Jones B E, Matthews C R
Department of Chemistry, Pennsylvania State University, University Park 16802, USA.
Protein Sci. 1995 Feb;4(2):167-77. doi: 10.1002/pro.5560040204.
The kinetic folding mechanism for Escherichia coli dihydrofolate reductase postulates two distinct types of transient intermediates. The first forms within 5 ms and has substantial secondary structure but little stability. The second is a set of four species that appear over the course of several hundred milliseconds and have secondary structure, specific tertiary structure, and significant stability (Jennings PA, Finn BE, Jones BE, Matthews CR, 1993, Biochemistry 32:3783-3789). Pulse labeling hydrogen exchange experiments were performed to determine the specific amide hydrogens in alpha-helices and beta-strands that become protected from exchange through the formation of stable hydrogen bonds during this time period. A significant degree of protection was observed for two subsets of the amide hydrogens within the dead time of this experiment (6 ms). The side chains of one subset form a continuous nonpolar strip linking six of the eight strands in the beta-sheet. The other subset corresponds to a nonpolar cluster on the opposite face of the sheet and links three of the strands and two alpha-helices. Taken together, these data demonstrate that the complex strand topology of this eight-stranded sheet can be formed correctly within 6 ms. Measurement of the protection factors at three different folding times (13 ms, 141 ms, and 500 ms) indicates that, of the 13 amide hydrogens displaying significant protection within 6 ms, 8 exhibit an increase in their protection factors from approximately 5 to approximately 50 over this time range; the remaining five exhibit protection factors > 100 at 13 ms. Only approximately half of the population of molecules form this set of stable hydrogen bonds. Thirteen additional hydrogens in the beta-sheet become protected from exchange as the set of native conformers appear, suggesting that the stabilization of this network reflects the global cooperativity of the folding reaction.
大肠杆菌二氢叶酸还原酶的动力学折叠机制假定存在两种不同类型的瞬时中间体。第一种在5毫秒内形成，具有大量二级结构但稳定性较差。第二种是一组四种物种，在几百毫秒的过程中出现，具有二级结构、特定三级结构和显著稳定性（詹宁斯PA、芬恩BE、琼斯BE、马修斯CR，1993年，《生物化学》32:3783 - 3789）。进行脉冲标记氢交换实验以确定α - 螺旋和β - 链中特定的酰胺氢，这些氢在这段时间内通过形成稳定氢键而免受交换。在该实验的死时间（6毫秒）内，观察到酰胺氢的两个子集有显著程度的保护。一个子集的侧链形成一条连续的非极性带，连接β - 折叠中八条链中的六条。另一个子集对应于折叠片相反面上的一个非极性簇，连接三条链和两个α - 螺旋。综上所述，这些数据表明这个八链折叠片的复杂链拓扑结构可以在6毫秒内正确形成。在三个不同折叠时间（13毫秒、141毫秒和500毫秒）测量保护因子表明，在6毫秒内显示出显著保护的13个酰胺氢中，有8个在此时间范围内其保护因子从约5增加到约50；其余5个在13毫秒时保护因子>100。只有大约一半的分子群体形成这组稳定的氢键。随着天然构象体的出现，β - 折叠中的另外13个氢免受交换，这表明这个网络的稳定反映了折叠反应的全局协同性。
