The unusually strong stabilizing effects of glycine betaine on the structure and function of the oxygen-evolving Photosystem II complex

Natural osmoregulatory substances (osmolytes) allow a wide variety of organisms to adjust to environments with high salt and/or low water content. In addition to their role in osmoregulation, some osmolytes protect proteins from denaturation and deactivation by, for example, elevated temperature and chaotropic compounds. A ubiquitous protein-stabilizing osmolyte is glycine betaine (N-trimethyl glycine). Its presence has been reported in bacteria, in particular cyanobacteria, in animals and in plants from higher plants to algae. In the present review we describe the experimental evidence related to the ability of glycine betaine to enhance and stabilize the oxygen-evolving activity of the Photosystem II protein complexes of higher plants and cyanobacteria. The osmolyte protects the Photosystem II complex against dissociation of the regulatory extrinsic proteins (the 18 kD, 23 kD and 33 kD proteins of higher plants and the 9 kD protein of cyanobacteria) from the intrinsic components of the Photosystem II complex, and it also stabilizes the coordination of the Mn cluster to the protein cleft. By contrast, glycine betaine has no stabilizing effect on partial photosynthetic processes that do not involve the oxygen-evolving site of the Photosystem II complex. It is suggested that glycine betaine might act, in part, as a solute that is excluded from charged surface domains of proteins and also as a contact solute at hydrophobic surface domains.     

光合放氧系统的结构(综述)

本文介绍近年来高等植物光合放氧系统结构的研究进展.     

Coordination sphere and structure of the Mn cluster of the oxygen-evolving complex in photosynthetic organisms

The great similarity between the binding of Fe(II) and the high-affinity Mn-binding site in the Mn-depleted PSII membranes (Semin et al. (1996) FEBS Lett. 375, 223–226) suggests that the coordination sphere of Mn in PSII is also suitable for iron. A comparison is performed between the primary amino acid sequences of D1 and D2 and diiron-oxo enzymes with the function of oxygen activation. All conservative motifs (EXXH) and residues binding and stabilizing the diiron cluster in diiron-oxo enzymes have been found in the C-terminal domains of D1 and D2 polypeptides. On the basis of these sequence similarities we suggest a structural model for the manganese cluster in the oxygen-evolving complex.     

Glycinebetaine stabilizes the association of extrinsic proteins with the photosynthetic oxygen-evolving complex.

The photosynthetic oxygen-evolving activity of the photosystem 2 complex, prepared from spinach, was labile when the complex was exposed to high-salt conditions under which the extrinsic proteins were dissociated from the complex. Glycinebetaine prevented the dissociation of the 18-kDa and the 23-kDa extrinsic proteins from the photosystem 2 complex in the presence of 1 M NaCl. It also prevented the dissociation of the 33-kDa extrinsic protein from the complex in the presence of 1 M MgCl2 or 1 M CaCl2. The oxygen-evolving activity of the photosystem 2 complex was stabilized by glycinebetaine when the complex was subjected to treatment with NaCl and MgCl2.     

Comparison of photosynthetic performances of marine picocyanobacteria with different configurations of the oxygen-evolving complex

The extrinsic PsbU and PsbV proteins are known to play a critical role in stabilizing the Mn₄CaO₅ cluster of the PSII oxygen-evolving complex (OEC). However, most isolates of the marine cyanobacterium Prochlorococcus naturally miss these proteins, even though they have kept the main OEC protein, PsbO. A structural homology model of the PSII of such a natural deletion mutant strain (P. marinus MED4) did not reveal any obvious compensation mechanism for this lack. To assess the physiological consequences of this unusual OEC, we compared oxygen evolution between Prochlorococcus strains missing psbU and psbV (PCC 9511 and SS120) and two marine strains possessing these genes (Prochlorococcus sp. MIT9313 and Synechococcus sp. WH7803). While the low light-adapted strain SS120 exhibited the lowest maximal O₂ evolution rates (P_max per divinyl-chlorophyll a, per cell or per photosystem II) of all four strains, the high light-adapted strain PCC 9511 displayed even higher P^Chl_max and P^PSII_max at high irradiance than Synechococcus sp. WH7803. Furthermore, thermoluminescence glow curves did not show any alteration in the B-band shape or peak position that could be related to the lack of these extrinsic proteins. This suggests an efficient functional adaptation of the OEC in these natural deletion mutants, in which PsbO alone is seemingly sufficient to ensure proper oxygen evolution. Our study also showed that Prochlorococcus strains exhibit negative net O₂ evolution rates at the low irradiances encountered in minimum oxygen zones, possibly explaining the very low O₂ concentrations measured in these environments, where Prochlorococcus is the dominant oxyphototroph.     

