Okabe Takahiro, Tsukamoto Seiichi, Fujiwara Kazuo, Shibayama Naoya, Ikeguchi Masamichi
Department of Bioinformatics, Soka University , 1-236 Tangi-machi, Hachioji, Tokyo 192-8577, Japan.
Biochemistry. 2014 Jun 17;53(23):3858-66. doi: 10.1021/bi5003647. Epub 2014 Jun 5.
Many studies have shown that during the early stages of the folding of a protein, chain collapse and secondary structure formation lead to a partially folded intermediate. Thus, direct observation of these early folding events is crucial if we are to understand protein-folding mechanisms. Notably, these events usually manifest as the initial unresolvable signals, denoted the burst phase, when monitored during conventional mixing experiments. However, folding events can be substantially slowed by first trapping a protein within a silica gel with a large water content, in which the trapped native state retains its solution conformation. In this study, we monitored the early folding events involving secondary structure formation of five globular proteins, horse heart cytochrome c, equine β-lactoglobulin, human tear lipocalin, bovine α-lactalbumin, and hen egg lysozyme, in silica gels containing 80% (w/w) water by CD spectroscopy. The folding rates decreased for each of the proteins, which allowed for direct observation of the initial folding transitions, equivalent to the solution burst phase. The formation of each initial intermediate state exhibited single exponential kinetics and Arrhenius activation energies of 14-31 kJ/mol.
许多研究表明，在蛋白质折叠的早期阶段，链塌陷和二级结构形成会导致部分折叠的中间体。因此，如果我们要理解蛋白质折叠机制，直接观察这些早期折叠事件至关重要。值得注意的是，在传统混合实验中监测时，这些事件通常表现为最初无法分辨的信号，即爆发阶段。然而，通过首先将蛋白质捕获在含水量高的硅胶中，折叠事件可以显著减慢，其中捕获的天然状态保留其溶液构象。在本研究中，我们通过圆二色光谱法监测了五种球状蛋白质（马心细胞色素c、马β-乳球蛋白、人泪乳铁蛋白、牛α-乳白蛋白和鸡卵溶菌酶）在含水量为80%（w/w）的硅胶中的涉及二级结构形成的早期折叠事件。每种蛋白质的折叠速率都降低了，这使得能够直接观察到相当于溶液爆发阶段的初始折叠转变。每个初始中间态的形成表现出单指数动力学，阿累尼乌斯活化能为14 - 31 kJ/mol。
