Rosen Laura E, Connell Katelyn B, Marqusee Susan
Biophysics Graduate Group, Institute for Quantitative Biosciences-Berkeley, University of California, Berkeley, CA 94720-3220.
Institute for Quantitative Biosciences-Berkeley, University of California, Berkeley, CA 94720-3220 Chemical Biology Graduate Program.
Proc Natl Acad Sci U S A. 2014 Oct 14;111(41):14746-51. doi: 10.1073/pnas.1410630111. Epub 2014 Sep 25.
The molten globule, a conformational ensemble with significant secondary structure but only loosely packed tertiary structure, has been suggested to be a ubiquitous intermediate in protein folding. However, it is difficult to assess the tertiary packing of transiently populated species to evaluate this hypothesis. Escherichia coli RNase H is known to populate an intermediate before the rate-limiting barrier to folding that has long been thought to be a molten globule. We investigated this hypothesis by making mimics of the intermediate that are the ground-state conformation at equilibrium, using two approaches: a truncation to generate a fragment mimic of the intermediate, and selective destabilization of the native state using point mutations. Spectroscopic characterization and the response of the mimics to further mutation are consistent with studies on the transient kinetic intermediate, indicating that they model the early intermediate. Both mimics fold cooperatively and exhibit NMR spectra indicative of a closely packed conformation, in contrast to the hypothesis of molten tertiary packing. This result is important for understanding the nature of the subsequent rate-limiting barrier to folding and has implications for the assumption that many other proteins populate molten globule folding intermediates.
熔球态是一种构象集合体，具有显著的二级结构，但三级结构只是松散堆积，有人认为它是蛋白质折叠过程中普遍存在的中间体。然而，很难评估瞬时存在的物种的三级堆积情况来验证这一假设。已知大肠杆菌核糖核酸酶H在折叠的限速屏障之前会形成一种中间体，长期以来人们一直认为这种中间体是熔球态。我们通过两种方法构建了处于平衡态的基态构象的中间体模拟物，来研究这一假设：一种是截短以生成中间体的片段模拟物，另一种是使用点突变选择性地破坏天然态的稳定性。光谱表征以及模拟物对进一步突变的响应与对瞬态动力学中间体的研究一致，表明它们模拟了早期中间体。与熔球态三级堆积的假设相反，这两种模拟物都能协同折叠，并呈现出表明紧密堆积构象的核磁共振谱。这一结果对于理解后续折叠限速屏障的性质很重要，并且对许多其他蛋白质存在熔球态折叠中间体这一假设也有影响。
