Micelle formation in the presence of photosystem I

AbstractThe correlation between membrane protein solubilisation and detergent aggregation in aqueous solution is studied for a series of n-alkyl-β-d-maltosides (CxG2 with x=10, 11, 12 being the number of carbon atoms in the alkyl chain) using the trimeric photosystem I core complex (PSIcc) of oxygenic photosynthesis from Thermosynechococcus elongatus as model protein. While protein solubilisation is monitored via the turbidity of the solution, the aggregation behavior of the detergent is probed via the fluorescence spectrum of the polycyclic aromatic hydrocarbon pyrene. In addition, changes of the fluorescence spectrum of PSIcc in response to formation of the detergent belt surrounding its hydrophobic surface are investigated. Solubilisation of PSIcc and aggregation of detergent into micelles or belts are found to be strictly correlated. Both processes are complete at the critical solubilisation concentration (CSC) of the detergent, at which the belts are formed. The CSC depends on the concentration of the membrane protein, [prot], and is related to the critical micelle concentration (CMC) by the empirical law ln(CSC/CMC)=n¯0 [prot], where the constant n¯0 = (2.0±0.3) μM−1 is independent of the alkyl chain length x. Formation of protein-free micelles below the CSC is not observed even for x=10, where a significant excess of detergent is present at the CSC. This finding indicates an influence of PSIcc on micelle formation that is independent of the binding of detergent to the hydrophobic protein surface. The role of the CSC in the optimisation of membrane protein crystallisation is discussed

The influence of poly(ethylene glycol) on the micelle formation of alkyl maltosides used in membrane protein crystallization

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG geförderten) Allianz- bzw. Nationallizenz frei zugänglich.This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively.With the aim of better understanding the phase behavior of alkyl maltosides (n-alkyl-beta-D-maltosides, C(n)G(2)) under the conditions of membrane protein crystallization, we studied the influence of poly(ethylene glycol) (PEG) 2000, a commonly used precipitating agent, on the critical micelle concentration (CMC) of the alkyl maltosides by systematic variation of the number n of carbon atoms in the alkyl chain (n = 10, 11, and 12) and the concentration of PEG2000 (chi) in a buffer suitable for the crystallization of cyanobacterial photosystem II. CMC measurements were based on established fluorescence techniques using pyrene and 8-anilinonaphthalene-1-sulfonate (ANS). We found an increase of the CMC with increasing PEG concentration according to ln(CMC/CMC0) = k(P)chi, where CMC0 is the CMC in the absence of PEG and k(P) is a constant that we termed the "polymer constant". In parallel, we measured the influence of PEG2000 on the surface tension of detergent-free buffer solutions. At PEG concentrations chi > 1% w/v, the surface pressure pi(s)(chi) = gamma(0) - gamma(chi) was found to depend linearly on the PEG concentration according to pi(s)(chi) = kappa chi + pi(s)(0), where gamma(0) is the surface tension in the absence of PEG. Based on a molecular thermodynamic modeling, CMC shifts and surface pressure due to PEG are related, and it is shown that k(P) = kappa c(n) + eta, where c(n) is a detergent-specific constant depending inter alia on the alkyl chain length n and eta is a correction for molarity. Thus, knowledge of the surface pressure in the absence of a detergent allows for the prediction of the CMC shift. The PEG effect on the CMC is discussed concerning its molecular origin and its implications for membrane protein solubilization and crystallization.DFG, SFB 429, Molekulare Physiologie, Energetik und Regulation primärer pflanzlicher StoffwechselprozesseDFG, SFB 498, Protein-Kofaktor-Wechselwirkungen in biologischen ProzessenDFG, SFB 1078, Proteinfunktion durch ProtonierungsdynamikDFG, EXC 314, Unifying Concepts in CatalysisBMBF, 031A154B, Basistechnologien Forschertandem: Nutzung von Sonnenenergie für die Bioelektrokatalyse - Entwicklung von Photo-Bioelektrodenstrukturen für die Synthes

Dynamic water bridging and proton transfer at a surface carboxylate cluster of photosystem II

