of purified YidC and lipids produced two-dimensional crystals that were highly dependent on the ionic strength and lipid composition for their growth. Electron cryo-microscopy on the frozen-hydrated crystals and image processing visualised structural details at about 10 Å resolution. Averaging two alternative projection structures in p2 and p121_a symmetry, respectively, yielded essentially the same features. Four YidC monomers form one unit cell (dimensions 82 x 71 Å, included angle 85 ° and 90 °, respectively) and seem to be arranged as two sets of dimers integrated in an anti-parallel fashion into the membrane. An area of low density in the centre of each YidC monomer resembles possibly a constriction of the membrane, which could have particular relevance for the integration of substrate proteins into the lipid bilayer.
Life-threatening fungal infections are becoming increasingly common for immunocompromised patients such as those with AIDS, or those undergoing organ transplantation or chemotheraphy, as well as for other health-vulnerable patients. Excellent targets for antifungal drugs are chitin synthases, which are essential for survival of the fungus and lacking in humans. To design new antifungal drugs, knowledge of the three-dimensional structure and mechanism of action of chitin synthases are crucial. Chitin synthases are members of an important family of enzymes that synthesize structural polysaccharides, such as cellulose, β(1,3)-glucan, β(1,4)-mannan and hyaluronan. Therefore, chitin synthases could be used as a model system to understand these more complex enzymes, which are also of major medical and commercial importance. Chitin synthase 2 from Saccharomyces cerevisiae (ScChS2), the protein under study, is an integral membrane protein that synthesizes the primary septum between mother and daughter cells in budding yeast. It is essential for proper cell separation and expected to be highly regulated. An important aspect is that ScChS2 shows 55% sequence identity and is functionally analogous to chitin synthase 1 from the human opportunistic pathogen Candida albicans, this enzyme is also essential for cell survival (Munro, Winter et al. 2001). ...
Electron tomography was used to investigate membrane proteins in a variety of contexts. A high-angle tilt holder, suitable for electron tomography was designed, constructed and characterised. 2D crystals of membrane proteins, NhaA and YidC, were examined as a resolution test, and a method established for determining planarity of crystals. A model for specific gold binding to NhaA crystals was also presented. ATP synthase, a membrane protein complex in mitochondria, were imaged in a frozen hydrated state. They were found to form ribbons of dimers at highly curved regions of the membrane. Dimers from bovine heart and rat liver were excised from the tomographic volumes and averaged. Based on the location of the dimers in the mitochondrion, a model was established whereby ATP synthase, a molecular motor driven by the proton motive force, benefits from the high curvature that it induces in the membrane. Whole yeast mitochondria, imaged by electron cryo-tomography, also contained long ribbons of dimeric ATP synthase. Multiple copies of an unknown membrane protein complex were visualised by electron cryo-tomography, excised and averaged. A general method for the identification of unknown proteins was presented to deal with this inevitable issue, as native tissues and organelles are imaged, and the structures of complexes determined in situ.
ATP synthases are multi-subunit membrane enzymes, which utilize the energy stored in a transmembrane electrochemical ion gradient to produce adenosine-5´-triphosphate (ATP), the universal energy carrier in biological systems. Research on these important enzymes goes back more than 50 years and has produced innumerable studies. The F-type ATP synthase consists of two functionally distinct, but tightly coupled subcomplexes, the water-soluble F1 and the membrane-embedded Fo complex. In its simplest form, F1 consists of five different subunits with a stoichiometry of α 3β3γδε, and harbors three catalytic centers in the α 3β3-headpiece, while Fo consists of three different subunits in a stoichiometry of ab2cn, where n varies between 8 to 15 depending on the species. From a mechanistic standpoint, the complex can also be divided into two different units, namely a stator, α3β3δ-ab2, and a rotor, γε-cn. The enzyme utilizes the energy stored in a transmembrane electrochemical gradient of protons, or in some cases Na+, to drive ATP synthesis. In particular, the downhill translocation of these ions across the Fo complex drives rotation of the γε-cn unit, which is then transduced to the active centers, catalyzing the phosphorylation of adenosine-5`-diphosphate (ADP) with inorganic phosphate (Pi), and the release of ATP....
The universal biological energy currency adenosine triphosphate (ATP) is synthesized by the F1Fo-ATP synthase in most living organisms. The overall structure and function of F-type ATPases is conserved in the different organisms. The F1Fo-ATP synthase consist of two domains; the soluble F1 complex has the subunit stoichiometry α3β3γδε and the membrane embedded Fo complex consists of subunits ab2c10-15 in its simplest form found in bacteria. F1 and Fo both function as reversible rotary motors that are connected by a central stalk (γε) and a peripheral stalk (b2δ).
