PROTEIN FOLDING KINETICS

在这个项目建议书中，折叠和展开反应 模型系统重组α-巨噬细胞集落刺激因子β (RHM-CSFbeta)将被表征。停止流动CD将用于 分析第一个可检测到的动力学形成的动力学 复性途径上的中间体(重组的单体rHM-CSFβ 二级结构)和硫醇的羧胺甲基化效应 关于恢复二级结构的问题。高效液相色谱与荧光强度 变化将被用来检查重新折叠过程中的缓慢时间 域(分钟-小时)，其中蛋白质等参数的影响 重折叠途径上的浓度将被检测。时间- 离散RHM-的氢/氢交换动力学依赖关系 CSFβ结构域在展开过程中将通过电喷雾质量来确定 快速混合淬火交换产生的多肽的光谱分析 实验。这些实验应该允许绘制动力学图。 RHM-MCSFβ折叠途径及其第一个详细分析 路径中的可检测的动力学步骤以及 溶剂可及蛋白结构域与蛋白质的形成/分解 动力学中间体。烷基化对化学损伤的影响 和硫醇的氧化和蛋氨酸的氧化在能级上 将分析折叠动力学，并将其与 修改。

In this project proposal, the folding and unfolding reactions of the model system recombinant alpha-macrophage colony stimulating factor beta (rhm-CSFbeta) will be characterized. Stopped flow CD will be used to analyze the kinetics of the formation of the first detectable kinetic intermediate on the refolding pathway (monomeric rhm-CSFbeta with reformed secondary structure) and the effects of carboxamido-methylation of thiols on the regaining of secondary structure. HPLC and fluorescence intensity changes will be used to examine the refolding process in the slow time domain (minutes-hours) where the effect of parameters such as protein concentration on the refolding pathway will be examined. The time- dependence of deuterium/hydrogen exchanges kinetics for discrete rhm- CSFbeta domains during unfolding will be determined by electrospray mass spectrometry on peptic peptides generated after rapid-mix-quench exchange experiments. These experiments should permit mapping of the kinetic pathway of rhm-MCSFbeta folding and a detailed analysis of the first detectable kinetic step in the pathway as well as a correlation between solvent accessible protein domains and the formation/decomposition of kinetic intermediates. The effects of chemical insult due to alkylation and oxidation of thiols and oxidation of methionine on the energetics on folding kinetics will be analyzed and correlated with the site of modifications.