The BRCA1 C-terminal domain: structure and function

The BRCA1 C-terminal region contains a duplicated globular domain termed BRCT that is found within many DNA damage repair and cell cycle checkpoint proteins. The unique diversity of this domain superfamily allows BRCT modules to interact forming homo/hetero BRCT multimers, BRCT-non-BRCT interactions, and interactions with DNA strand breaks. The sequence and functional diversity of the BRCT superfamily suggests that BRCT domains are evolutionarily convenient interaction modules.     

Functional and structural study on chelator-induced suppression of S2/S1 FTIR spectrum in photosynthetic oxygen-evolving complex

Chelating agents have been shown to induce characteristic changes in the light-minus-dark Fourier transform infrared (FTIR) difference spectrum for the S(2)/S(1) difference in the oxygen-evolving complex (OEC). Addition of various ethylenediamine-N,N,N',N'-tetraacetic acid (EDTA)-type chelators, such as EDTA, O,O'-bis(2-aminoethyl)ethyleneglycol-N,N,N',N'-tetraacetic acid (EGTA), trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CyDTA), or N-(2-hydroxyethyl)ethylenediamine-N,N',N'-triacetic acid (HEDTA), to Ca(2+)-depleted PS II membranes resulted in the suppression of typical S(2)/S(1) vibrational features, including the symmetric (1365(+)/1404(-) cm(-1)) and the asymmetric (1587(+)/1566(-) cm(-1)) carboxylate stretching vibrations, as well as the amide I and II modes of the backbone polypeptides. In contrast, the addition of ethylenediamine-N,N'-diacetic acid (EDDA) showed less inhibitory effects. The effects of the chelators depended on the number of the carboxylate groups; chelators with more than two carboxymethyl groups were effective in altering the FTIR spectrum. The bridging structure that connects the two nitrogen atoms also influenced the inhibitory effects. However, the effects were not necessarily correlated with the stability constants of the chelators to Mn(2+). The vibrational modes that were suppressed by EDTA were almost completely restored by subsequent washing with Chelex-treated Ca(2+)-free buffer medium, indicating that the spectral changes are attributable to the reversible association of chelators with the Ca(2+)-depleted OEC. Nevertheless, prolonged incubation with chelators led to the impairment of the O(2)-evolving capability, with differences in the effectiveness, in the order that is consistent with that for the suppression effects on FTIR spectra. Chelators with carboxylate and/or carboxymethyl groups bound to a single nitrogen [nitrilotriacetic acid (NTA) and iminodiacetic acid (IDA)] or carbon (citric acid) were relatively ineffective for the suppression. A chelator that includes four phosphate groups, ethylenediamine-N,N,N',N'-tetrakis(methylenephosphonic) acid (EDTPO), also showed suppression effects on both the carboxylate and amide modes. Based on these findings, a possible mode of interaction between the chelators and the Mn cluster is discussed.     

丙氨酸消旋酶C-端结构域重组体的构建及其功能初探

[目的]将嗜碱芽孢杆菌丙氨酸消旋酶OF4DadX的N-端结构域分别与多个不同种属的丙氨酸消旋酶C-端结构域重组,探究丙氨酸消旋酶C-端结构域功能.[方法]利用基因拼接构建丙氨酸消旋酶重组基因,通过镍亲和层析纯化酶蛋白,采用D-氨基酸氧化酶偶联法检测重组酶蛋白的酶学特性,借助分子筛和HPLC液相色谱分析其聚合状态及动力学...     

The interaction of ammonia with the photosynthetic oxygen-evolving system

The reaction of ammonia with the oxygen-evolving system was investigated using EPR. Two sites with distinct binding properties were found. One site, previously known to be responsible for the modification by ammonia of the multiline EPR signal from the S₂ state and believed to be accessible in this state only, was found to bind ammonia also in the S₁ state although weaker. The second binding site, identified by the effect of bound ammonia on the shape and position of the g = 4.1 EPR signal, was also found to be accessible in both the S₁ and S₂ states. The apparent dissociation constants for ammonia at the two sites in the S₁ and S₂ states were determined. In neither state did the binding the ammonia account for the observed inhibition of oxygen evolution, suggesting that binding to other S states plays an important role in the inhibition. Chloride, which is known to interfere with ammonia-induced inhibition of oxygen evolution, was found to compete with ammonia at the site associated with the modification of the g = 4.1 EPR signal. The broadening of the hyperfine lines of the multiline EPR signal, seen in the presence of ¹⁷O-labeled water, was still observed after the modification of the signal by ammonia. This indicates that ammonia has not completely displaced water bound to the catalytic site in the S₂ state. The results of the binding studies are interpreted in terms of a two state — two site model, where the two states are identified by their EPR signals, the multiline and the g = 4.1 signal, respectively, and the two sites identified by the effects of ammonia on these signals and where the equilibrium between the two states is regulated by the binding of ligands to the sites.     

