Protein Structure and Folding at Low Temperature

这一建议涉及蛋白质折叠和稳定性的两个方面：部分折叠物种的性质，特别是熔融球状状态，以及决定特定折叠途径的因素。长期目标是了解多肽的氨基酸序列如何引导折叠到天然状态，并决定蛋白质构象的稳定性。一种相对致密的蛋白质构象状态，通常被称为熔融球状状态，已被证明是某些蛋白质再折叠的中间产物，并且在某些蛋白质的某些变性条件下是稳定的。本研究的目标之一是增加对熔融球的分子细节和物理性质的了解。将研究平衡和瞬态再折叠条件下的熔融球态。此外，折叠途径将通过使用来自分子不同区域的特定信号在环境和零下温度下的停止流动动力学实验中绘制出来。各种生物物理技术将被用于从几种不同的蛋白质中结构表征熔融球状态（为了建立普遍性）；这些包括FTIR，圆二色性（CD），荧光和流体动力半径测量。此外，动力学实验使用停止流动荧光和CD将进行。

This proposal concerns two aspects of protein folding and stability: the nature of partially-folded species, especially the molten globule state, and the factors which determine a particular folding pathway. The long-term goals are to learn how the amino acid sequence of a polypeptide guides the folding to the native state, and determines the stability of protein conformations. A relatively compact protein conformational state, often referred to as the molten globule state, has been shown to be an intermediate in the refolding of certain proteins, and to be stable under certain denaturation conditions for some proteins. One of the goals of this research is to increase the understanding about the molecular details and physical properties of molten globules. The molten globule state will be studied under both equilibrium and transient refolding conditions. In addition the pathway of folding will be mapped out by using specific signals from different regions of the molecule in stopped-flow kinetics experiments at both ambient and subzero temperatures. A variety of biophysical techniques will be used to structurally characterize the molten globule state from several different proteins (in order to establish generalities); these include FTIR, circular dichroism (CD), fluorescence and hydrodynamic radius measurements. In addition kinetic experiments using stopped-flow fluorescence and CD will be performed.