Tan Y J, Oliveberg M, Otzen D E, Fersht A R
Cambridge Centre for Protein Engineering, UK.
J Mol Biol. 1997 Jun 20;269(4):611-22. doi: 10.1006/jmbi.1997.1043.
There are four peptidyl-proline bonds in the 64-residue protein chymotrypsin inhibitor 2 (CI2), all of which are in the trans conformation in the native structure. The isomerisation of one or more of these peptidyl-proline bonds to the cis conformation in the denatured state gives rise to heterogeneity, leading to both fast and slow-folding species. The refolding of the fast-folding species, which has all trans peptidyl-proline bonds, is much faster than that of the slow-folding species, which have one or more cis peptidyl-proline bonds. In CI2, the slow-folding species can be classified into two groups by their rates of refolding, temperature-dependence, pH-dependence and [GdmCl]-dependence of the rate constants and the effect of peptidyl-prolyl isomerase on the rate constants. The replacement of Pro6 by Ala removes one of the slow refolding phases, suggesting that the cis peptidyl-Pro6 conformation is solely responsible for one of the slow-folding species. Pro6 is located in a region of the protein where non-random interactions have been found in a series of N-terminal fragments of CI2 (residues 1 to 13, 1 to 25, 1 to 28 and 1 to 40). In addition, NMR studies on a mutant fragment, (1-40)T3A, have confirmed that this non-native interaction is associated with the bulky side-chain of Trp5. The atypical rate of cis to trans isomerisation of the peptidyl-Pro bond is indicative of the presence of a similar hydrophobic cluster in the physiological denatured state of intact CI2.
在由64个残基组成的蛋白质胰凝乳蛋白酶抑制剂2（CI2）中存在四个肽基脯氨酸键，在天然结构中它们均处于反式构象。在变性状态下，这些肽基脯氨酸键中的一个或多个异构化为顺式构象会导致异质性，从而产生快速折叠和慢速折叠的物种。具有所有反式肽基脯氨酸键的快速折叠物种的重折叠速度比具有一个或多个顺式肽基脯氨酸键的慢速折叠物种快得多。在CI2中，慢速折叠物种可根据其重折叠速率、速率常数的温度依赖性、pH依赖性和[GdmCl]依赖性以及肽基脯氨酰异构酶对速率常数的影响分为两组。用丙氨酸取代Pro6消除了一个慢速重折叠阶段，这表明顺式肽基-Pro6构象是造成一种慢速折叠物种的唯一原因。Pro6位于蛋白质的一个区域，在CI2的一系列N端片段（残基1至13、1至25、1至28和1至40）中发现了非随机相互作用。此外，对突变片段(1-40)T3A的核磁共振研究证实，这种非天然相互作用与Trp5的大侧链有关。肽基-Pro键从顺式到反式的异构化的非典型速率表明在完整CI2的生理变性状态中存在类似的疏水簇。