Structural changes of D1 C-terminal alpha-carboxylate during S-state cycling in photosynthetic oxygen evolution

Changes in the chemical structure of alpha-carboxylate of the D1 C-terminal Ala-344 during S-state cycling of photosynthetic oxygen-evolving complex were selectively measured using light-induced Fourier transform infrared (FTIR) difference spectroscopy in combination with specific [(13)C]alanine labeling and site-directed mutagenesis in photosystem II core particles from Synechocystis sp. PCC 6803. Several bands for carboxylate symmetric stretching modes in an S(2)/S(1) FTIR difference spectrum were affected by selective (13)C labeling of the alpha-carboxylate of Ala with l-[1-(13)C]alanine, whereas most of the isotopic effects failed to be induced in a site-directed mutant in which Ala-344 was replaced with Gly. Labeling of the alpha-methyl of Ala with l-[3-(13)C]alanine had much smaller effects on the spectrum to induce isotopic bands due to a symmetric CH(3) deformation coupled with the alpha-carboxylate. The isotopic bands for the alpha-carboxylate of Ala-344 showed characteristic changes during S-state cycling. The bands appeared prominently upon the S(1)-to-S(2) transition and to a lesser extent upon the S(2)-to-S(3) transition but reappeared at slightly upshifted frequencies with the opposite sign upon the S(3)-to-S(0) transition. No obvious isotopic band appeared upon the S(0)-to-S(1) transition. These results indicate that the alpha-carboxylate of C-terminal Ala-344 is structurally associated with a manganese ion that becomes oxidized upon the S(1)-to-S(2) transition and reduced reversely upon the S(3)-to-S(0) transition but is not associated with manganese ion(s) oxidized during the S(0)-to-S(1) (and S(2)-to-S(3)) transition(s). Consistently, l-[1-(13)C]alanine labeling also induced spectral changes in the low frequency (670-350 cm(-1)) S(2)/S(1) FTIR difference spectrum.     

Studies on photosynthetic oxygen-evolving complex by means of Fourier transform infrared spectroscopy: calcium and chloride cofactors

Structural roles of functional Ca²⁺ and Cl⁻ ions in photosynthetic oxygen-evolving complexes (OEC) were studied using low- (640–350 cm⁻¹) and mid- (1800–1200 cm⁻¹) frequency S₂/S₁ Fourier transform infrared (FTIR) difference spectroscopy. Studies using highly active Photosystem (PS) II core particles from spinach enabled the detection of subtle spectral changes. Ca²⁺-depleted and Ca²⁺-reconstituted particles produced very similar mid- and low-frequency spectra. The mid-frequency spectrum was not affected by reconstitution with ⁴⁴Ca isotope. In contrast, Sr²⁺-substituted particles showed unique spectral changes in the low-frequency Mn–O–Mn mode at 606 cm⁻¹ as well as in the mid-frequency carboxylate stretching modes. The mid-frequency spectrum of Cl⁻-depleted OEC exhibited marked changes in the carboxylate stretching modes and the suppression of protein modes compared with that of Cl⁻-reconstituted OEC. However, Cl⁻-depletion did not exert significant effects on the low-frequency spectrum.     

Mechanism of photosynthetic water oxidation in the dimeric oxygen-evolving complex of chloroplast photosystem II

Based on the analysis of the molecular organization and properties of an isolated oxygen-evolving complex of photosystem II of plant chloroplasts, a mechanism of water oxidation and oxygen release during photosynthesis was proposed. It is suggested that the photolysis of water occurs in a dimeric oxygen-evolving complex consisting of two core complexes. In the region of contact of these complexes, a hydrophobic "boiler" is formed where the conditions for screening and stabilization of Z-linanded manganese cations accumulating positive charges for the oxidation of water molecules are created. A prerequisite to the photolysis of water is the formation of a binuclear [Mn(3+)-OH ... HO-Mn3+] hydroxyl-manganese associate, which appears in the dimeric oxygen-evolving complex after the first two light flashes as a result of photohydrolysis of photochemically oxidized Z-liganded manganese cations. The process is accompanied by the release of the first water protons to the medium. The photosynthetic oxidation of water hydroxyls occurs at the next stage and is considered as synchronous detachment of four electrons from two bound OH-groups of the associate upon photooxidation of Mn3+ cations to Mn4+ cations after two subsequent light flashes. This process is accompanied by the disproportionation of electron density and the formation of a bond between oxygen atoms of hydroxyls followed by the evolution of molecular oxygen and protons, and regeneration of two starting Mn2+ cations and the primary state of the system.     

