Using circular dichroism collected as a function of temperature to determine the thermodynamics of protein unfolding and binding interactions
Using circular dichroism collected as a function of temperature to determine the thermodynamics of protein unfolding and binding interactions
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DOI：
10.1038/nprot.2006.204
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发表时间：
2006-01-01
期刊：
影响因子：
14.8
通讯作者：
Greenfield, Norma J.
中科院分区：
文献类型：
--
作者：
Greenfield, Norma J.
圆二色性（CD）是一种很好的光谱技术，用于跟踪蛋白质的展开和折叠作为温度的函数。它的主要应用之一是确定突变和配体对蛋白质和多肽稳定性的影响。如果CD随温度的变化是可逆的，则可以通过分析数据来确定展开的范霍夫焓和熵、展开跃迁的中点和展开的自由能。蛋白质-蛋白质和蛋白质-配体相互作用的结合常数也可以从展开曲线中估计出来。作为温度函数的CD光谱分析对于确定蛋白质是否具有展开中间体也很有用。测量五种折叠蛋白的光谱及其在单一波长下的展开曲线需要大约8小时。
Circular dichroism (CD) is an excellent spectroscopic technique for following the unfolding and folding of proteins as a function of temperature. One of its principal applications is to determine the effects of mutations and ligands on protein and polypeptide stability. If the change in CD as a function of temperature is reversible, analysis of the data may be used to determined the van't Hoff enthalpy and entropy of unfolding, the midpoint of the unfolding transition and the free energy of unfolding. Binding constants of protein-protein and protein-ligand interactions may also be estimated from the unfolding curves. Analysis of CD spectra obtained as a function of temperature is also useful to determine whether a protein has unfolding intermediates. Measurement of the spectra of five folded proteins and their unfolding curves at a single wavelength requires similar to 8 h.