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，特别是熔融的球状状态，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自然状态，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重折叠的中间产物，并且对某些蛋白质在一定的变性条件下是稳定的。这项研究的目标之一是增加对熔融球体的分子细节和物理性质的了解。在平衡重折叠和瞬时重折叠两种条件下，都将研究熔融球状状态。此外，在常温和零度以下的停流动力学实验中，将通过使用来自分子不同区域的特定信号来绘制折叠路径。将使用各种生物物理技术从结构上表征几种不同蛋白质的熔融球状状态(以建立共性)；这些技术包括FTIR、圆二色谱(CD)、荧光和流体动力学半径测量。此外，还将使用停流荧光和CD进行动力学实验。

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.