Early events of folding of β-structure-rich protein

蛋白质通过特定途径折叠。折叠研究的目的是寻找其折叠途径。我们研制了以圆二色谱、X射线散射和荧光为探针的低温停流装置。我们研究了各种类型的蛋白质。α-螺旋蛋白的结果显示α-螺旋形成的折叠速率如此之快，以至于我们无法测量α-螺旋形成的速率。相比之下，主要由β结构组成的蛋白质在其最早阶段就具有丰富的α螺旋。我们重点研究了SH 3的折叠过程，SH 3在天然状态下以β-构象存在，在折叠的最早阶段以富含α-螺旋的中间体存在。然后，我们研究了其他富含β结构的蛋白质，发现无一例外地出现了中间体。我们比较了爆发的振幅与其他参数，并发现爆发的振幅与螺旋分数计算程序Helix 2有很好的关系。该程序主要基于螺旋-卷曲转变理论，并结合相邻残基的实验数据。通过SH 3的一个残基突变（丙氨酸45变为甘氨酸），我们发现在pH 3时，构象由富含β构象转变为富含α构象。这表明beta到alpha的转换比预期的更频繁。

Protein folds via definite pathway. The purpose of the folding study is to find its folding pathway. We have developed cryo-stopped-flow apparatus with circular dichrosism, x-ray scattering and fluoresecence as probes. We have investigated various types of proteins. Results with alpha-helical protein show the folding rate of alpha-helix formation is so rapid that we cannot measure the rate of alpha-helix formation. In contrast, proteins, mainly consisting of beta-structure, take alpha-helix-rich intrmeditate at its earliest stage. We have focused the folding of SH3, which takes beta-conformation at its native state.SH3 takes alpha-helix-rich intermediate at its earliest stage of folding. We then investigated other beta-structure-rich proteins, and found the intermediate appeared without exception. We compared the amplitude of the burst with other parameters, and found the burst amplitude has good relationship with the helical fraction calculated by a program, Helix2. The program is mainly based on helix-coil transition theory with experimental data of adjacent residues. This suggest that the initial folding cores are formed by the interaction of short-lived alpha-helices.By the one residue mutation of SH3 (Alanine 45 to Glycine), we found the conformation changed from beta-rich to alpha-rich at pH3. This indicates the conversion of beta to alpha occurs more frequently than expected.