Slow motion analysis of the fokling reactions of proteins within wet silica gels

蛋白质被包裹在湿的、光学透明的、多孔的硅胶基质中，大大延长了折叠时间，从而可以使用光谱技术直接观察整个折叠过程。在本研究中，我们重点研究了以下三种模型蛋白的折叠反应。马细胞色素C湿硅胶，细胞色素c折叠几小时、几天、几周或更长时间，视老化时间而定。在硅胶中的重折叠过程中，如在水溶液中观察到的那样，崩溃和螺旋的形成是以一种逐步的方式发生的。动力学和瞬变光谱分析揭示了一个由四个不同的中间体组成的序列，这些中间体的折叠度逐渐增加，其中两个与以前在溶液中描述的非常相似，即早期坍塌的中间体和熔融的球状中间体。另外两种是可能逃脱常规动力学方法检测的预坍塌和预熔化的球体中间体。牛泛素：当泛素在水溶液中以明显的两种状态折叠时，当被困在湿硅胶中时，部分结构的中间体被填充在泛素的折叠路径上。这一发现表明了一种有趣的可能性，即我们的方法能够稳定和捕获可能存在于折叠途径上但不被传统技术检测到的途径上的“隐藏的”折叠中间体。牛β-乳球蛋白在湿硅胶中β-乳球蛋白的复性也非常缓慢。通过对瞬时CD谱的分析，我们可以探索早期的折叠事件，这些折叠事件包括非自然的a-螺旋形成，自然的β-Sheet核心形成，以及进一步的a-螺旋到(β-Sheet)的转变。这些结果说明了我们的方法在不显著干扰折叠途径的情况下对快速折叠事件进行高分辨率分析的潜在用途。

Entrapment of proteins in wet, optically transparent, porous silica gel matrices has enabled a dramatic expansion of the folding time, allowing direct observation of the entire folding pathway using spectroscopic techniques. In this study, we have focused on the folding reactions of the following three model proteins.1. Horse cytochrome cIn wet silica gels, cytochrome c folds over a period of hours, days, weeks, or longer, depending on the aging time. During refolding in silica gels, collapse and helix formation occur in a stepwise manner, as observed in aqueous solution. Analysis of kinetics and transient spectra reveals a sequence of four distinct intermediates with progressively increasing degree of folding, two of which closely resemble those previously characterized in solution, namely, the early collapsed and the molten globule intermediates. The other two are the pre-collapsed and pre-molten globule intermediates that may escape detection by conventional kinetic methods.2. Bovine ubiquitinWhile ubiquitin folds in an apparent two-state manner in aqueous solution, a partially structured intermediate is populated on the folding pathway of ubiquitin when entrapped in wet silica gels. This finding suggests an interesting possibility that our approach is able to stabilize and capture on-pathway "hidden" folding intermediates that may exist on the folding pathway but escape detection by conventional techniques.3. Bovineβ-lactoglobulinThe refolding of β-lactoglobulin was also very slow in wet silica gels. Analysis of the transient CD spectra allows us to probe the early folding events that consist of non-native a-helix formation, native β-sheet core formation, and further a-helix to (β-sheet transition. These results illustrate the potential usefulness of our approach for high-resolution analysis of rapid folding events without significantly perturbing the folding pathway.