STRUCTURE FUNCTION & FOLDING OF AN RNA BINDING PROTEIN

描述：(改编自申请人的摘要)本提案调查 更好地了解蛋白质的结构、稳定性、折叠和活性 蛋白质中的缺陷会导致疾病，以及蛋白质的稳定性和 活动可能会受到操纵。模型系统是最好的 鉴定了四股螺旋束蛋白Rop。申请者将分析 热力学性质和高分辨晶体结构的确定 Rop的一系列变种，具有完全重新包装的疏水核心。 这些研究将提供对物理的分子基础的洞察 天然蛋白质所显示的性质，并说明这些性质是如何 可能会感到不安。它们还将揭示指定 螺旋-螺旋缔合的取向。因为Rop可以折叠和展开 特别缓慢的是，申请人将使用实时核磁共振来表征 折叠中间体的性质更具结构细节，提供了 对我们理解中间体在 蛋白质折叠。ROP通过相互作用调节Cole1质粒的复制 有发夹情结的。申请人将研究Rop-RNA相互作用， 并特别强调识别能够增强 亲和力或改变交互作用的专一性。这些研究将是 在Rop的高分辨率结构的背景下解释， 发夹配对，以及罗普-发夹配对复合体。在这些研究过程中， 一系列生化、分子生物学和生物物理技术将 包括蛋白质的表达和纯化，电泳法， 圆二色谱、荧光、核磁共振和x射线结晶学。

DESCRIPTION: (adapted from applicant's abstract) This proposal investigates protein structure, stability, folding and activity to better understand how defects in proteins give rise to disease and how a protein's stability and activity may be manipulated. The model system is the extremely well characterized four helix bundle protein, Rop. The applicant will analyze the thermodynamic properties and determine the high resolution crystal structures of a series of variants of Rop with completely repacked hydrophobic cores. These studies will provide insights into the molecular basis of the physical properties displayed by natural proteins and illustrate how these properties can be perturbed. They will also reveal the interactions which specify the orientation of helix-helix associations. Because Rop folds and unfolds particularly slowly, the applicant will use real-time NMR to characterize the nature of folding intermediates in greater structural detail, providing an important contribution to our understanding of the role of intermediates in protein folding. Rop regulates the replication of ColE1 plasmids by interacting with hairpin-pair complexes. The applicant will study the Rop-RNA interaction, with particular emphasis on the identification of mutations that enhance the affinity or alter the specificity of the interaction. These studies will be interpreted in the context of the high resolution structures of Rop, the hairpin pair, and the Rop-hairpin pair complex. In the course of these studies, a range of biochemical, molecular biological and biophysical techniques will be employed, including protein expression and purification, electrophoresis, circular dichroism, fluorescence, NMR, and x-ray crystallography.