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的许多演示或推定的新功能具有 出现了。 果蝇和酵母上的基因实验确立了 肌动蛋白细胞骨架的功能中的斑纹蛋白。 生化研究 建议将profilin作为磷酸肌醇的成分双重作用 信号转导机械和从膜到肌动蛋白的使者 和其他配体。 另外，结合聚-L-丙啉的能力是 profilins中的共同特征。 该系统也是测试的理想选择 由Freire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 蛋白质折叠/展开仅使用埋藏的极性变化 区域作为特定于结构的信息。 三角洲，三角洲和三角洲 数量预测血管紧张素II与抗体的结合 通过这个模型。 我们计划通过使用它来探测其有效性范围 分析富含脯氨酸的肽与profilin的结合。 因此，我们提出 到： 完善并比较profilin I的NMR和X射线结构 acanthamoeba。 比较acanthamoeba和脊椎动物叶子蛋白的结构 用于系统发育分析。 鉴定高亲和力脯氨酸 - 棒状肽和与结合的蛋白质 使用基于结构的荧光蛋白上的聚丙烯结合位点 profilin和噬菌体显示方法的衍生物，确定结构 通过NMR或晶体学的这些肽与叶酸蛋白的复合物的复合物。 表征叶胶蛋白与多产和与多产的结合的能量学和与 脯氨酸丰富的配体；确定测量值是否由 Murphy和Freire的SBTA模型。 Acanthamoeba Actophorin是肌动蛋白单体家族的成员 结合/肌动蛋白细丝切断蛋白质，包括肌动蛋白去聚合 因子，destrin，cofilin和depactin。 生理功能和 这些蛋白质的作用机理仍在研究中。 到 为这些蛋白质的高级研究提供了结构性基础，我们 建议： 通过疯狂的相相来完成并完善Actopatophorin晶体结构 方法。 结晶并确定脊椎动物辅助蛋白的结构以进行比较 与actophorin。 将Actophorin的复合物与sadp-肌动蛋白结晶。 通过建模肌动蛋白来探索肌动蛋白丝的机理 Actophorin复合物。 我们将尝试使许多肌动蛋白结晶 结合蛋白。