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

将蛋白质包裹在湿的、光学透明的、多孔的硅胶基质中，使折叠时间大大延长，从而可以使用光谱技术直接观察整个折叠途径。在本研究中，我们重点研究了以下三种模式蛋白的折叠反应。马细胞色素c湿硅胶，细胞色素c在数小时，数天，数周或更长时间内折叠，这取决于老化时间。在硅胶中的再折叠过程中，坍塌和螺旋形成以逐步的方式发生，正如在水溶液中观察到的那样。动力学和瞬态光谱分析揭示了四种不同的中间体序列，其折叠程度逐渐增加，其中两种中间体与先前在溶液中表征的中间体非常相似，即早期坍塌和熔融球状中间体。另外两种是预坍缩和预熔融的球状中间体，它们可能逃脱常规动力学方法的检测。虽然泛素在水溶液中以明显的双态方式折叠，但在湿硅胶中包裹时，泛素的折叠途径上填充了部分结构的中间体。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路径上的“隐藏”折叠中间体，这些中间体可能存在于折叠路径上，但无法被传统技术检测到。牛β-乳球蛋白β-乳球蛋白在湿硅胶中的再折叠也非常缓慢。通过对瞬态CD谱的分析，我们可以探索由非原生a-螺旋形成、原生β-薄片核心形成以及进一步的a-螺旋向β-薄片转变组成的早期折叠事件。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有用性。

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.