Multi-Dimensional Studies of Protein Folding

这项研究将侧重于小球形蛋白质展开的热力学，重点是收集多个扰动轴（如温度、压力、变性剂浓度、pH值）上的平衡展开数据，并将这些数据集纳入全局非线性最小二乘，以便对蛋白质展开的两态模型进行更严格的测试，以确定一整套热力学参数（如焓、熵、自由能、热、热、热）。热容，体积变化，扰动的pka和可能的高阶参数，如(V/(P)，可压缩性的差异。一个组合的圆二色/荧光计将用于收集展开数据，同时监测两种类型的信号。色氨酸类似物，包括5-羟色氨酸和氟色氨酸，将被纳入蛋白质中，为监测展开提供额外的位点特异性光谱探针。要研究的蛋白质包括野生型葡萄球菌核酸酶（用作一个良好的模型），它的一些突变体（包括一组电荷变化突变体），一个全β蛋白，白细胞介素1-(，以及其他一些β型的小球状蛋白。除了这些热力学研究之外，还将努力开发新的策略来解决和量化展开平衡中的中间态人口。其中一种策略将涉及蛋白质的快速冻结/淬火（在各种初始条件下），随后使用固态19F-nmr来识别中间体的峰（化学交换被冻结）。第二种策略将涉及在中低温下使用毛细管电泳来分离平衡态的物种。除了与理解蛋白质展开的热力学相关的基本问题外，对于研究人员来说，能够描述蛋白质的稳定性仍然具有实际重要性，例如，在比较一组突变蛋白质时。如何以及是否可以根据蛋白质的稳定性来解释数据取决于所假设的展开模型（例如，双态等）。本研究的目的是测试所选蛋白质的两态模型的极限，并制定确定平衡展开中间体存在的实验策略。通过探索双态模型的局限性，希望对如何解释蛋白质稳定性数据有所了解。

9808635 Eftink This research will focus on the thermodynamics of the unfolding of small, globular proteins, with emphasis on collecting equilibrium unfolding data over multiple perturbation axes (e.g., temperature, pressure, denaturant concentration, pH) and subjecting such data sets to global nonlinear least-squares in order to perform more rigorous tests of the two-state model for protein unfolding, to determine a full set of thermodynamic parameters (e.g., enthalpy, entropy, free energy, heat capacity, and volume changes, perturbed pKas and possibly higher order parameters such as the ((V/(P, the difference in compressibility). A combined circular dichroism/fluorometer will be used to collect unfolding data, with both types of signals being simultaneously monitored. Tryptophan analogues, including 5-hydroxytryptophan and the fluorotryptophans, will be incorporated into proteins to provide additional site-specific spectroscopic probes for monitoring unfolding. The proteins to be studied include wild type Staphylococcal nuclease (used as a well behaved model), some of its mutants ( including a set of charge change mutants), an all beta protein, interleukin 1-(, and a few other small globular proteins of the beta type. In addition to these thermodynamic studies, effort will be made to develop new strategies to resolve and quantitate the population of intermediate states in an unfolding equilibrium. One such strategy will involve rapid freeze/quenching of proteins (at various initial conditions), with subsequent solid-state 19F-nmr to identify peaks for an intermediate (chemical exchange being halted by the freezing). A second strategy will involve the use of capillary electrophoresis at moderately low temperature to separate species in an equilibrium. In addition to the fundamental issues related to understanding the thermodynamics of protein unfolding, it continues to be of practical importance for researchers to be able to describe the stability of proteins, for example, when compar ing a set of mutant proteins. How and whether data can be interpreted in terms of the stability of a protein depends on the unfolding model that is assumed (e.g., two-state, etc). The goal of this research is to test the limits of the two-state model for selected proteins and to develop experimental strategies for determining the presence of an equilibrium unfolding intermediate. By exploring the limitations of the two-state model, it is hoped to achieve some understanding as to how to interpret protein stability data.