STRUCTURE/INTERACTIONS OF ACTINS AND ACTIN-BINDING PROTEIN

肌动蛋白和肌动蛋白结合蛋白的结构/相互作用

• 批准号: 6271817
• 负责人: EATON E LATTMAN
• 金额: $ 17.7万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-06-01 至 1999-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6271817
• 关键词: Acanthamoeba; X ray crystallography; actin binding protein; actins; binding proteins; calorimetry; chemical binding; computer simulation; crosslink; crystallization; evolution; fluorescent dye /probe; inositol phosphates; ligands; mathematical model; myosins; nuclear magnetic resonance spectroscopy; proline; protein purification; protein structure; structural biology; thermodynamics; thymosin; vaccinia virus

Profilin is the prototype of small proteins that sequester actin monomers. Recently a host of demonstrated or putative new functions for profilin have emerged. Genetic experiments on flies and yeast established a role for profilin in the function of the actin cytoskeleton. Biochemical studies suggest a dual role for profilin as a component of the phosphoinositide signal transduction machinery and as a messenger from the membrane to actin and other ligands. In addition, the ability to bind poly-L-proline is a common feature among profilins. This system is also ideal for testing the Structure-Based Thermodynamic Analysis (SBTA) model developed by Freire and others. This model predicts the thermodynamic parameters describing protein folding/unfolding using only the change in buried polar and apolar area as structure-specific information. The deltaH, delta S and deltaG for the binding of angiotensin II to an antibody were quantitatively predicted by this model. We plan to probe its range of validity by using it to analyze the binding of proline-rich peptides to profilin. We thus propose to: Refine and compare the NMR and x-ray structures of profilin I from Acanthamoeba. Compare the structures of Acanthamoeba and vertebrate profilin as a basis for phylogenetic analysis. Identify high affinity proline-rick peptides and proteins that bind to the polyproline binding site on profilin using a structure based fluorescent derivatives of profilin and phage display methods, determine the structure of the complex of these peptides with profilin by NMR or crystallography. Characterize the energetics of binding of profilin to polyproline and to the proline rich ligands; determine if the measured values are predicted by the SBTA model of murphy and Freire. Acanthamoeba actophorin is a member of a family of actin monomer binding/actin filament severing proteins that includes actin depolymerizing factor, destrin, cofilin, and depactin. The physiological functions and mechanisms of action of these proteins is still under investigations. To provide a structural basis for advanced studies of these proteins, we propose to: Complete and refine the actophorin crystal structure by the MAD phasing method. Crystalize and determine the structure of vertebrate cofilin for comparison with actophorin. Crystalize the complex of actophorin with SADP-actin. Explore the mechanism of actin-filament severing by modeling of the actin- actophorin complex. We will attempt to crystalize a number of actin- binding proteins.

Profilin 是隔离肌动蛋白单体的小蛋白的原型。 最近，Profilin 已被证明或推定有许多新功能 出现了。 果蝇和酵母的基因实验确立了 Profilin 在肌动蛋白细胞骨架的功能中的作用。 生化研究 表明 profilin 作为磷酸肌醇的组成部分具有双重作用 信号转导机制并作为从膜到肌动蛋白的信使 和其他配体。 此外，结合聚-L-脯氨酸的能力是 配置文件之间的共同特征。 该系统也非常适合测试 Freire 和开发的基于结构的热力学分析 (SBTA) 模型 其他的。 该模型预测了描述的热力学参数 仅使用埋藏极性和非极性的变化进行蛋白质折叠/展开 区域作为特定于结构的信息。 deltaH、delta S 和 deltaG 为 定量预测血管紧张素 II 与抗体的结合 通过这个模型。 我们计划通过使用它来探索其有效性范围 分析富含脯氨酸的肽与 profilin 的结合。 因此我们建议 到： 精炼并比较 Profilin I 的 NMR 和 X 射线结构 棘阿米巴。 比较棘阿米巴和脊椎动物的profilin的结构作为基础 用于系统发育分析。 鉴定与结合的高亲和力富含脯氨酸的肽和蛋白质 使用基于结构的荧光在 profilin 上的聚脯氨酸结合位点 Profolinin 和噬菌体展示方法的衍生物，确定结构 通过NMR或晶体学分析这些肽与profilin的复合物。 表征 profilin 与聚脯氨酸结合的能量学并 富含脯氨酸的配体；确定测量值是否由下式预测 墨菲和弗莱雷的 SBTA 模型。 棘阿米巴肌动蛋白是肌动蛋白单体家族的成员 结合/肌动蛋白丝切断蛋白，包括肌动蛋白解聚 因子、destrin、cofilin 和 depactin。 生理功能和 这些蛋白质的作用机制仍在研究中。 到 为这些蛋白质的高级研究提供结构基础，我们 建议： 通过 MAD 定相完成并完善肌动蛋白晶体结构 方法。 结晶并确定脊椎动物丝切蛋白的结构以进行比较 与肌动蛋白。 使肌动蛋白与 SADP-肌动蛋白复合物结晶。 通过肌动蛋白丝的建模探索肌动蛋白丝断裂的机制 肌动蛋白复合物。 我们将尝试将一些行动具体化 结合蛋白。