For ATP synthesis, the electrochemical energy formed by a proton or sodium ion gradient is required. The ion translocation across the Fo subcomplex induces torque in the motor part of the enzyme (cnγε), which causes conformational changes in the α3β3 domain leading to ATP synthesis from ADP and inorganic phosphate (Pi) catalyzed in the β-subunits. ATP hydrolysis causes a reverse torque in the Fo subcomplex triggering uphill ion translocation from cytoplasm to periplasm, and the enzyme functions as an ion pump.
The ATP synthesis mechanism is well understood, since several high-resolution structures of F1 are available. In contrast, the ion translocation mechanism across the membrane, mediated by the Fo subcomplex, is not understood in its structural detail.
Subunit a and the c-ring form an ion pathway, but subunit b is needed to form an active ion translocation pathway in both H+- and Na+-dependent systems. Several high-resolution structures of c-rings have provided insights in the ion translocation mechanism. The different ion translocation models based on biochemical, biophysical and structural analysis are in agreement in the fact that ions are translocated through a periplasmic ion access pathway in subunit a to the middle of the membrane and there to the binding site of a c-subunit. After almost a whole rotation of the c-ring the ion returns into the a-c interface, where it can be released to the cytoplasm. In the different models the cytoplasmic access pathway has been proposed to be located in subunit a, at the a-c interface or within the c-ring. The driving force of torque generation has been proposed to be the pH gradient or membrane potential. Several biochemical studies show that a conserved arginine in helix four of subunit a (R226 in Ilyobacter tartaricus or R210 in Escherichia coli)plays a critical role in the ion translocation. The arginine has been proposed to function as an electrostatic separator between the cytoplasmic and periplasmic pathways and as a mediator of the ion exchange into the c-ring ion-binding site.
Structural data of a related enzyme (V1Vo-ATPase from Thermus thermophilus) has provided insight into the helical arrangement of the ion translocating subunits I and Lring (related to subunit a and the c-ring). These structures indicated a small interface between subunit I and the L-ring, and two four-helix bundles in the N-terminal domain of subunit I were proposed to build the periplasmic and cytoplasmic ion pathways. To comprehend the ion-translocation and torque generation mechanism in F1Fo-ATP synthase, structural data of an intact a-c complex is needed.
The goal of this work was to obtain structural data of subunit a, most preferably in a complex with the c-ring or additionally with subunit b. Therefore, a new purification procedure for the I. tartaricus Fo-subcomplex, heterologously expressed in E. coli cells, was established. The purified Fo was characterized biochemically and by Laserinduced liquid bead ion desorption mass spectrometry (LILBID-MS). These analyses showed that pure and completely assembled Fo containing all its subunits in the correct stoichiometry (ab2c11) was obtained. The purified Fo complex was stable at 4°C for several months and at room temperature in the presence of lipids for several weeks. A lipid analysis was performed by thin-layer chromatography (TLC) to investigate the qualitative lipid composition of I. tartaricus whole lipid extract and various I. tartaricus F1Fo isolates. The whole lipid extract contained PC, PG and PE lipids and probably cardiolipin. PC, PG and PE lipids were bound to wild type I. tartaricus F1Fo, whereas recombinant I. tartaricus F1Fo did not have any bound lipids, but was able to bind the synthetic lipids POPC and POPG if they were provided during the purification.
For subsequent structural studies the purified Fo was subjected to two-dimensional (2D) crystallization trials. Vesicles and sheets tightly packed with protein and crystals with a rare plane group for I. tartaricus c11 (p121) were obtained. The c-ring was visible in the CCD images, and immunogold-labeling revealed the presence of the His-tagged a-subunit in the reconstituted vesicles. Furthermore, atomic force microscopy (AFM) imaging showed protein densities next to the c-rings, which protruded less from the membrane (0.4±0.1 nm) than the c-ring (0.7±0.1 nm). These protein densities presumably belonged to subunit a.
Cryo-electronmicroscopy (cryo-EM) was used to collect data of the p121 crystals and a merged projection density map was calculated to 7.0 Å resolution. The unit cell of the crystals (81 × 252 Å) contained two asymmetric units with three c-rings in each and next to the c11-rings new prominent densities were visible. In each extra density up to 7 transmembrane helices were visible, belonging to the stator subunit a and/or subunit b. To elucidate whether there are conserved elements in the three extra densities non-crystallographic averaging was applied using a single-particle approach.
Six possible arrangements for the c-rings and the extra densities were identified and used for the averaging. The extra densities were enhanced only in one of the possible arrangements. The average showed a four-helix bundle and a fifth helix in close proximity to the c-ring. Two more helices were present in each position but their position was ambivalent. The data obtained in this work provides the first insight in the helical arrangement in the a-c interface of F1Fo-ATP synthase.
