MACROMOLECULAR STRUCTURE

大分子结构

• 批准号: 3810736
• 负责人: A C STEVEN
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN 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/3810736
• 关键词: Herpesviridae; Raman spectrometry; X ray crystallography; bacteriophage T4; carbohydrate structure; conformation; crosslink; cytoskeletal proteins; cytoskeleton; electron microscopy; fibrous protein; gel electrophoresis; glycoprotein structure; human immunodeficiency virus 1; image processing; immunocytochemistry; keratin; laboratory mouse; macromolecule; molecular weight; newborn animals; protein folding; protein sequence; protein structure; scanning electron microscopy; virus envelope; virus protein

The structures and mechanisms of assembly of biological macromolecular, complexes, and subcellular structures are studied, primarily by high-resolution electron microscopy and computer image processing. Two projects completed during the past year concern (1) characterization of a major conformational change in a viral capsid protein that occurs when the precursor capsid is transformed into the mature virion; and (2) determination of the molecular weights, oligomeric status, carbohydrate contents, and two-dimensional mass-map of the gp160/gp120 surface glycoprotein of human immunodeficiency virus, type 1 (HIV-1) . 1) After polymerization of bacteriophage T4 precursor capsid is complete, its surface lattice undergoes a radical, cooperative, conformational change that results in an 18% increase in size, and striking morphological, functional, and immunological differences. We have investigated the conformational basis of this event by using laser Raman spectroscopy to determine the respective secondary structures of the precursor and mature states. Upon expansion, the alpha-helix content of the major capsid protein is reduced from 36% to 21%, and its beta-sheet content increases from 33% to 46%. We conclude that the conformational change involves a fundamental re-folding of a substantial portion of the protein. 2) Scanning transmission electron microscopy has been applied to preparations of gp160/gp120 of HIV-1 purified from a recombinant viral expression system. From these data, we have established that the precursor molecule, gp160, is a dimer of 125 kDa subunits, each of which comprises 91 kDa of protein together with, on average, 34 kDa of oligosaccharides. Thus the nominal SDS-PAGE-derived molecular weight of 160 kDa is a major overestimate, presumably on account of its high carbohydrate contents. Gp120 and gp41, the maturation cleavage products of gp160, have monomer masses of 89 kDa and 36 kDa respectively, indicating that gp120 contains virtually all the carbohydrates present on gp160. Moreover, gp160 monomers dimerize primarily through interactions between their gp41 moieties.

生物大分子的结构和组装机制， 复合物和亚细胞结构的研究，主要是通过 高分辨率电子显微镜和计算机图像处理。两 过去一年完成的项目涉及：（1） 病毒衣壳蛋白的一种主要构象变化， 前体衣壳转化为成熟病毒体;和（2） 测定分子量、低聚状态、碳水化合物 gp 160/gp 120表面的二维质量图 人类免疫缺陷病毒1型（HIV-1）糖蛋白。1)后 当噬菌体T4前体衣壳的聚合完成时， 表面晶格经历了一个激进的，合作的，构象的变化 这导致了18%的大小增加，以及惊人的形态学， 功能和免疫学差异。我们已经调查了 通过使用激光拉曼光谱， 确定前体和成熟的各自的二级结构 states.扩增后，主要衣壳蛋白的α-螺旋含量 从36%降低到21%，β-折叠含量从33%增加到20%。 到46%。我们的结论是，构象变化涉及一个基本的 蛋白质的大部分的再折叠。2)扫描 透射电子显微镜已应用于制备 从重组病毒表达系统纯化的HIV-1的gp 160/gp 120。 根据这些数据，我们已经确定前体分子gp 160是 125 kDa亚基的二聚体，每个亚基包含91 kDa的蛋白质 以及平均34 kDa的寡糖。因此， 160 kDa的SDS-PAGE衍生分子量是一个主要的高估， 大概是因为它的高碳水化合物含量。GP 120和GP 41， gp 160的成熟裂解产物具有89 kDa的单体质量 和36 kDa，这表明gp 120几乎包含了所有的蛋白质。 存在于GP 160上的碳水化合物。此外，gp 160单体主要二聚化 通过它们的GP 41部分之间的相互作用。