Site-Specific Probing of Folding and Unfolding of Yeast Iso-1-Cytochrome c By Dielectric Resonator-Based Flow and Stopped-Flow EPR

ScholesMCB 9817598本研究的目的是发展和应用本实验室开发的独特的动力学自旋标记EPR（电子顺磁共振）方法来观察球形蛋白质的早期毫秒折叠。一个直接的焦点是球状蛋白，酵母异l-细胞色素c。所采用的装置包括基于介质谐振器的流动和停流EPR。自旋标记法的优点是可以将标记物连接到蛋白质中任何可以突变为半胱氨酸的位置，并且已经建立了进行这种突变的表达系统。迄今为止，研究早期折叠事件的两种常用方法是脉冲标记NMR光谱和快速流动荧光光谱。这种自旋标记方法既不依赖于氢键（如NMR方法），也不依赖于细胞色素c中存在的单一荧光色氨酸。虽然脉冲标记核磁共振和荧光结果将提供一个背景，结合时间分辨率的停流和流动物理方法和标签放置在以前未研究的位置提出了一个单一的机会，以解决有关蛋白质折叠的早期事件的根本争议。争论的焦点是早期折叠是一种普遍的疏水性折叠还是通过一系列的亚结构进行的。这种争论当然不仅限于细胞色素c;它是迄今为止在细胞色素c中最简单最好的描述。一般的疏水性折叠在许多位置的自旋探针的报告类似，而在不同位置的折叠亚结构的进展将被不同的，并在不同位置的探针顺序报告。为了测试这一发现在细胞色素c中的普遍性，未来的计划将是探测其他蛋白质系统的早期折叠事件。（电子顺磁共振）和动力学设备以一种独特的方式了解蛋白质折叠在其早期阶段是如何发生的。蛋白质是氨基酸结构单元的线性序列，对于蛋白质折叠成具有生物学功能的三维结构的时间过程，目前还没有一致的解释。一个标记程序被使用，它把探针放在单个氨基酸位点，以判断这些位点是否参与折叠。然后，在折叠开始后的几毫秒内，该装置将被用来检查标记的氨基酸是否确实已经折叠。这项研究的具体目标是在不同的蛋白质工程位点使用探针，以解决蛋白质在折叠开始时是否经历普遍折叠的问题，或者折叠是否从蛋白质的一个部分到下一个部分进行。目前还不知道折叠模型与顺序折叠模型是否是正确的，这种结合局部探针的动力学方法旨在确定实际发生的情况。

ScholesMCB 9817598The objective of this study is to develop and apply the unique kinetic spin labelEPR (electron paramagnetic resonance) methods developed in this laboratory toobserve the early millisecond folding of globular proteins. An immediate focus ison the globular protein, yeast iso-l-cytochrome c. The apparatus to be employedinvolves dielectric resonator-based flow and stopped-flow EPR. The spin labelmethod has the advantage that the label can be attached to any position in a protein thatcan be mutated to cysteine, and the expression system for making suchmutations has been established. To date the two common methods for studyingearly folding events have been pulse label NMR spectroscopy and rapid flowfluorescence spectroscopy. This spin label method is dependent on neitherhydrogen bonding (as is the NMR method), nor the existence of thesingle fluorescing tryptophan in cytochrome c. While the pulse label NMRand fluorescence results will provide a background, the combination of timeresolution of stopped-flow and flow physical methods and label placement atpreviously unstudied positions presents a singular opportunity to resolve afundamental controversy concerning the early events in protein folding. Thecontroversy is whether the early folding is a general hydrophobic collapseor whether early folding progresses through a sequence of substructures.This controversy is of course not limited to cytochrome c; it is simplybest delineated so far in cytochrome c. The general hydrophobic collapsewould be reported similarly by spin probes at numerous positions; whereas aprogression of folding substructures at different locations would bereported differently and sequentially by probes at different locations. Totest the generality of this finding in cytochrome c, the plan for thefuture will be to probe other protein systems for their early foldingevents.This study applies a new biophysical technique that relies on newmicrowave (electron paremagnetic resonance) and kinetic equipment tounderstand in a unique way how protein folding occurs in its early stages.A protein is a linear sequence of amino acid building blocks, and there isno consistent explanation yet for the time course for proteins to fold totheir biologically functional 3-dimensional structure. A labeling procedureis used which puts probes at individual amino acid sites to tell if thesites are involved in folding. The apparatus will then be used to examinequickly within milliseconds after folding starts whether thelabeled amino acid is indeed already folded. The specific goal of thisstudy, using probes at separate protein-engineered sites, is to resolve thequestion of whether a protein undergoes a universal collapse when foldingstarts or whether folding progresses in a sequence from one part of theprotein to the next. It is presently not known whether the collapse modelversus the sequential folding model is the proper one, and this kineticmethod combined with localized probes is intended to determine whatactually happens.