固氮酶晶体学研究进展

生物大分子的功能取决于它的空间结构.X射线衍射分析是获得生物大分子结构信息的常用方法.本文对固氮酶晶体的生长及其X射线衍射分析的主要进展简要地进行了介绍和评论.最后展望了今后的发展及问题.     

蛋白质分子的构象运动及其功能意义

 <正> 自从五十年代末成功地运用X射线衍射分析技术测定肌红蛋白分子的晶体结构以来,我们关于蛋白质在三维水平的结构及其与功能关系的认识,已经历了两个阶段。第一个阶段包括60年代初至70年代中期。在这一期间,解决了蛋白质晶体结构测定方法的一般化问题,使以同晶置换法为主的一套X射线衍射分析技术成为研究生物大分子三维结构的成熟手段,给     

转移核糖核酸的三维结构

核酸类物质的晶体结构的研究,迄今只有酵母苯丙氨酸转移核糖核酸(tRNA~(Phe))获得了原子水平的结果。这是利用X射线衍射晶体结构分析技术完成的。1968年,5—6个实验室宣称拿到了可供X射线衍射用的单晶。实际上,那种晶体有序程度很差,衍射分辨率很低,     

应用于生物科学研究的一门新技术——中子散射分析技术

近十年来,中子散射分析技术已经发展成为研究物质结构的重要方法,最近又把这个方法应用到生物科学研究中去。研究生物的细微结构,一般采用电子显微镜和X射线衍射技术。应用电子显微镜,来检查细胞、细胞的组成部分。用X射线衍射技术来研究蛋白质等生物大分子内的原子空间排列。可是在这两个范畴之间,尚有一个空隙:细胞的某些部分,用电子显微镜检测嫌小,用X射线衍射研究原子细节嫌大。而这个中间水平的结构资料,却又往往非常重要。象核糖体就是一个例子。核糖体是一切细胞里都有的、非常重要的细胞器,它由55个蛋白质分子和三个RNA分子所组成。如果不首先了解这些蛋白质、RNA分子是如何进行装配的,我们几乎无法了解核糖体在蛋白质合成中如何执行其任务。可是核糖体在电子显微镜图象中,只能是一个轮廓,从这些图象中获取的结构资料是     

X射线衍射法在研究蛋白质动态过程中的应用

介绍了X射线衍射技术在研究蛋白质动态过程中的应用.首先介绍了用常规X射线衍射法和劳埃X射线衍射法等数据采样法研究反应时间为几分钟的蛋白质催化反应.然后介绍了通过选择不匹配底物,不适宜酸度,选择温度和酸度的跳跃,金属和光化学瞬时激发达到反应的同步来研究反应时间为几秒钟的蛋白质催化反应.     

X射线辐射引起的apo-GAPDH酶晶体衍射谱的反常变化

apo-GAPDH晶体X射线衍射实验发现,有些衍射谱的强度随着X射线照射时间增加而出现反常变化.联系蛋白质构象的可变性做了初步探讨.     

生物学研究中利用强X射线源的衍射技术

X射线衍射技术应用于生物学研究,六十年代主要是采用固定靶或旋转靶X射线源,研究蛋白质和核酸等的静态晶体结构,以从分子解剖学的角度认识有关的生命现象。但是,这种技术方法对于较复杂的生物体系和生命现象中发生在分予水平的瞬时动态过程,诸如酶促反应巾酶分子结构变化与功能的关系,肌肉收缩过程巾肌动蛋白和肌球蛋白的变化规律等分了生理学变化,却无从观祭。因此,近年来开始利用电子同步加速器的强X射线作为衍射光源,来研究肌肉运动和酶反应等复杂体系的动态过程。目前,国际上已建立了若干可供生物学研究应用的电子同步加速器强X射线源。     

结构基因组学中的衍射相位问题

简要介绍了结构基因组学研究中,用于测定蛋白质结构的X射线分析在解决衍射相位问题方面的最新进展。     

微生物处理对秸秆结构的影响

使用红外光谱、X射线衍射及扫描电镜对4种经固态培养的微生物降解稻草秸秆的效果进行了研究,并与原稻草粉进行结构对比分析。结果表明：经过高碳低氮培养,秸秆木质素均有明显降解,其中黄孢原毛平革菌达到37.17%。扫描电镜发现秸秆的表面形态和内部结构发生了明显改变,红外光谱中芳香环与醚键吸收明显减少,X射线衍射得到的结晶度最大下降了5.60%。     

