THERMODYNAMIC AND KINETIC STUDIES OF PROTEIN STRUCTURE AND ENZYMIC MECHANISMS

蛋白质结构和酶机制的热力学和动力学研究

• 批准号: 2572897
• 负责人: P MC PHIE
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2572897
• 关键词: amphotericin B; chemical kinetics; circular dichroism; conformation; enzyme activity; enzyme mechanism; high performance liquid chromatography; histidine; protein folding; protein sequence; protein structure; sulfotransferase; thermodynamics; tryptophan synthase

This laboratory is interested in the relationship between protein sequence, structure and the mechanisms of protein folding and enzymatic reactions. Histidine Rich Proteins - The malaria parasite, Plasmodium falciparum, produces large amounts of several histidine rich proteins of unknown structure. Their high concentrations of histidine distort their circular dichroic spectra, so that, unlike those of normal proteins they cannot be interpreted in terms of secondary structures. We are reinvestigating the behavior of poly L-histidine as a function of pH and solvent additives, using modern methods of spectral deconvolution and analysis. We are establishing a set of reference spectra under defined conditions, which can be used in analysis of the structures of these unusual proteins. Structure of Tryptophan Synthase - (with S. Ashraf Ahmed and Edith Wilson Miles, LBP) We are investigating the conformational states of the tryptophan synthase alpha2/beta2 complex and of the separate alpha and beta subunits. We find that mutations, cosolvents and cations alter the structure and function. Our analysis of the effects of thermally induced unfolding of the alpha2/beta2 complex on the UV-visible spectra and activity show the presence of an inactive, partially unfolded intermediate. Structure of Mammalian Sulphotransferases. (with D. Marshall and W.B.Jakoby, LBM) During preparation of a cloned phenol sulphotransferase, two distinct forms of the enzyme were isolated. One form (B) behaved like enzyme previously isolated from rat liver; the second form (A) showed reduced activity in the sulphation reaction and was able to catalyze the transfer of sulphate without added cofactor, PAP (adenosine 3', 5' bisphosphate). Forms A and B can be interconverted by treatment with oxidized/reduced glutathione, DTNB or mercaptoethanol. This indicates the involvement of one or more disulphide bonds at the active site of the enzyme. Peptide mapping and HPLC are being used to locate these bond(s). Interaction of Amphotericin B with Membranes. (with R. A. Brutyan, LBM) The antifungal drug Amphotericin B undergoes a large enhancement in its CD spectrum on binding to membranes. It's activity is inhibited by the organic cation TEA, but the continued presence of optical activity rules out the possibility of dissociation as an explanation of this inhibition.

这个实验室对蛋白质之间的关系很感兴趣。 蛋白质折叠和折叠的序列、结构和机制 酶促反应。 富含组氨酸的蛋白质-疟疾寄生虫，恶性疟原虫， 产生大量的富含组氨酸的未知蛋白质 结构。他们高浓度的组氨酸扭曲了他们的 圆二色光谱，因此，与正常蛋白质的光谱不同 它们不能用二级结构来解释。我们是 聚合L组氨酸与pH关系的再研究 和溶剂添加剂，使用现代光谱去卷积方法 和分析。我们正在建立一套以下的参考光谱 定义的条件，可用于分析的结构 这些不寻常的蛋白质。色氨酸合成酶的结构-(与S. 艾哈迈德和伊迪丝·威尔逊·迈尔斯，LBP)我们正在调查 色氨酸合成酶α2/β2复合体的构象状态 以及不同的α和β亚基。我们发现突变， 助溶剂和阳离子改变了结构和功能。我们的分析 热诱导的α2/β2去折叠的影响 络合物的紫外-可见光谱和活性表明存在一个 不活跃，部分展开的中间体。 哺乳动物硫转移酶的结构。(与D.马歇尔和 W.B.Jakoby，LBM)在制备克隆苯酚的过程中 分离得到了两种不同形态的硫酸盐转移酶。 一种形式(B)表现出以前从大鼠肝脏中分离出的酶的行为； 第二种形式(A)在硫化反应中的活性降低 并能够在不添加任何添加剂的情况下催化硫酸盐的转移 辅因子，PAP(腺苷3‘，5’二磷酸)。表格A和B可以是 通过氧化/还原谷胱甘肽、DTNB或 硫醇。这表明有一个或多个 二硫键位于酶的活性部位。多肽图谱 和高效液相色谱用于确定这些键的位置(S)。 两性霉素B与膜的相互作用(与R.A.Brutyan一起， LBM)抗真菌药物两性霉素B经历了很大的增强 在其CD光谱中关于与膜的结合。它的活性被抑制了 受有机阳离子茶的影响，但光学的持续存在 活动排除了解离的可能性作为一种解释 这种抑制。