Crystal structure of yeast Sis1 peptide-binding fragment and Hsp70 Ssa1 C-terminal complex

Heat shock protein (Hsp) 40 facilitates the critical role of Hsp70 in a number of cellular processes such as protein folding, assembly, degradation and translocation in vivo. Hsp40 and Hsp70 stay in close contact to achieve these diverse functions. The conserved C-terminal EEVD motif in Hsp70 has been shown to regulate Hsp40-Hsp70 interaction by an unknown mechanism. Here, we provide a structural basis for this regulation by determining the crystal structure of yeast Hsp40 Sis1 peptide-binding fragment complexed with the Hsp70 Ssa1 C-terminal. The Ssa1 extreme C-terminal eight residues, G634PTVEEVD641, form a beta-strand with the domain I of Sis1 peptide-binding fragment. Surprisingly, the Ssa1 C-terminal binds Sis1 at the site where Sis1 interacts with the non-native polypeptides. The negatively charged residues within the EEVD motif in Ssa1 C-terminal form extensive charge-charge interactions with the positively charged residues in Sis1. The structure-based mutagenesis data support the structural observations.     

FTIR detection of structural changes in a histidine ligand during S-state cycling of photosynthetic oxygen-evolving complex

Changes in structural coupling between the Mn cluster and a putative histidine ligand during the S-state cycling of the oxygen-evolving complex (OEC) have been detected directly by Fourier transform infrared (FTIR) spectroscopy in photosystem (PS) II core particles from the cyanobacterium Synechocystis sp. PCC6803, in which histidine residues were selectively labeled with l-[(15)N(3)]histidine. The bands sensitive to the histidine-specific isotope labeling appeared at 1120-1090 cm(-)(1) in the spectra induced upon the first-, second-, and fourth-flash illumination, for the S(2)/S(1), S(3)/S(2), and S(1)/S(0) differences, at similar frequencies with different sign and/or intensity depending on the respective S-state transitions. However, no distinctive band was observed in the third-flash induced spectrum for the S(0)/S(3) difference. The results indicate that a single histidine residue coupled with the structural changes of the OEC during the S-state cycling is responsible for the observed histidine bands, in which the histidine modes changed during the S(0)-to-S(1) transition are reversed upon the S(1)-to-S(2) and S(2)-to-S(3) transitions. The 1186(+)/1178(-) cm(-)(1) bands affected by l-[(15)N(3)]histidine labeling were observed only for the S(2)/S(1) difference, but those affected by universal (15)N labeling appeared prominently showing a clear S-state dependency. Possible origins of these bands and changes in the histidine modes during the S-state cycling are discussed.     

Solution structure of the RIM1alpha PDZ domain in complex with an ELKS1b C-terminal peptide

PDZ domains are widespread protein modules that commonly recognize C-terminal sequences of target proteins and help to organize macromolecular signaling complexes. These sequences usually bind in an extended conformation to relatively shallow grooves formed between a beta-strand and an alpha-helix in the corresponding PDZ domains. Because of this binding mode, many PDZ domains recognize primarily the C-terminal and the antepenultimate side-chains of the target protein, which commonly conform to motifs that have been categorized into different classes. However, an increasing number of PDZ domains have been found to exhibit unusual specificities. These include the PDZ domain of RIMs, which are large multidomain proteins that regulate neurotransmitter release and help to organize presynaptic active zones. The RIM PDZ domain binds to the C-terminal sequence of ELKS with a unique specificity that involves each of the four ELKS C-terminal residues. To elucidate the structural basis for this specificity, we have determined the 3D structure in solution of an RIM/ELKS C-terminal peptide complex using NMR spectroscopy. The structure shows that the RIM PDZ domain contains an unusually deep and narrow peptide-binding groove with an exquisite shape complementarity to the four ELKS C-terminal residues in their bound conformation. This groove is formed, in part, by a set of side-chains that is conserved selectively in RIM PDZ domains and that hence determines, at least in part, their unique specificity.     

