WATER AS LIGAND - PREFERENTIAL BINDING AND EXCLUSION OF DENATURANTS IN PROTEIN UNFOLDING
WATER AS LIGAND - PREFERENTIAL BINDING AND EXCLUSION OF DENATURANTS IN PROTEIN UNFOLDING
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DOI：
10.1021/bi00156a001
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发表时间：
1992-10-20
期刊：
影响因子：
2.9
通讯作者：
TIMASHEFF, SN
中科院分区：
文献类型：
--
作者：
TIMASHEFF, SN
用尿素或盐酸胍(GDN-HCl)1等试剂变性蛋白质通常需要3-8M的高浓度变性剂。在这些浓度下，变性剂使典型球状蛋白的折叠状态不稳定约10-20千卡/摩尔(佩斯，1975)。这种自由能贡献(SAG)是当蛋白质展开时蛋白质和变性剂之间额外相互作用的结果。主要数据通常以过渡曲线的形式获得，如图1A所示(Thomson等人，1989)。通常用几种方法分析这样的曲线：(I)Wyman(1964)曲线图，log Kd与对数变性剂浓度，其中是N^D反应的平衡常数；斜率有时等于变性时与蛋白质结合的额外变性剂分子的数量An[参见Tanford(1970)和Pace(1975,1986)]；(Ii)线性外推，AG与变性剂浓度(Greene&Pace，1974)，它给出一个特征斜率m，它与Wyman曲线图(Pace，1975)的参数An直接相关；(Iii)变性剂结合模型(Auné&Tanford，1969)，其中SAG设为变性剂与新暴露在蛋白质展开上的基团的结合自由能；(Iv)Tanford模型(Tanford，1964,1970)，该模型规定变性自由能增量SAG等于新暴露的第I类基团在展开时从水到变性溶液的转变自由能之和5gtr：
The denaturation of proteins by agents such as urea or guanidine hydrochloride (Gdn-HCl) 1 usually requires high concentrations, 3-8 M, of denaturant. At these concentrations, denaturants destabilize the folded state of a typical globular protein by about 10-20 kcal/mol (Pace, 1975). This free energy contribution (SAG) is the result of additional inter-actions between the protein and the denaturant when the protein becomes unfolded. The primary data are normally obtained in the form of transition curves such as the one shown in Figure 1A (Thomson et al., 1989). Such curves have been usually analyzed by several approaches:(i) The Wyman (1964) plot, log Kd vs log denaturant concentration, where is the equilibrium constant for the N^ D reaction; the slope has been at times equated to the number, An, of additional denaturant molecules binding to the protein on denaturation [for review, see Tanford (1970) and Pace (1975, 1986)];(ii) The linear extrapolation, AG vs denaturant concentration (Greene & Pace, 1974), which gives a characteristic slope, m, that is directly related to the parameter An of the Wyman plot (Pace, 1975);(iii) The denaturant binding model (Auné & Tanford, 1969), in which SAG is set equal to the free energy of binding of denaturant to groups newly exposed on protein unfolding;(iv) Tanford’s model (Tanford, 1964, 1970), which states that the denaturation free energy increment, SAG, is equal to the sum of the transition free energies from water to denaturant solution, 5gtr,„of newly exposed groups of type i on unfolding: