Center for Theoretical Biological Physics and Department of Chemistry and Biochemistry, University of California at San Diego, 9500 Gilman Drive, La Jolla, California 92093, USA.
Acc Chem Res. 2010 May 18;43(5):652-60. doi: 10.1021/ar9002703.
The covalently bound heme cofactor plays a dominant role in the folding of cytochrome c. Because of the complicated inorganic chemistry of the heme, some might consider the folding of cytochrome c to be a special case, following principles different from those used to describe the folding of proteins without cofactors. Recent investigations, however, demonstrate that common models describing folding for many proteins work well for cytochrome c when heme is explicitly introduced, generally providing results that agree with experimental observations. In this Account, we first discuss results from simple native structure-based models. These models include attractive interactions between nonadjacent residues only if they are present in the crystal structure at pH 7. Because attractive nonnative contacts are not included in native structure-based models, their energy landscapes can be described as "perfectly funneled". In other words, native structure-based models are energetically guided towards the native state and contain no energetic traps that would hinder folding. Energetic traps are denoted sources of "frustration", which cause specific transient intermediates to be populated. Native structure-based models do, however, include repulsion between residues due to excluded volume. Nonenergetic traps can therefore exist if the chain, which cannot cross over itself, must partially unfold so that folding can proceed. The ability of native structure-based models to capture this kind of motion is partly responsible for their successful predictions of folding pathways for many types of proteins. Models without frustration describe the sequence of folding events for cytochrome c well (as inferred from hydrogen-exchange experiments), thereby justifying their use as a starting point. At low pH, the experimentally observed folding sequence of cytochrome c deviates from that at pH 7 and from models with perfectly funneled energy landscapes. Here, alternate folding pathways are a result of "chemical frustration". This frustration arises because some regions of the protein are destabilized more than others due to the heterogeneous distribution of titratable residues that are protonated at low pH. Beginning with native structure-based terms, we construct more complex models by adding chemical frustration. These more complex models only modestly perturb the energy landscape, which remains, overall, well funneled. These perturbed models can accurately describe how alternative folding pathways are used at low pH. At alkaline pH, cytochrome c populates distinctly different structural ensembles. For instance, lysine residues are deprotonated and compete for the heme ligation site. The same models that can describe folding at low pH also predict well the structures and relative stabilities of intermediates populated at alkaline pH. The success of models based on funneled energy landscapes suggest that cytochrome c folding is driven primarily by native contacts. The presence of heme appears to add chemical complexity to the folding process, but it does not require fundamental modification of the general principles used to describe folding. Moreover, its added complexity provides a valuable means of probing the folding energy landscape in greater detail than is possible with simpler systems.
亚铁血红素辅因子通过共价键结合，在细胞色素 c 的折叠中起着主导作用。由于血红素的无机化学性质复杂，有些人可能认为细胞色素 c 的折叠是一个特殊情况，遵循的原则不同于那些用于描述没有辅因子的蛋白质折叠的原则。然而，最近的研究表明，当明确引入血红素时，描述许多蛋白质折叠的常见模型通常可以很好地适用于细胞色素 c，并且通常与实验观察结果一致。在本报告中，我们首先讨论了基于简单天然结构的模型的结果。这些模型仅包括非相邻残基之间的吸引力相互作用，如果它们在 pH 值为 7 时存在于晶体结构中。由于基于天然结构的模型不包括非天然的吸引力接触，因此它们的能量景观可以被描述为“完美的漏斗形”。换句话说，基于天然结构的模型在能量上被引导到天然状态，并且没有能量陷阱会阻碍折叠。能量陷阱被表示为“挫折”的来源，这会导致特定的瞬时中间体被占据。然而，基于天然结构的模型确实包括由于排除体积而引起的残基之间的排斥。如果链不能交叉自身，那么部分展开以进行折叠，因此可以存在非能量陷阱。基于天然结构的模型能够捕获这种运动的能力部分解释了它们成功预测许多类型蛋白质折叠途径的原因。没有挫折的模型很好地描述了细胞色素 c 的折叠事件序列（如从氢交换实验推断），从而证明了它们作为起点的使用是合理的。在低 pH 值下，实验观察到的细胞色素 c 的折叠序列与 pH 值为 7 时的折叠序列以及具有完美漏斗形能量景观的模型不同。在这里，替代折叠途径是“化学挫折”的结果。这种挫折是由于蛋白质的某些区域由于在低 pH 值下质子化的可滴定残基的不均匀分布而比其他区域更不稳定。从基于天然结构的术语开始，我们通过添加化学挫折来构建更复杂的模型。这些更复杂的模型仅适度地扰动能量景观，总体上仍然保持很好的漏斗形。这些受扰模型可以准确描述在低 pH 值下如何使用替代折叠途径。在碱性 pH 值下，细胞色素 c 呈现出明显不同的结构集合。例如，赖氨酸残基去质子化并争夺血红素的配位位置。可以描述低 pH 值下折叠的相同模型也很好地预测了碱性 pH 值下存在的中间体的结构和相对稳定性。基于漏斗形能量景观的模型的成功表明，细胞色素 c 的折叠主要由天然接触驱动。血红素的存在似乎为折叠过程增加了化学复杂性，但它不需要对用于描述折叠的一般原则进行根本性的修改。此外，其增加的复杂性提供了一种有价值的手段，可以比更简单的系统更详细地探测折叠能量景观。
