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

色氨酸光物理学

• 批准号: 6120161
• 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: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2000-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6120161
• 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 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.

这个项目的长期目标是了解分子