Proton-transfer proteins are often exposed to the bulk clusters of carboxylate groups that might bind protons transiently. This raises important questions as to how the carboxylate groups of a protonated cluster interact with each other and with water, and how charged protein groups and hydrogen-bonded waters could have an impact on proton transfers at the cluster. We address these questions by combining classical mechanical and quantum mechanical computations with the analysis of cyanobacterial photosystem II crystal structures from Thermosynechococcus elongatus. The model system we use consists of an interface between PsbO and PsbU, which are two extrinsic proteins of photosystem II. We find that a protonated carboxylate pair of PsbO is part of a dynamic network of protein–water hydrogen bonds which extends across the protein interface. Hydrogen-bonded waters and a conserved lysine sidechain largely shape the energetics of proton transfer at the carboxylate cluster

Preferential pathways for light-trapping involving β-ligated chlorophylls

AbstractThe magnesium atom of chlorophylls (Chls) is always five- or six-coordinated within chlorophyll–protein complexes which are the main light-harvesting systems of plants, algae and most photosynthetic bacteria. Due to the presence of stereocenters and the axial ligation of magnesium the two faces of Chls are diastereotopic. It has been previously recognized that the α-configuration having the magnesium ligand on the opposite face of the 17-propionic acid moiety is more frequently encountered and is more stable than the more seldom β-configuration that has the magnesium ligand on the same face [T.S. Balaban, P. Fromme, A.R. Holzwarth, N. Krauβ, V.I. Prokhorenko, Relevance of the diastereotopic ligation of magnesium atoms in chlorophylls in Photosystem I, Biochim. Biophys. Acta (Bioenergetics), 1556 (2002) 197–207; T. Oba, H. Tamiaki, Which side of the π-macrocycle plane of (bacterio)chlorophylls is favored for binding of the fifth ligand? Photosynth. Res. 74 (2002) 1–10]. In photosystem I only 14 Chls out of a total of 96 are in a β-configuration and these occupy preferential positions around the reaction center. We have now analyzed the α/β dichotomy in the homodimeric photosystem II based on the 2.9 Å resolution crystal structure [A. Guskov, J. Kern, A. Gabdulkhakov, M. Broser, A. Zouni, W. Saenger, Cyanobacterial photosystem II at 2.9 Å resolution: role of quinones, lipids, channels and chloride, Nature Struct. Mol. Biol. 16 (2009) 334–342] and find that out of 35 Chls in each monomer only 9 are definitively in the β-configuration, while 4 are uncertain. Ab initio calculations using the approximate coupled-cluster singles-and-doubles model CC2 [O. Christiansen, H. Koch, P. Jørgensen, The second-order approximate coupled cluster singles and doubles model CC2, Chem. Phys. Lett. 243 (1995) 409–418] now correctly predict the absorption spectra of Chls a and b and conclusively show for histidine, which is the most frequent axial ligand of magnesium in chlorophyll–protein complexes, that only slight differences (<4 nm) are encountered between the α- and β-configurations. Significant red shifts (up to 50 nm) can, however, be encountered in excitonically coupled β–β-Chl dimers. Surprisingly, in both photosystems I and II very similar “special” β–β dimers are encountered at practically the same distances from P700 and P680, respectively. In purple bacteria LH2, the B850 ring is composed exclusively of such tightly coupled β-bacteriochlorophylls a. A statistical analysis of the close contacts with the protein matrix (<5 Å) shows significant differences between the α- and β-configurations and the subunit providing the axial magnesium ligand. The present study allows us to conclude that the excitation energy transfer in light-harvesting systems, from a peripheral antenna towards the reaction center, may follow preferential pathways due to structural reasons involving β-ligated Chls

Protein crystallization and initial neutron diffraction studies of the photosystem II subunit PsbO

The PsbO protein of photosystem II stabilizes the active-site manganese cluster and is thought to act as a proton antenna. To enable neutron diffraction studies, crystals of the β-barrel core of PsbO were grown in capillaries. The crystals were optimized by screening additives in a counter-diffusion setup in which the protein and reservoir solutions were separated by a 1% agarose plug. Crystals were cross-linked with glutaraldehyde. Initial neutron diffraction data were collected from a 0.25 mm3 crystal at room temperature using the MaNDi single-crystal diffractometer at the Spallation Neutron Source, Oak Ridge National Laboratory

Modeling of variant copies of subunit D1 in the structure of photosystem II from Thermosynechococcus elongatus