The light-harvesting complex of photosystem II (LHC-II) is the major antenna complex in plant photosynthesis. It accounts for roughly 30% of the total protein in plant chloroplasts, which makes it arguably the most abundant membrane protein on Earth, and binds about half of plant chlorophyll (Chl). The complex assembles as a trimer in the thylakoid membrane and binds a total of 54 pigment molecules, including 24 Chl a, 18 Chl b, 6 lutein (Lut), 3 neoxanthin (Neo) and 3 violaxanthin (Vio). LHC-II has five key roles in plant photosynthesis. It: (1) harvests sunlight and transmits excitation energy to the reaction centres of photosystems II and I, (2) regulates the amount of excitation energy reaching each of the two photosystems, (3) has a structural role in the architecture of the photosynthetic supercomplexes, (4) contributes to the tight appression of thylakoid membranes in chloroplast grana, and (5) protects the photosynthetic apparatus from photo damage by non photochemical quenching (NPQ). A major fraction of NPQ is accounted for its energy-dependent component qE. Despite being critical for plant survival and having been studied for decades, the exact details of how excess absorbed light energy is dissipated under qE conditions remain enigmatic. Today it is accepted that qE is regulated by the magnitude of the pH gradient (ΔpH) across the thylakoid membrane. It is also well documented that the drop in pH in the thylakoid lumen during high-light conditions activates the enzyme violaxanthin de-epoxidase (VDE), which converts the carotenoid Vio into zeaxanthin (Zea) as part of the xanthophyll cycle. Additionally, studies with Arabidopsis mutants revealed that the photosystem II subunit PsbS is necessary for qE. How these physiological responses switch LHC-II from the active, energy transmitting to the quenched, energy-dissipating state, in which the solar energy is not transmitted to the photosystems but instead dissipated as heat, remains unclear and is the subject of this thesis. From the results obtained during this doctoral work, five main conclusions can be drawn concerning the mechanism of qE: 1. Substitution of Vio by Zea in LHC-II is not sufficient for efficient dissipation of excess excitation energy. 2. Aggregation quenching of LHC-II does not require Vio, Neo nor a specific Chl pair. 3. With one exception, the pigment structure in LHC-II is rigid. 4. The two X-ray structures of LHC-II show the same energy transmitting state of the complex. 5. Crystalline LHC-II resembles the complex in the thylakoid membrane. Models of the aggregation quenching mechanism in vitro and the qE mechanism in vivo are presented as a corollary of this doctoral work. LHC-II aggregation quenching in vitro is attributed to the formation of energy sinks on the periphery of LHC-II through random interaction with other trimers, free pigments or impurities. A similar but unrelated process is proposed to occur in the thylakoid membrane, by which excess excitation energy is dissipated upon specific interaction between LHC-II and a PsbS monomer carrying Zea. At the end of this thesis, an innovative experimental model for the analysis of all key aspects of qE is proposed in order to finally solve the qE enigma, one of the last unresolved problems in photosynthesis research.
This thesis presents a 5.9 Å map of yeast FAS obtained by cryo-electron microscopy using single particle analysis (SPA). The EM-map has been analyzed both by quantitative and qualitative analysis to aid in understanding of the structure and dynamics of yeast FAS. This study approaches the factors limiting the resolution in EM (>20 Å) and further discusses the possibilities of achieving higher-resolutions (<10 Å) in cryo-EM by single particle analysis. Here, SPA is highlighted as a powerful tool for understanding the structure and dynamics of macro-molecular complexes at near native conditions. Though SPA has been used over the last four decades, the low-resolution range (20-30 Å) of the method has limited its use in structural biology. Over the last decade, sub nanometer resolution (<10 Å) structures solved by SPA have been reported --both in studies involving symmetric particles, such as GroEL (D7) and asymmetric particles, such as ribosomes (C1). Recently, near-atomic resolution in the range of 3.8-4.2 Å has been achieved in cases of highly symmetric icosahedral viral capsid structures as well. The yeast FAS structure (D3) presented here is one of two low symmetry structures submitted to the EM-database in a resolution range of 5-6 Å; the other being GroEL (D7). Fatty acid synthase (FAS) is the key enzyme for the biosynthesis of fatty acids in living organisms. There are two types of FAS, namely the type II FAS system in prokaryotes, consisting of a set of individual enzymes, and type I FAS found in eukaryotes as a multienzyme complex. Yeast fatty acid synthase (FAS) is a 2.6 MDa barrel-shaped multienzyme complex, which carries out cyclic synthesis of fatty acids. By electron cryomicroscopy of single particles we obtained a 3D map of yeast FAS at 5.9 Å resolution. Compared to the crystal structures of fungal FAS, the EM map reveals major differences and new features that indicate a considerably different arrangement of the complex in solution, as well as a high degree of variance inside the barrel. Distinct density regions in the reaction chambers next to each of the catalytic domains fit well with the substratebinding acyl carrier protein (ACP) domain. In each case, this resulted in the expected distance of ~18 Å from the ACP substrate binding site to the active site of the catalytic domains. The multiple, partially occupied positions of the ACP within the reaction chamber provide direct insight into the proposed substrate-shuttling mechanism of fatty acid synthesis in this large cellular machine.