古人类骨中羟磷灰石的XRD和喇曼光谱分析

人骨残骸是生物考古的主要对象,而骨骼污染鉴别是样品选择的依据,也是生物考古的前提。利用X射线衍射（XRD）和喇曼光谱相结合的方法,通过对新疆克雅河圆沙古城遗传出土的人类骨骼中羟磷灰石的分析,来辨析骨骼污染程度。研究结果表明,两种方法的有机结合,准确反映了骨骼中羟磷石结晶度的变化,从而可简单、较为有效地鉴别古代人类骨骼样品的污染。     

Insights into nucleotide signal transduction in nitrogenase: structure of an iron protein with MgADP bound

Coupling the energy of nucleoside triphosphate binding and hydrolysis to conformational changes is a common mechanism for a number of proteins with disparate cellular functions, including those involved in DNA replication, protein synthesis, and cell differentiation. Unique to this class of proteins is the dimeric Fe protein component of nitrogenase in which the binding and hydrolysis of MgATP controls intermolecular electron transfer and reduction of nitrogen to ammonia. In the work presented here, the MgADP-bound (or "off") conformational state of the nitrogenase Fe protein has been captured and a 2.15 A resolution X-ray crystal structure is presented. The structure described herein reveals likely mechanisms for long-range communication from the nucleotide-binding sites for controlling the affinity of association with the MoFe protein component. Two pathways, termed switches I and II, appear to be integral to this nucleotide signal transduction mechanism. In addition, the structure provides the basis for the changes in the biophysical properties of the [4Fe-4S] cluster observed when Fe protein binds nucleotides. The structure of the MgADP-bound Fe protein provides important insights into the respective contributions of nucleotide interaction and complex formation in defining the conformational states that are the keys to nitrogenase catalysis.     

Probing the MgATP-bound conformation of the nitrogenase Fe protein by solution small-angle X-ray scattering

The MgATP-bound conformation of the Fe protein of nitrogenase from Azotobacter vinelandii has been examined in solution by small-angle X-ray scattering (SAXS) and compared to existing crystallographically characterized Fe protein conformations. The results of the analysis of the crystal structure of an Fe protein variant with a Switch II single-amino acid deletion recently suggested that the MgATP-bound state of the Fe protein may exist in a conformation that involves a large-scale reorientation of the dimer subunits, resulting in an overall elongated structure relative to the more compact structure of the MgADP-bound state. It was hypothesized that the Fe protein variant may be a conformational mimic of the MgATP-bound state of the native Fe protein largely on the basis of the observation that the spectroscopic properties of the [4Fe-4S] cluster of the variant mimicked in part the spectroscopic signatures of the native nitrogenase Fe protein in the MgATP-bound state. In this work, SAXS studies reveal that the large-scale conformational differences between the native Fe protein and the variant observed by X-ray crystallography are also observed in solution. In addition, comparison of the SAXS curves of the Fe protein nucleotide-bound states to the nucleotide-free states indicates that the conformation of the MgATP-bound state in solution does not resemble the structure of the variant as initially proposed, but rather, at the resolution of this experiment, it resembles the structure of the nucleotide-free state. These results provide insights into the Fe protein conformations that define the role of MgATP in nitrogenase catalysis.     

Inhibition of nitrogenase activity by ammonium chloride in Azotobacter vinelandii.

In Azotobacter vinelandii cells, the short-term inhibition of nitrogenase activity by NH4Cl was found to depend on several factors. The first factor is the dissolved oxygen concentration during the assay of nitrogenase. When cells are incubated with low concentrations of oxygen, nitrogenase activity is low and ammonia inhibits strongly. With more oxygen, nitrogenase activity increases. Cells incubated with an optimum amount of oxygen have maximum nitrogenase activity, and the extent of inhibition by ammonia is small. With higher amounts of oxygen, the nitrogenase activity of the cells is decreased and strongly inhibited by ammonia. The second factor found to be important for the inhibition of nitrogenase activity by NH4Cl was the pH of the medium. At a low pH, NH4+ inhibits more strongly than at a higher pH. The third factor that influenced the extent of ammonia inhibition was the respiration rate of the cells. When cells are grown with excess oxygen, the respiration rate of the cells is high and inhibition of nitrogenase activity by ammonia is small. Cells grown under oxygen-limited conditions have a low respiration rate and NH4Cl inhibition of nitrogenase activity is strong. Our results explain the contradictory reports described in the literature for the NH4Cl inhibition of nitrogenase in A. vinelandii.     

Formation and characterization of a transition state complex of Azotobacter vinelandii nitrogenase

A stable complex is formed between the nitrogenase proteins of Azotobacter vinelandii, aluminium fluoride and MgADP. All nitrogenase activities are inhibited. The complex formation was found to be reversible. An incubation at 50°C recovers nitrogenase activity. The complex has been characterized with respect to protein and nucleotide composition and redox state of the metal-sulphur clusters. Based on the inhibition by aluminium fluoride together with MgADP, it is proposed that a stable transition state complex of nitrogenase is isolated.     

