LARGE-SCALE MEASUREMENT OF PROTEIN THERMODYNAMIC PARAMETERS

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 测量蛋白质随着化学变性剂浓度的增加而展开的程度可以用来推断其热力学稳定性。T7噬菌体在相对高浓度的变性剂中是稳定的。我们利用这一事实对WW结构域的突变体进行了大规模的变性实验。我们将噬菌体展示的WW结构域变异体的文库暴露于化学变性剂胍的浓度增加，然后进行了多肽结合的选择。通过考察每种变异体在浓度增加时的表现，我们可以计算出每种变异体的相对热力学稳定性。由此得到的WW结构域的稳定性图应该提供了一个前所未有的关于控制该结构域稳定性的区域的视图，并可能阐明蛋白质稳定性的一般原理。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Measuring the unfolding of a protein in response to increasing concentrations of chemical denaturants can be used to derive its thermodynamic stability. T7 phage are stable in relatively high concentrations of denaturant. We have exploited this fact to perform denaturation experiments on mutants of a WW domain on a massive scale. We exposed a library of phage-displayed WW domain variants to increasing concentrations of the chemical denaturant guanidine and then performed a selection for peptide binding. By examining the performance of each variant in increasing concentrations of guanidine, we can calculate the relative thermodynamic stability of each variant. The resulting stability map of the WW domain should offer an unprecedented view of regions governing the stability of this domain and may illuminate general principles of protein stability.