STRUCTURE/INTERACTIONS OF ACTINS AND ACTIN-BINDING PROTEIN

Profilin是一种能隔离肌动蛋白单体的小蛋白质原型。 最近，许多已证实或推定的profilin新功能已被发现， 涌现 对苍蝇和酵母的遗传实验确立了 profilin在肌动蛋白细胞骨架的功能。 生化研究 提示作为磷酸肌醇组分的profilin具有双重作用 信号转导机制和作为信使从膜到肌动蛋白 和其它配体。 此外，结合聚-L-脯氨酸的能力是一个重要因素。 这是profilins的共同特征。 该系统也是测试 基于结构的热力学分析（SBTA）模型由Freire开发， 他人 该模型预测的热力学参数描述 蛋白质折叠/去折叠仅使用埋藏极性和非极性的变化 作为特定结构的信息。 Δ H、Δ S和Δ G 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它的有效性范围， 分析富含脯氨酸的肽与profilin的结合。 因此，我们建议 收件人： 改进并比较来自于C12 H12 O 12的profilin I的NMR和X射线结构。 阿米巴原虫 比较棘阿米巴和脊椎动物profilin的结构作为基础 用于系统发育分析。 鉴定结合到蛋白质的高亲和力脯氨酸-里克肽和蛋白质。 使用基于结构的荧光标记在profilin上的聚脯氨酸结合位点 profilin的衍生物和噬菌体展示方法，确定结构 这些肽与profilin的复合物。 表征profilin与聚脯氨酸和与聚脯氨酸的结合的能量学。 富含脯氨酸的配体;确定测量值是否通过 Murphy和Freire的SBTA模型。 阿米巴肌动蛋白是肌动蛋白单体家族的一员 包括肌动蛋白解聚的结合/肌动蛋白丝切断蛋白 因子、destrin、cofilin和depactin。 生理功能和 这些蛋白的作用机制仍在研究中。 到 为这些蛋白质的进一步研究提供了结构基础，我们 提议： 通过MAD定相完成并细化肌动蛋白晶体结构 法 脊椎动物丝切蛋白的结晶和结构测定以进行比较 与actophorin。 使肌动蛋白与SADP-肌动蛋白的复合物结晶。 探讨肌动蛋白丝切断的机制， 肌动蛋白复合物 我们将尝试结晶一些肌动蛋白- 结合蛋白

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.