POTRA Domains,Extracellular Lid,and Membrane Composition Modulate the Conformational Stability of the β Barrel Assembly Factor BamA

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High-Resolution Cryoelectron Microscopy Structure of the Cyclic Nucleotide-Modulated Potassium Channel MloK1 in a Lipid Bilayer

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Time-resolved step-scan FTIR difference spectroscopy for the study of photosystem I with different benzoquinones incorporated into the A1 binding site

Time-resolved step-scan FTIR difference spectroscopy has been used to study photosystem I (PSI) with plastoquinone-9 (PQ) and two other benzoquinones (2,6-dimethyl-1,4-benzoquinone and 2,3,5,6-tetrachloro-1,4-benzoquinone) incorporated into the A₁ binding site. By subtracting a (P700⁺A₁^????P700A₁) FTIR difference spectrum for PSI with the native phylloquinone (PhQ) incorporated from corresponding spectra for PSI with different benzoquinones (BQs) incorporated, FTIR double difference spectra are produced, that display bands associated with vibrational modes of the quinones, without interference from features associated with protein vibrational modes.Molecular models for BQs involved in asymmetric hydrogen bonding were constructed and used in vibrational mode frequency calculations. The calculated data were used to aid in the interpretation and assignment of bands in the experimental spectra. We show that the calculations capture the general trends found in the experimental spectra.By comparing four different FTIR double difference spectra we are able to verify unambiguously bands associated with phyllosemiquinone in PSI at 1495 and 1415?cm^?1. We also resolve a previously unrecognized band of phyllosemiquinone at 1476?cm^?1 that calculations suggest is due in part to a C₄$\stackrel{?}{?}$O stretching mode.For PSI with PQ incorporated, calculations and experiment taken together indicate that the C₁$\stackrel{?}{?}$O and C₄$\stackrel{?}{?}$O vibrational modes of the semiquinone give rise to bands at 1487 and 1444?cm^?1, respectively. This is very distinct compared to PSI with PhQ incorporated.From the calculated and experimental spectra, we show that it is possible to distinguish between two possible orientations of PQ in the A₁ protein binding site.     

Roles of the F-domain in [FeFe] hydrogenase

The role of accessory Fe-S clusters of the F-domain in the catalytic activity of M3-type [FeFe] hydrogenase and the contribution of each of the two Fe-S surface clusters in the intermolecular electron transfer from ferredoxin are both poorly understood. We designed, constructed, produced and spectroscopically, electrochemically and biochemically characterized three mutants of Clostridium acetobutylicum CaHydA hydrogenase with modified Fe-S clusters: two site-directed mutants, HydA_C100A and HydA_C48A missing the FS4C and the FS2 surface Fe-S clusters, respectively, and a HydA_ΔDA mutant that completely lacks the F-domain. Analysis of the mutant enzyme activities clearly demonstrated the importance of accessory clusters in retaining full enzyme activity at potentials around and higher than the equilibrium 2H⁺/H₂ potential but not at the lowest potentials, where all enzymes have a similar turnover rate. Moreover, our results, combined with molecular modelling approaches, indicated that the FS2 cluster is the main gate for electron transfer from reduced ferredoxin.     

Mixed Domains Enhance Charge Generation and Extraction in Bulk‐Heterojunction Solar Cells with Small‐Molecule Donors

The interplay between nanomorphology and efficiency of polymer‐fullerene bulk‐heterojunction (BHJ) solar cells has been the subject of intense research, but the generality of these concepts for small‐molecule (SM) BHJs remains unclear. Here, the relation between performance; charge generation, recombination, and extraction dynamics; and nanomorphology achievable with two SM donors benzo[1,2‐b:4,5‐b]dithiophene‐pyrido[3,4‐b]‐pyrazine BDT(PPTh₂)₂, namely SM1 and SM2, differing by their side‐chains, are examined as a function of solution additive composition. The results show that the additive 1,8‐diiodooctane acts as a plasticizer in the blends, increases domain size, and promotes ordering/crystallinity. Surprisingly, the system with high domain purity (SM1) exhibits both poor exciton harvesting and severe charge trapping, alleviated only slightly with increased crystallinity. In contrast, the system consisting of mixed domains and lower crystallinity (SM2) shows both excellent exciton harvesting and low charge recombination losses. Importantly, the onset of large, pure crystallites in the latter (SM2) system reduces efficiency, pointing to possible differences in the ideal morphologies for SM‐based BHJ solar cells compared with polymer‐fullerene devices. In polymer‐based systems, tie chains between pure polymer crystals establish a continuous charge transport network, whereas SM‐based active layers may in some cases require mixed domains that enable both aggregation and charge percolation to the electrodes.     

