STRUCTURE FUNCTION & FOLDING OF AN RNA BINDING PROTEIN

描述：（改编自申请人的摘要）本提案研究 蛋白质结构、稳定性、折叠和活性，以更好地了解 蛋白质的缺陷会引起疾病，蛋白质的稳定性和 活动可以被操纵。该模型系统是非常好的 鉴定了四螺旋束蛋白Rop.申请人将分析 热力学性质和确定高分辨率晶体结构 的一系列变体的Rop与完全重新包装的疏水核心。 这些研究将提供深入了解的分子基础的物理 天然蛋白质显示的特性，并说明这些特性如何 可能会受到干扰。它们还将揭示指定 螺旋-螺旋缔合的方向。因为Rop会折叠和展开 特别是缓慢地，申请人将使用实时NMR来表征 折叠中间体的性质在更大的结构细节，提供了一个 对我们理解中间体的作用的重要贡献， 蛋白质折叠ROP通过相互作用调节ColE 1质粒的复制 发夹对复合物。申请人将研究Rop-RNA相互作用， 特别强调识别增强免疫原性的突变， 亲和力或改变相互作用的特异性。这些研究报告将 在Rop的高分辨率结构的背景下解释， 发夹对和Rop-发夹对复合物。在这些研究的过程中， 一系列生物化学、分子生物学和生物物理学技术将被 包括蛋白表达和纯化，电泳， 圆二色性、荧光、NMR和X射线晶体学。

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.