MUTAGENIC STUDY OF YEAST ACTIN CONFORMATIONAL CHANGES

酵母肌动蛋白构象变化的诱变研究

• 批准号: 6519149
• 负责人: Peter A. Rubenstein
• 金额: $ 30.19万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-07-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6519149
• 关键词: Saccharomyces cerevisiae; actins; adenosine triphosphate; adenosinetriphosphatase; conformation; cysteine; fluorescence resonance energy transfer; fungal genetics; fungal proteins; gene mutation; hydrolysis; hydropathy; molecular cloning; polymerization; protein biosynthesis; protein structure function; site directed mutagenesis; temperature sensitive mutant

We are trying to ascertain the molecular basis underlying actin filament formation and turnover. Actin must form a filament to fulfill its biological roles. It must bind to ATP or ADP along with a divalent cation to maintain its native structure. Finally, the hydrolysis of the nucleotide concomitant with polymerization and subsequent release of the Pi creates ADP-F actin which is a less stable filament than ATP- or ADP- Pi F actin. Thus, actin filament cycling may depend on the rate of Pi release from the filament. Although the structure of G-actin is known to atomic resolution, the filament structure has not been directly determined. Holmes et al. proposed a model of F-actin based on the structure of the monomer coupled with fiber diffraction data from oriented actin gels. The model predicts a reorientation of a loop between subdomains 3 and 4, containing a hydrophobic "plug", to allow it to interact with a hydrophobic pocket composed of subdomains 2 and 4 of adjacent monomers in the opposing strand. Such a bridge would greatly enhance interstrand stabilization of the structure. However, this aspect of the model has not been experimentally verified. We have altered the plug of yeast actin by site-directed mutagenesis to begin to test the plug-pocket interaction theory. To continue, we will place a cysteine in a proposed conformationally active plug position, L269. We will attach a fluorescent probe and study the behavior of the plug in the G and F states using steady state fluorescence and fluorescence resonance energy transfer. We will create mutants at H73 to test a theory proposing this residue as an important factor in determining the rate of Pi release. The cold-sensitive polymerization defect associated with these mutants suggests they may weaken the subdomain 2 component of the pocket of the plug-pocket interaction. We will test this by examining the factors needed to stabilize these mutant filaments against the cold. We will combine the H73 and L269C mutations to examine the behavior of the loop in the monomer under conditions where it would normally polymerize. Finally, we will use genetics to determine which proteins, in vivo, restore polymerizability to our polymerization- defective mutants. This work should provide new insight concerning the molecular basis underlying F-actin assembly and disassembly.

我们正试图确定肌动蛋白细丝的分子基础。 阵型和周转。肌动蛋白必须形成细丝才能完成其 生物角色。它必须与ATP或ADP结合，并与二价体结合 阳离子以保持其固有的结构。最后，水解物的 核苷酸伴随着聚合和随后的释放 PI产生ADP-F肌动蛋白，它是一种比ATP或ADP-更不稳定的细丝- PI F肌动蛋白。因此，肌动蛋白细丝循环可能取决于PI的速率 从灯丝上松开。尽管已知G-肌动蛋白的结构 对于原子分辨率来说，丝状结构还没有直接得到 下定决心。Holmes等人。在此基础上提出了F-肌动蛋白模型 与光纤衍射数据相耦合的单体结构 定向肌动蛋白凝胶。该模型预测循环的重定向 在亚域3和亚域4之间，包含疏水“插头”，以允许 它与一个疏水口袋相互作用，该口袋由亚域2和 4在相反链中的相邻单体。这样一座桥将会 大大增强了结构的线间稳定性。然而， 该模型的这一方面尚未得到实验验证。我们有 通过定点突变改变酵母肌动蛋白的塞子开始 来检验即插即用相互作用理论。要继续，我们将放置 建议构象活性插头位置的半胱氨酸，L269。 我们将安装一个荧光探头并研究插头的行为 在G和F状态下使用稳态荧光和荧光 共振能量传递。我们将在H73上创造突变体来测试 提出将这一残留物作为决定 PI释放率。与冷敏聚合缺陷相关的 这些突变体表明它们可能会削弱亚区2的成分 即插即用交互的口袋。我们将通过以下方式进行测试 研究稳定这些突变细丝以对抗 寒冷的天气。我们将结合H73和L269C突变来检查 单体中的环在以下条件下的行为 通常是聚合的。最后，我们将使用遗传学来确定 蛋白质，在体内，恢复我们聚合的聚合性- 有缺陷的突变体。这项工作应该提供关于 F-肌动蛋白组装和拆解的分子基础。