Quantification of excitation energy distribution between photosystems based on a mechanistic model of photosynthetic electron transport

Absorbed light energy is converted into excitation energy. The excitation energy is distributed to photosystems depending on the wavelength and drives photochemical reactions. A non‐destructive, mechanistic and quantitative method for estimating the fraction of the excitation energy distributed to photosystem II (f) was developed. For the f values for two simultaneously provided actinic lights (ALs) with different spectral distributions to be estimated, photochemical yields of the photosystems were measured under the ALs and were then fitted to an electron transport model assuming the balance between the electron transport rates through the photosystems. For the method to be tested using leaves with different properties in terms of the long‐term and short‐term acclimation (adjustment of photosystem stoichiometry and state transition, respectively), the f values for red and far‐red light (R and FR) were estimated in leaves grown (~1 week) under white light without and with supplemental FR and adapted (~10 min) to R without and with supplemental FR. The f values for R were clearly greater than those for FR and those of leaves grown with and adapted to supplemental FR tended to be higher than the controls. These results are consistent with previous studies and therefore support the validity of the proposed method.     

Phosphopeptide binding to the N-terminal SH2 domain of the p85 alpha subunit of PI 3'-kinase: a heteronuclear NMR study.

The N-terminal src-homology 2 domain of the p85 alpha subunit of phosphatidylinositol 3' kinase (SH2-N) binds specifically to phosphotyrosine-containing sequences. Notably, it recognizes phosphorylated Tyr 751 within the kinase insert of the cytoplasmic domain of the activated beta PDGF receptor. A titration of a synthetic 12-residue phosphopeptide (ESVDY*VPMLDMK) into a solution of the SH2-N domain was monitored using heteronuclear 2D and 3D NMR spectroscopy. 2D-(15N-1H) heteronuclear single-quantum correlation (HSQC) experiments were performed at each point of the titration to follow changes in both 15N and 1H chemical shifts in NH groups. When mapped onto the solution structure of the SH2-N domain, these changes indicate a peptide-binding surface on the protein. Line shape analysis of 1D profiles of individual (15N-1H)-HSQC peaks at each point of the titration suggests a kinetic exchange model involving at least 2 steps. To characterize changes in the internal dynamics of the domain, the magnitude of the (15N-1H) heteronuclear NOE for the backbone amide of each residue was determined for the SH2-N domain with and without bound peptide. These data indicate that, on a nanosecond timescale, there is no significant change in the mobility of either loops or regions of secondary structure. A mode of peptide binding that involves little conformational change except in the residues directly involved in the 2 binding pockets of the p85 alpha SH2-N domain is suggested by this study.     

Heparin binding domain peptides of antithrombin III: analysis by isothermal titration calorimetry and circular dichroism spectroscopy.

The serine proteinase inhibitor antithrombin III (ATIII) is a key regulatory protein of intrinsic blood coagulation. ATIII attains its full biological activity only upon binding polysulfated oligosaccharides, such as heparin. A series of synthetic peptides have been prepared based on the proposed heparin binding regions of ATIII and their ability to bind heparin has been assessed by CD spectrometry, by isothermal titration calorimetry, and by the ability of the peptides to compete with ATIII for binding heparin in a factor Xa procoagulant enzyme assay. Peptide F123-G148, which encompasses both the purported high-affinity pentasaccharide binding region and an adjacent, C-terminally directed segment of ATIII, was found to bind heparin with good affinity, but amino-terminal truncations of this sequence, including L130-G148 and K136-G148 displayed attenuated heparin binding activities. In fact, K136-G148 appears to encompass only a low-affinity heparin binding site. In contrast, peptides based solely on the high-affinity binding site (K121-A134) displayed much higher affinities for heparin. By CD spectrometry, these high-affinity peptides are chiefly random coil in nature, but low microM concentrations of heparin induce significant alpha-helix conformation. K121-A134 also effectively competes with ATIII for binding heparin. Thus, through the use of synthetic peptides that encompass part, if not all, of the heparin binding site(s) within ATIII, we have further elucidated the structure-function relations of heparin-ATIII interactions.     

基因表达调控中的核因子作用

利用病毒和动物系统对基因表达调控进行了广泛和深入的研究,发现了顺式作用调节序列,鉴定了序列专一的DNA结合蛋白,DNA与蛋白质相互识别、结合及蛋白质与蛋白质相互作用中起作用的蛋白质结构域,并且对调节蛋白基因的克隆和序列进行了分析．基因表达调控领域又由于植物基因调控机制取得的发展而得到了补充,文章着重介绍植物基因中的DNA与蛋白质间的作用;植物调节蛋白基因的分离;这一领域的今后研究方向及展望．