Multi-Dimensional Studies of Protein Folding

点击翻译按钮获取中文摘要

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.