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

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。 单色氨酸突变体将在 所述色氨酸残基策略性地位于整个分子中， 认为适合研究以下疾病分子基础的研究中心 具体的结构和功能特性。 例如， 用于研究聚合反应的分子机理， 放置在假定的分子间相互作用的位点附近， 丝，而突变体用于研究原肌球蛋白/肌动蛋白 相互作用将被放置在假定的原肌球蛋白结合附近 研究中心。 研究了这些单分子的物理和动力学行为， 色氨酸突变体将通过稳态研究（在 PI的实验室）和时间分辨（在RLBL完成）强度和偏振 单体（G-肌动蛋白）中蛋白质的各向异性测量， 丝状（F-肌动蛋白）的形式，并在相互作用与生理 配体如毒鬼笔环肽和肌动蛋白结合蛋白 原肌球蛋白（不含糖蛋白）和凝溶胶蛋白（ 结合化学计量< 1：100）。 结果将被解释为 根据已知的G-肌动蛋白的三维结构， F-actin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 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.