Mo-independent nitrogenase 3 is advantageous for diazotrophic growth of Azotobacter vinelandii on solid medium containing molybdenum.

Competition experiments between wild-type Azotobacter vinelandii and a mutant lacking Mo-independent nitrogenase 3 indicate that nitrogenase 3 provides an advantage during diazotrophic growth on agar media containing 100 to 500 nM Na2MoO4 but not in liquid media under the same conditions. Expression of nitrogenase 3 in wild-type cells growing on agar surfaces was verified with an anfH-lacZ fusion and by detection of nitrogenase 3 subunits. These results show that nitrogenase 3 is important for diazotrophic growth on agar medium at molybdenum concentrations that are not limiting for Mo-dependent diazotrophic growth in liquid medium.     

New insights into structure-function relationships in nitrogenase: A 1.6 A resolution X-ray crystallographic study of Klebsiella pneumoniae MoFe-protein.

The X-ray crystal structure of Klebsiella pneumoniae nitrogenase component 1 (Kp1) has been determined and refined to a resolution of 1.6 A, the highest resolution reported for any nitrogenase structure. Models derived from three 1.6 A resolution X-ray data sets are described; two represent distinct oxidation states, whilst the third appears to be a mixture of both oxidized and reduced states (or perhaps an intermediate state). The structures of the protein and the iron-molybdenum cofactor (FeMoco) appear to be largely unaffected by the redox status, although the movement of Ser beta90 and a surface helix in the beta subunit may be of functional significance. By contrast, the 8Fe-7S P-cluster undergoes discrete conformational changes involving the movement of two iron atoms. Comparisons with known component 1 structures reveal subtle differences in the FeMoco environment, which could account for the lower midpoint potential of this cluster in Kp1. Furthermore, a non-proline- cis peptide bond has been identified in the alpha subunit that may have a functional role. It is within 10 A of the FeMoco and may have been overlooked in other component 1 models. Finally, metal-metal and metal-sulphur distances within the metal clusters agree well with values derived from EXAFS studies, although they are generally longer than the values reported for the closely related protein from Azotobacter vinelandii. A number of bonds between the clusters and their ligands are distinctly longer than the EXAFS values, in particular, those involving the molybdenum atom of the FeMoco.     

The activity of two forms of nitrogenase from Rhodopseudomonas capsulata in the presence of different electron donors

Abstract The active form of Rhodopseudomonas capsulata nitrogenase is active in vitro when dithionite or ferredoxins from this bacterium are used as electron donors. The presence of the activating nitrogenase enzyme and Mn²⁺ ions is needed for functioning of the inactive form of Rh. capsulata nitrogenase in vitro with the use of dithionite as an electron donor. The use of Rh. capsulata ferredoxins as electron donors in vitro makes the inactive form of nitrogenase fully active as is the case in vivo.     

Effect of light intensity and inhibitors of nitrogen assimilation on NH4+ inhibition of nitrogenase activity in Rhodospirillum rubrum and Anabaena sp

Nitrogenase activity in Rhodospirillum rubrum was inhibited by NH4+ more rapidly in low light than in high light. Furthermore, the nitrogenase of cells exposed to phosphorylation uncouplers was inhibited by NH4+ more rapidly than was the nitrogenase of controls without an uncoupler. These observations suggest that high levels of photosynthate inhibit the nitrogenase inactivation system. L-Methionine-DL-sulfoximine, a glutamine synthetase inhibitor, prevented NH4+ from inhibiting nitrogenase activity, which suggests that NH4+ must be processed at least to glutamine for inhibition to occur. An inhibitor of glutamate synthase activity, 6-diazo-5-oxo-L-norleucine, inhibited nitrogenase activity in the absence of NH4+, but only in cells exposed to low light. The mechanism of 6-diazo-5-oxo-L-norleucine inhibition appeared to be the same as that induced by NH4+, because nitrogenase activity could be restored in vitro by activating enzyme and Mn2+. The inhibitor data suggest that the glutamine pool or a molecule that responds to it activates the Fe protein-modifying (or protein-inactivating) system and that the accumulation of this (unidentified) molecule is retarded when the cells are exposed to high light. It was confirmed here that Anabaena nitrogenase is also inhibited by NH4+, but only when the cells are incubated under low light. This inhibition, however, unlike that in R. rubrum, could be completely reversed in high light, suggesting that the mechanisms of nitrogenase inhibition by NH4+ in these two phototrophs are different.