Sarkar S K, Young P E, Sullivan C E, Torchia D A
Proc Natl Acad Sci U S A. 1984 Aug;81(15):4800-3. doi: 10.1073/pnas.81.15.4800.
Heretofore the complexity of natural abundance spectra has precluded the use of 13C NMR to detect cis peptide bonds in proteins. We have incorporated [4-13C]proline into chicken calvaria collagen and report here well-resolved C gamma signals, arising from cis and trans X-Pro and X-Hyp peptide bonds (where X is any amino acid residue) in the 13C NMR spectrum of the thermally unfolded protein. Measurement of 13C signal areas shows that 16% of the X-Pro and 8% of X-Hyp bonds are cis in the unfolded collagen. These results strongly support the conclusion drawn from kinetic studies that cis-trans isomerization of peptide bonds is the rate-limiting step in helix propagation after nucleation. Our method can be applied to other proteins as well and should aid in testing the generality of the hypothesis of Brandts, Halvorson, and Brennan that cis-trans isomerization is the rate-limiting step in protein folding when proline is present.
迄今为止，天然丰度谱的复杂性使得无法使用碳-13核磁共振（13C NMR）来检测蛋白质中的顺式肽键。我们已将[4-13C]脯氨酸掺入鸡颅骨胶原蛋白中，并在此报告了热变性蛋白的13C NMR谱中，由顺式和反式X-脯氨酸（X-Pro）及X-羟脯氨酸（X-Hyp）肽键（其中X为任何氨基酸残基）产生的分辨率良好的Cγ信号。对13C信号面积的测量表明，在未折叠的胶原蛋白中，16%的X-Pro键和8%的X-Hyp键为顺式。这些结果有力地支持了动力学研究得出的结论，即肽键的顺反异构化是成核后螺旋延伸的限速步骤。我们的方法也可应用于其他蛋白质，并且应有助于检验布兰特斯、哈尔沃森和布伦南提出的假说的普遍性，该假说认为当存在脯氨酸时，顺反异构化是蛋白质折叠的限速步骤。
