Determining effects of environmental conditions on protein folding using single m

描述(申请人提供)：蛋白质折叠是现代生物学中一个尚未解决的重大问题。更好地了解蛋白质折叠机制将有助于解释为什么一些蛋白质错误折叠，导致阿尔茨海默氏症、帕金森氏病、II型糖尿病和一些癌症(1)等疾病。为了了解环境和细胞条件在蛋白质折叠中的作用以及哪些条件可能导致错误折叠，了解环境因素如温度、溶质和应变的影响是重要的。这项提议的目的是探索力和溶质两个环境因素对使用光学镊子进行蛋白质折叠的影响。光学镊子被用来探测单个蛋白质分子沿特定拉力轴(2)作用力的反应。溶质可以对涉及大规模构象变化的蛋白质过程产生重大而特定的影响(3)，因此是光镊子实验的自然微扰。溶质效应取决于过程中埋藏或暴露的表面的数量和类型(�AsA)，因此可用于探测构象变化。这里概述的实验将探索变性尿素和稳定剂甘氨酸甜菜碱(GB)对src SH3结构域折叠的影响。1.通过绘制GB和尿素的力与溶质浓度的折叠与未折叠的相图，表征变性剂和渗透压对机械折叠的影响。2.通过测定尿素和GB对src SH3结构域折叠的影响，探索单分子力折叠实验与标准系综实验的不同之处。溶质效应与�AsA的折叠有关，�AsA的任何差异都应该揭示这些实验之间变性状态系综结构程度的差异。此外，由于光学镊子实验是在单个蛋白质分子上进行的，它们可以用来观察单个蛋白质的行为如何偏离平均整体行为，从而潜在地揭示可能导致错误折叠的罕见折叠事件。3.确定不同的折叠途径，通过确定尿素和GB对src SH3结构域的折叠或解折叠速度的影响，当使用光钳沿两个不同的轴拉伸时。这些速率与�AsA折叠进入和离开过渡态有关，因此可以用来表征折叠机制。溶质还可能足以扰乱折叠过程，从而揭示替代的折叠路径。

DESCRIPTION (provided by applicant): Protein folding is a major unsolved problem in modern biology. A better understanding of the protein folding mechanism will help elucidate why some proteins misfold, leading to disorders like Alzheimers disease, Parkinson's disease, type II diabetes and some cancers (1). In order to understand the role of the environment and cellular conditions in protein folding and what conditions might lead to misfolding, it is important to understand the effect of environmental factors like temperature, solutes and strain. The goal of this proposal is to explore the effect of two environmental factors, force and solutes, on protein folding using optical tweezers. Optical tweezers are used to probe the response of single protein molecules to force along a specific pulling axis (2). Solutes can have large and specific effects on protein processes involving large-scale conformational change (3) and so are a natural perturbant for optical tweezers experiments. Solute effects depend on the amount and type of surface buried or exposed in a process (�ASA) and so can be used to probe conformational changes. The experiments outlined here will explore the effects of the denaturant urea and the stabilizing osmolyte glycine betaine (GB) on folding of the src SH3 domain using optical tweezers. 1. Characterize how denaturants and osmolytes affect mechanical folding by developing a folded vs unfolded phase diagram of force vs solute concentration for GB and urea. 2. Explore how folding in single molecule force experiments differs from standard ensemble experiments by determining the effect of urea and GB on folding the src SH3 domain using both techniques. Solute effects are related to �ASA for folding and any differences in �ASA should reveal differences in the extent of structure in the denatured state ensemble between these experiments. Also, because optical tweezers experiments are performed on single protein molecules, they can be used to look at how behavior of individual proteins deviates from average ensemble behavior, potentially revealing rare folding events that could lead to misfolding. 3. Characterize different folding pathways, by determining the effect of urea and GB on the folding or unfolding rate of the src SH3 domain when pulled along two different axes using optical tweezers. These rates are related to �ASA for folding to and from the transition state so can be used to characterize the folding mechanism. Solutes could also potentially perturb the folding process enough to reveal alternate folding pathways.