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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.
Die Schmelztemperaturen und Schmelzenthalpien verschiedener homologer und isomerer Reihen zeigen Alternanzverhalten. In einem Vergleich von über 140 Datenreihen wurde versucht, einen möglichen Zusammenhang von Molekülstruktureigenschaften und dem Auftreten von Alternanzen der genannten physikalischen Eigenschaften darzulegen.
Structural analysis of the enzyme N-formylmethanofuran:tetrahydromethanopterin formyltransferase
(2008)
Archaea represent a third domain of life and some archaea exhibit a high degree of tolerance to extreme environmental conditions. Several members are methanogens and present in many anaerobic environments. Most methanogens are able to maintain growth simply on H2 and CO2 via the enzymatically catalyzed reaction 4H2 + CO2 > CH4 + 2 H2O. The archaeon Methanopyrus kandleri grows optimally at temperatures of 84°C to 110°C, pH values of 5.5 to 7.0 and NaCl concentrations 0.2% to 4%. The enzyme N-formylmethanofuran tetrahydromethanopterin formyltransferase (MkFTR) catalyzes the transfer of a formyl group from the cofactor N-formylmethanofuran (FMF) to the cofactor tetrahydromethanopterin (H4MPT), the second step of the above reaction. X-ray crystallographic analysis yielded insights into the structure and function of MkFTR, (1) the MkFTR monomer exhibits a pseudo-two fold structure suggestive of an evolutionary gene duplication. (2) The structure is a D2 homo-tetramer with prominent cleft-like surface features. Analysis of the interface contacts showed that the tetramer is best described as a dimer of dimers. The clefts were associated with the monomer:monomer interface and were weakly occupied by extra electron density which might be attributed to the H4MPT analog folate. (3) This suggested that the clefts are active sites and their association with oligomer interfaces suggested a basis for the dependence of activity on oligomerization. (4) The thermal stability of MkFTR most likely arises from the greater number of H- and ionic-bonds within the monomer and between monomers with respect to mesophilic protein structures. (5) The structure showed a large number of surface exposed negatively charged, glutamate and aspartate residues. These residues explain the salt dependent oligomerization, as only at high enough salt concentration is the electrostatic charge compensated by cation binding and neutralized allowing oligomerization. (6) These residues also improve the solubility of MkFTR at high salt concentration by increased charge repulsion. (7) Comparison of MkFTR structures from low and hight salt conditions showed that surface glutamate residues bind slightly more water molecules at high salt conditions further contributing to MkFTR solubility at high salt concentration.
My graduate thesis is on the "Structural studies of membrane transport proteins". Transporters are membrane proteins that have multiple membrane-spanning a-helices. They are dynamic and diverse proteins, undergoing a large conformational change and transporting wide range of susbtrates. Based on their energy source they can be classified into primary and secondary transport systems. Primary transport systems are driven by the use of chemical (ATP) or light energy, while secondary transporters utilize ion gradients to transport substrates. I began my PhD dissertation on secondary transporters by two-dimensional crystallization and electron crystallographic analysis and recently my focus also has shifted towards 3D crystallization. The following projects constitute my PhD thesis: 1) 2D crystallization of MjNhaP1 and pH induced structural change: MjNhaP1, a Na+/H+ antiporter that is regulated by pH has been implicated in homeostasis of H+ and Na+ in Methanococcus jannaschii, a hyperthermophilic archaeon that grows optimally at 85°C. MjNhaP1 was cloned and expressed in E. coli. Two-dimensional crystals were obtained from purified protein at pH4. Electron cryo-microscopy yielded an 8Å projection map. The map of MjNhaP1 shows elongated densities in the centre of the dimer and a cluster of density peaks on either side of the dimer core, indicative of a bundle of 4-6 membrane-spanning helices. The effect of pH on the structure of MjNhaP1was studied in situ in 2D crystals revealing a major change in density within the helix bundle relative to the dimer interface. This change occurred at pH6 and above. The two conformations at low and high pH most likely represent the closed and open states of the antiporter, respectively. This is the first instance where a conformational change associated with the regulation of a secondary transporter appears to map structurally. Reconstruction of 3D map and high-resolution structure by x-ray crystallography would be necessary to understand the mechanism of ion transport and regulation by pH. 2) 2D crystallization of Proline transporter: Proline transporter (PutP) from E.coli belongs the sodium-solute symporter family that includes disease related sodium dependent glucose and iodide transporter in humans. Sodium and proline are co-transported with a stoichiometry of 1:1. Purified PutP was reconstituted to yield 2D crystals that were hexagonal in nature. The 2D crystals had tendency to stack indicating their willingness to form 3D crystals. A projection map of PutP from negatively stained crystals showed trimeric arrangement of protein. Other members of the SSF family have been shown to be monomers. My analysis of oligomeric state of PutP in detergent by blue native gel indicates a monomer in detergent solution. It is likely that PutP can function as a monomer but at higher concentration and in lipid bilayer it tends to form trimer. 3) Oligomeric state and crystallization of carnitine transporter from E.coli: E.coli carnitine transporter (CaiT) belongs to the BCCT (Betaine, Carnitine and Choline) superfamily that transports molecules with quaternary amine groups. CaiT is predicted to span the membrane 12 times and acts as a L-carnitine/g-butyrobetaine exchanger. Unlike other members in this transporter family, it does not require an ion gradient and does not respond to osmotic stress. Over-expression of the protein yielded ~2mg of protein/L of culture. The structure and oligomeric state of the protein were analyzed in detergent and lipid bilayers. Blue native gel electrophoresis indicated that CaiT was a trimer in detergent solution. Gel filtration and cross-linking studies further support this. Reconstitution of CaiT into lipid bilayers resulted in 2D crystals. Analysis of negatively stained 2D crystals confirmed that CaiT is a trimer in the membrane. Initial 3D crystallization trials have been successful and currently, the crystals diffract to 6Å and are being improved. 4) Monomeric porin OmpG: OmpG is a bacterial outer membrane b-barrel protein. It is monomeric and its size (33kDa) places it as a prime candidate for a structural solution, using the recently developed method of solid state NMR (work in collaboration with Prof.Hartmut Oskinat, FMP, Berlin). A long-term aim would be to study porins as templates for designing nanopores, for DNA sequencing and identification. I have expressed OmpG in inclusion bodies and refolded at an efficiency of >90% into a functional form using detergent. OmpG was then crystallized by 2D crystallization yielding an 8Å projection map whose structure was similar to native protein. In addition, these crystals were used for structure determination by solid state NMR. An initial spectrum of heavy isotopically labeled OmpG has allowed identification of specific amino acid residues including threonine and proline. Additionally, I obtained 3D crystals in detergent that diffract to 5.5Å and are being improved.