Hooke S D, Radford S E, Dobson C M
Oxford Centre for Molecular Sciences, New Chemistry Laboratory, University of Oxford.
Biochemistry. 1994 May 17;33(19):5867-76. doi: 10.1021/bi00185a026.
Pulsed hydrogen exchange labeling has been used in conjunction with circular dichroism in the near and far UV to study the refolding of human lysozyme from its guanidinium chloride denatured state. Human lysozyme differs in sequence by 51 residues and one insertion from the hen protein, which has previously been studied under identical conditions by similar methods [Radford, S. E., Dobson, C. M., & Evans, P. A. (1992) Nature 358, 302-307]. The two proteins show marked differences in their folding kinetics. First, the overall rate of refolding of human lysozyme is 4-fold faster than that of the hen protein. Second, although protection of amides in the alpha-domain develops faster than that of amides in the beta-domain in both proteins, unlike hen lysozyme stabilization of the secondary structural elements of the alpha-domain in human lysozyme does not occur in a fully cooperative manner. Rather, amide hydrogens in two alpha-helices located near to the N-terminus and in the 3(10) helix close to the C-terminus of the protein are protected from exchange significantly faster than those in the remaining two alpha-helices in the alpha-domain of the protein. Third, stopped flow CD measurements show that both proteins develop extensive secondary structure during the dead time of these experiments (ca. 2 ms); this is accompanied by formation of tertiary interactions, probably involving tryptophan residues, only in the human enzyme. These results suggest that although the fundamental folding process is similar in the two proteins, human lysozyme differs in that it forms a stable subdomain involving the two N-terminal alpha-helices and the C-terminal 3(10) helix in the first few milliseconds of folding, and that at least some tryptophan residues are ordered before the formation of the native state. This indicates that the details of the folding of homologous proteins may differ as a consequence of amino acid substitutions and suggests that the study of mutant and variant proteins can provide clues as to the determinants of folding.
脉冲氢交换标记已与近紫外和远紫外圆二色性结合使用，以研究人溶菌酶从其氯化胍变性状态的重折叠过程。人溶菌酶与鸡溶菌酶在序列上有51个残基不同，还有一个插入片段，鸡溶菌酶此前已在相同条件下用类似方法进行过研究[拉德福德，S. E.，多布森，C. M.，& 埃文斯，P. A.（1992年）《自然》358，302 - 307]。这两种蛋白质在折叠动力学上表现出显著差异。首先，人溶菌酶的整体重折叠速率比鸡溶菌酶快4倍。其次，尽管两种蛋白质中α结构域中酰胺的保护比β结构域中酰胺的保护发展得更快，但与鸡溶菌酶不同，人溶菌酶α结构域二级结构元件的稳定化并非以完全协同的方式发生。相反，位于蛋白质N端附近的两个α螺旋以及靠近C端的3(10)螺旋中的酰胺氢比蛋白质α结构域中其余两个α螺旋中的酰胺氢受到交换保护的速度明显更快。第三，停流圆二色性测量表明，在这些实验的死时间（约2毫秒）内，两种蛋白质都形成了广泛的二级结构；这伴随着三级相互作用的形成，可能涉及色氨酸残基，且仅在人溶菌酶中发生。这些结果表明，尽管两种蛋白质的基本折叠过程相似，但人溶菌酶的不同之处在于，它在折叠的最初几毫秒内形成了一个稳定的亚结构域，该亚结构域涉及两个N端α螺旋和C端3(10)螺旋，并且至少一些色氨酸残基在天然状态形成之前就已有序排列。这表明同源蛋白质折叠的细节可能因氨基酸取代而不同，并表明对突变体和变体蛋白质的研究可以为折叠的决定因素提供线索。
