Mohan P M Krishna, Chakraborty Swagata, Hosur Ramakrishna V
Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai, 400 005, India.
J Biomol NMR. 2009 May;44(1):1-11. doi: 10.1007/s10858-009-9311-5. Epub 2009 Mar 24.
Understanding protein stability at residue level detail in the native state ensemble of a protein is crucial to understanding its biological function. At the same time, deriving thermodynamic parameters using conventional spectroscopic and calorimetric techniques remains a major challenge for some proteins due to protein aggregation and irreversibility of denaturation at higher temperature values. In this regard, we describe here the NMR investigations on the conformational stabilities and related thermodynamic parameters such as local unfolding enthalpies, heat capacities and transition midpoints in DLC8 dimer, by using temperature dependent native state hydrogen exchange; this protein aggregates at high (>65 degrees C) temperatures. The stability (free energy) of the native state was found to vary substantially with temperature at every residue. Significant differences were found in the thermodynamic parameters at individual residue sites indicating that the local environments in the protein structure would respond differently to external perturbations; this reflects on plasticity differences in different regions of the protein. Further, comparison of this data with similar data obtained from GdnHCl dependent native state hydrogen exchange indicated many similarities at residue level, suggesting that local unfolding transitions may be similar in both the cases. This has implications for the folding/unfolding mechanisms of the protein.
在蛋白质的天然态系综中，从残基水平细节上理解蛋白质稳定性对于理解其生物学功能至关重要。同时，对于一些蛋白质而言，由于蛋白质聚集以及在较高温度下变性的不可逆性，使用传统光谱学和量热技术推导热力学参数仍然是一项重大挑战。在这方面，我们在此描述了通过使用温度依赖性天然态氢交换对DLC8二聚体的构象稳定性以及相关热力学参数（如局部解折叠焓、热容和转变中点）进行的核磁共振研究；该蛋白质在高温（>65摄氏度）下会聚集。发现天然态的稳定性（自由能）在每个残基处都随温度有显著变化。在各个残基位点的热力学参数中发现了显著差异，这表明蛋白质结构中的局部环境对外部扰动的响应会有所不同；这反映了蛋白质不同区域的可塑性差异。此外，将该数据与从盐酸胍依赖性天然态氢交换获得的类似数据进行比较，发现在残基水平上有许多相似之处，这表明在这两种情况下局部解折叠转变可能相似。这对蛋白质的折叠/解折叠机制具有启示意义。