Relationship between local structure and stability in hen egg white lysozyme mutant with alanine substituted for glycine

We prepared five mutant lysozymes in which glycines whose dihedral angles are located in the region of the left-handed helix, Gly49, Gly67, Gly71, Gly102 and Gly117, were mutated to an alanine residue. From analyses of their thermal stabilities using differential scanning calorimetry, most of them were more destabilized than the native lysozyme, except for the G102A mutant, which has a stability similar to that of the native lysozyme at pH 2.7. As for the destabilized mutant lysozymes, their X-ray crystallographic analyses showed that their global structures did not change but that the local structures changed slightly. By examining the dihedral angles at the mutation sites based on X-ray crystallographic results, it was found that the dihedral angles at these mutation sites tended to adopt favorable values in a Ramachandran plot and that the extent and direction of their shifts from the original value had similar tendencies. Therefore, the change in dihedral angles may be the cause of the slight local structural changes around the mutation site. On the other hand, regarding the mutation of G102A, the global structure was almost identical with that of the native structure but the local structure was drastically changed. Therefore, it was suggested that the drastic local conformational change might be effective in releasing the unfavorable interaction of the native state at the mutation site.     

Changes in structural and functional properties of oxygen-evolving complex induced by replacement of D1-glutamate 189 with glutamine in photosystem II: ligation of glutamate 189 carboxylate to the manganese cluster

A carboxylate group of D1-Glu-189 in photosystem II has been proposed to serve as a direct ligand for the manganese cluster. Here we constructed a mutant that eliminates the carboxylate by replacing D1-Glu-189 with Gln in the cyanobacterium Synechocystis sp. PCC 6803, and we examined the resulting effects on the structural and functional properties of the oxygen-evolving complex (OEC) in photosystem II. The E189Q mutant grew photoautotrophically, and isolated photosystem II core particles evolved oxygen at approximately 70% of the rate of control wild-type particles. The E189Q OEC showed typical S(2) state electron spin resonance signals, and the spin center distance between the S(2) state manganese cluster and the Y(D) (D2-Tyr-160), detected by electron-electron double resonance spectroscopy, was not affected by this mutation. However, the redox potential of the E189Q OEC was considerably lower than that of the control OEC, as revealed by the elevated peak temperature of the S(2) state thermoluminescence bands. The mutation resulted in specific changes to bands ascribed to the putative carboxylate ligands for the manganese cluster and to a few carbonyl bands in mid-frequency (1800 to 1100 cm(-1)) S(2)/S(1) Fourier transform infrared difference spectrum. Notably, the low frequency (650 to 350 cm(-1)) S(2)/S(1) Fourier transform infrared difference spectrum was also uniquely changed by this mutation in the frequencies for the manganese cluster core vibrations. These results suggested that the carboxylate group of D1-Glu-189 ligates the manganese ion, which is influenced by the redox change of the oxidizable manganese ion upon the S(1) to S(2) transition.     

Analysis of tritium-labeled glycine and alanine using 3H-NMR

A method for analysis of three-component isotopic mixtures of tritium-labelled glycine and alanine in D2O solutions has been developed on the basis of high resolution 3H NMR spectra at 266.8 MHz. Determined were composition of the mixtures in molar per cent, as well as geminal and vicinal coupling constants (2JGly3H,H = -16.4 +/- 0.2 Hz; 2JAla3H,H = -14.0 +/- 0.5 Hz; 3JAla3H,H = 7.6 +/- 0.2 Hz) and isotopic shifts (0.21 +/- 0.001 ppm for glycine; 0.026 +/- 0.001 ppm for alanine).     

Genetic replacement of cyclin D1 function in mouse development by cyclin D2

D cyclins (D1, D2, and D3) are components of the core cell cycle machinery in mammalian cells. It is unclear whether each of the D cyclins performs unique, tissue-specific functions or the three proteins have virtually identical functions and differ mainly in their pattern of expression. We previously generated mice lacking cyclin D1, and we observed that these animals displayed hypoplastic retinas and underdeveloped mammary glands and a presented developmental neurological abnormality. We now asked whether the specific requirement for cyclin D1 in these tissues reflected a unique pattern of D cyclin expression or the presence of specialized functions for cyclin D1 in cyclin D1-dependent compartments. We generated a knock-in strain of mice expressing cyclin D2 in place of D1. Cyclin D2 was able to drive nearly normal development of retinas and mammary glands, and it partially replaced cyclin D1's function in neurological development. We conclude that the differences between these two D cyclins lie mostly in the tissue-specific pattern of their expression. However, we propose that subtle differences between the two D cyclins do exist and they may allow D cyclins to function in a highly optimized fashion. We reason that the acquisition of multiple D cyclins may allow mammalian cells to drive optimal proliferation of a diverse array of cell types.