TRYPTOPHAN PHOTOPHYSICS & ROTATIONAL DYNAMICS: SINGLE TRP MUTANTS OF YEAST ACTIN

这个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 光物理、猝灭和转动动力学的研究 酵母肌动蛋白的单一色氨酸突变体。酵母肌动蛋白的一个突变体 这四种原生色氨酸都已突变为 苯丙氨酸或酪氨酸是通过位点定向生成的 突变；由于该突变株在酵母中是活的，突变蛋白 功能正常。单个色氨酸突变体将在 哪些色氨酸残基战略性地放置在整个分子中 被认为适合研究其分子基础的位置 特定的结构和功能特性。例如，变种人 用于研究聚合反应的分子机理将是 放置在假定的分子间相互作用的位置附近 微丝，而突变体用于研究原肌球蛋白/肌动蛋白 相互作用将放置在假定的原肌球蛋白结合附近 网站(S)。这些单分子的光物理和动力学行为 色氨酸突变体将通过稳态进行研究(在 PI的实验室)和时间分辨的(在RLBL进行的)强度和偏振 单体(G-肌动蛋白)和蛋白质各向异性的测量 丝状肌动蛋白(F-肌动蛋白)的形成及其与生理的相互作用 毒素阴沟素和肌动蛋白结合蛋白等配体 原肌球蛋白(不含色氨酸)和明胶蛋白(起作用 结合化学计量比为1：100)。将对结果进行解释 根据已知的G-肌动蛋白的三维结构和 提出了F-肌动蛋白的三维结构。荧光各向异性数据 将用于改进分子动力学计算 G-肌动蛋白。

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 i nvestigate 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. The fluorescence anisotropy data obtained will be used to refine molecular dynamics calculations on G-actin.