Forge V, Hoshino M, Kuwata K, Arai M, Kuwajima K, Batt C A, Goto Y
Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka, 565-0871, Japan.
J Mol Biol. 2000 Mar 3;296(4):1039-51. doi: 10.1006/jmbi.1999.3515.
The refolding of beta-lactoglobulin, a beta-barrel protein consisting of beta strands betaA-betaI and one major helix, is unusual because non-native alpha-helices are formed at the beginning of the process. We studied the refolding kinetics of bovine beta-lactoglobulin A at pH 3 using the stopped-flow circular dichroism and manual H/(2)H exchange pulse labeling coupled with heteronuclear NMR. The protection pattern from the H/(2)H exchange of the native state indicated the presence of a stable hydrophobic core consisting of betaF, betaG and betaH strands. The protection pattern of the kinetic intermediate obtained about one second after initiating the reaction was compared with that of the native state. In this relatively late kinetic intermediate, which still contains some non-native helical structure, the disulfide-bonded beta-hairpin made up of betaG and betaH strands was formed, but the rest of the molecule was fluctuating, where the non-native alpha-helices may reside. Subsequently, the core beta-sheet extends, accompanied by a further alpha-helix to beta-sheet transition. Thus, the refolding of beta-lactoglobulin exhibits two elements: the critical role of the core beta-sheet is consistent with the hierarchic mechanism, whereas the alpha-helix to beta-sheet transition suggests the non-hierarchic mechanism.
β-乳球蛋白是一种由β链βA-βI和一个主要螺旋组成的β桶状蛋白，其重折叠过程不同寻常，因为在该过程开始时会形成非天然α螺旋。我们使用停流圆二色性以及手动H/（2）H交换脉冲标记结合异核核磁共振研究了pH 3条件下牛β-乳球蛋白A的重折叠动力学。天然状态下H/（2）H交换的保护模式表明存在一个由βF、βG和βH链组成的稳定疏水核心。将反应开始约一秒后获得的动力学中间体的保护模式与天然状态的保护模式进行了比较。在这个相对较晚的动力学中间体中，其仍含有一些非天然螺旋结构，由βG和βH链组成的二硫键连接的β发夹结构已形成，但分子的其余部分处于波动状态，非天然α螺旋可能位于此处。随后，核心β折叠片层扩展，同时伴随着进一步的α螺旋向β折叠片层的转变。因此，β-乳球蛋白的重折叠表现出两个特点：核心β折叠片层的关键作用与层次机制一致，而α螺旋向β折叠片层的转变则表明了非层次机制。