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG geförderten) Allianz- bzw. Nationallizenz frei zugänglich.This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively.In the cyanobacterium Thermosynechococcus elongatus BP-1, living in hot springs, the light environment directly regulates expression of genes that encode key components of the photosynthetic multi-subunit protein-pigment complex photosystem II (PSII). Light is not only essential as an energy source to power photosynthesis, but leads to formation of aggressive radicals which induce severe damage of protein subunits and organic cofactors. Photosynthetic organisms develop several protection mechanisms against this photo-damage, such as the differential expression of genes coding for the reaction center subunit D1 in PSII. Testing the expression of the three different genes (psbAI, psbAII, psbAIII) coding for D1 in T. elongatus under culture conditions used for preparing the material used in crystallization of PSII showed that under these conditions only subunit PsbA1 is present. However, exposure to high-light intensity induced partial replacement of PsbA1 with PsbA3. Modeling of the variant amino acids of the three different D1 copies in the 3.0 Å resolution crystal structure of PSII revealed that most of them are in the direct vicinity to redox-active cofactors of the electron transfer chain. Possible structural and mechanistic consequences for electron transfer are discussed.DFG, SFB 498, Protein-Kofaktor-Wechselwirkungen in biologischen ProzessenEC/FP6/516510/EU/Linking molecular genetics and bio-mimetic chemistry - a multidisciplinary approach to achieve renewable hydrogen production/SOLAR-

Structural insights into the light-driven auto-assembly process of the water- oxidizing Mn4CaO5-cluster in photosystem II

In plants, algae and cyanobacteria, Photosystem II (PSII) catalyzes the light- driven splitting of water at a protein-bound Mn4CaO5-cluster, the water- oxidizing complex (WOC). In the photosynthetic organisms, the light-driven formation of the WOC from dissolved metal ions is a key process because it is essential in both initial activation and continuous repair of PSII. Structural information is required for understanding of this chaperone-free metal-cluster assembly. For the first time, we obtained a structure of PSII from Thermosynechococcus elongatus without the Mn4CaO5-cluster. Surprisingly, cluster-removal leaves the positions of all coordinating amino acid residues and most nearby water molecules largely unaffected, resulting in a pre- organized ligand shell for kinetically competent and error-free photo-assembly of the Mn4CaO5-cluster. First experiments initiating (i) partial disassembly and (ii) partial re-assembly after complete depletion of the Mn4CaO5-cluster agree with a specific bi-manganese cluster, likely a di-µ-oxo bridged pair of Mn(III) ions, as an assembly intermediate

pH‐dependent protonation of surface carboxylate groups in PsbO enables local buffering and triggers structural changes

Photosystem II (PSII) catalyzes the splitting of water, releasing protons and dioxygen. Its highly conserved subunit PsbO extends from the oxygen‐evolving center (OEC) into the thylakoid lumen and stabilizes the catalytic Mn4CaO5 cluster. The high degree of conservation of accessible negatively charged surface residues in PsbO suggests additional functions, as local pH buffer or by affecting the flow of protons. For this discussion, we provide an experimental basis, through the determination of pKa values of water‐accessible aspartate and glutamate side‐chain carboxylate groups by means of NMR. Their distribution is strikingly uneven, with high pKa values around 4.9 clustered on the luminal PsbO side and values below 3.5 on the side facing PSII. pH‐dependent changes in backbone chemical shifts in the area of the lumen‐exposed loops are observed, indicating conformational changes. In conclusion, we present a site‐specific analysis of carboxylate group proton affinities in PsbO, providing a basis for further understanding of proton transport in photosynthesis

Where Water is Oxidized to Dioxygen: Structure of thePhotosynthetic Mn4Ca Cluster

Oxidation of water to dioxygen is catalyzed withinphotosystem II (PSII) by a Mn4Ca cluster, the structure of which remainselusive. Polarized extended X-ray absorption fine structure (EXAFS)measurements on PSII single crystals constrain the Mn4Ca cluster geometryto a set of three similar high-resolution structures. Combining polarizedEXAFS and X-ray diffraction data, the cluster was placed within PSIItaking into account the overall trend of the electron density of themetal site and the putative ligands. The structure of the cluster fromthe present study is unlike either the 3.0 or 3.5 Angstrom resolutionX-ray structures, and other previously proposed models