TRYPTOPHAN PHOTOPHYSICS IN SINGLE TRP MUTANTS OF YEAST ACTIN

酵母肌动蛋白单一 TRP 突变体中的色氨酸光物理学

• 批准号: 6281095
• 负责人: R D LUDESCHER
• 金额: $ 0.05万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-01 至 1999-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6281095
• 关键词: biological products; biomedical resource; proteins

The long term goal of this project is to understand the molecular basis for filament formation and function in the cytoskeletal protein actin. The project seeks to achieve this goal through fluorescence studies of the photophysics, quenching, and rotational dynamics of single tryptophan mutants of yeast actin. A mutant of yeast actin in which all of the four native tryptophans have been mutated to either phenylalanine or tyrosine has been generated using site directed mutagenesis; since this mutant is viable in yeast, the mutant protein functions normally. Single tryptophan mutants will be generated in which trp residues are place strategically throughout the molecule at sites deemed appropriate to investigate the molecular basis for specific structural and functional properties. For example, mutants used to investigate the molecular mechanism of polymerization will be placed near sites of putative intermolecular interaction in the filament, while mutants used to investigate the tropomyosin/actin interaction will be placed near the putative tropomyosin binding site(s). The photophysical and dynamical behavior of these single tryptophan mutants will be studied through steady-state (done in the PI's lab) and time-resolved (done at RLBL) intensity and polarization anisotropy measurements of the protein in the monomeric (G-actin) and filamentous (F-actin) forms and during interaction with physiological ligands such as the toxin phalloidin and the actin binding proteins tropomyosin (which has no tryptophans) and gelsolin (which functions at binding stoichiometry of < 1:100). The results will be interpreted in terms of the known three-dimensional structure of G-actin and the proposed 3-D structure of F-actin. A collaboration will be initiated with a laboratory involved in molecular dynamics calculations on G-actin in order to develop molecular level interpretations of the fluorescence anisotropy data.

该项目的长期目标是了解分子 细胞骨架蛋白中丝形成和功能的基础 肌动蛋白。 该项目试图通过荧光来实现这一目标 研究的天体物理学，淬火，和旋转动力学的 酵母肌动蛋白的单色氨酸突变体。 酵母肌动蛋白突变体， 这四个土生土长的印第安人都变异成了 苯丙氨酸或酪氨酸已经使用定点 诱变;由于该突变体在酵母中是可行的，突变蛋白质 功能正常。 将产生单一色氨酸突变体，其中色氨酸残基被 在整个分子中的被认为是 适合于研究特定结构的分子基础 和功能特性。 例如，用于研究 聚合的分子机理将被置于 假定的分子间相互作用的细丝，而突变体 用于研究原肌球蛋白/肌动蛋白的相互作用将被放置 在假定的原肌球蛋白结合位点附近。 物理学和 这些单色氨酸突变体的动力学行为将被研究 通过稳态（在PI实验室完成）和时间分辨（在 RLBL）的强度和偏振各向异性测量。 单体（G-肌动蛋白）和丝状（F-肌动蛋白）形式的蛋白质， 在与生理配体如毒素相互作用期间 鬼笔环肽和肌动蛋白结合蛋白原肌球蛋白（没有 凝溶胶蛋白（其功能是结合化学计量比为 < 1：100）。 结果将根据已知的 G-肌动蛋白的三维结构和提出的三维结构 F-actin。 将与一个实验室开展合作 参与G-肌动蛋白的分子动力学计算， 发展荧光各向异性的分子水平解释 数据