Chyan C L, Wormald C, Dobson C M, Evans P A, Baum J
Department of Chemistry, Rutgers University, Piscataway, New Jersey 08855-0939.
Biochemistry. 1993 Jun 1;32(21):5681-91. doi: 10.1021/bi00072a025.
A partially folded state of guinea pig alpha-lactalbumin (the A-state or molten globule state), formed by denaturation at low pH, has been studied using hydrogen exchange methods. The overall distribution of exchange kinetics, measured by 1-D NMR, suggests that fewer than 20 amides in the structure are involved in highly persistent residual structure, although CD results suggest that many other parts of the chain are folded, for a significant proportion of the time, into less stable structural elements. The pH-jump experiments show that some amides that are strongly protected from exchange in the native state become freely accessible in the A-state but that conversely a majority, at least, of those that are slow to exchange in the A-state retain that protection in the native state. This suggests that the persistent structure in the A-state is native-like although the possibility that nonnative like structural elements persist cannot be eliminated. Resonance assignments for key residues in the NMR spectrum of the native state have enabled us to use the pH-jump method also to identify the majority of the most protected amides in the A-state: they are located in two hydrophobic segments, corresponding to the B- and C-helices of the native structure. This strongly suggests that the most persistent structure of the A-state includes these regions. A variety of lines of evidence, including fluorescence quenching data and, most remarkably, very effective protection from exchange of an indole NH in a tryptophan side chain, suggest that some form of hydrophobic core in the helical domain of the native structure persists in the A-state, although without the stereochemical rigidity of the native tertiary structure. The other domain of the native structure, including the beta-sheet, appears not to contain structural elements which persist to the same extent in the A-state, emphasizing that the molten globule is highly heterogeneous, in terms of the stability and specificity of both backbone and side chain interactions.
利用氢交换方法研究了通过在低pH值下变性形成的豚鼠α-乳白蛋白的部分折叠状态（A态或熔球态）。通过一维核磁共振测量的交换动力学的总体分布表明，结构中参与高度持久的残余结构的酰胺少于20个，尽管圆二色性结果表明，在相当长的一段时间内，链的许多其他部分折叠成不太稳定的结构元件。pH跃变实验表明，一些在天然状态下受到强烈保护不发生交换的酰胺在A态中变得可自由交换，但相反，至少大多数在A态中交换缓慢的酰胺在天然状态下仍保持这种保护。这表明A态中的持久结构类似天然结构，尽管不能排除非天然样结构元件持续存在的可能性。天然状态核磁共振谱中关键残基的共振归属使我们能够使用pH跃变方法来识别A态中大多数受保护程度最高的酰胺：它们位于两个疏水片段中，对应于天然结构的B螺旋和C螺旋。这强烈表明A态中最持久的结构包括这些区域。包括荧光猝灭数据在内的各种证据，最显著的是色氨酸侧链中吲哚NH的交换受到非常有效的保护，表明天然结构螺旋结构域中的某种形式的疏水核心在A态中持续存在，尽管没有天然三级结构的立体化学刚性。天然结构的另一个结构域，包括β折叠，似乎不包含在A态中以相同程度持续存在的结构元件，这强调了熔球在主链和侧链相互作用的稳定性和特异性方面是高度异质的。
