Folding and dynamics of protein chimeras build from two different folds

蛋白质片段的折叠是新蛋白质折叠的出现和现有折叠的多样化的一种合理机制。大自然是否选择了使后代蛋白中的错误折叠和聚集最小化的片段进行繁殖？此外，新蛋白质的稳定性和折叠是相等的或不对称的“部分之和”还是独立于亲本蛋白质？在重组过程中编码的任何片段特异性信息，以最大限度地减少蛋白质折叠中的非生产性途径，可能会丢失突变漂移。然而，通过嵌合融合现有片段获得的合成蛋白质的折叠机制和构象动力学的研究可以为自然界如何选择错误折叠和聚集提供线索。在这种情况下，我们研究的折叠和动力学的嵌合蛋白质的亚结构域大小的蛋白质片段的融合。一种这样的嵌合体是CheYHisF，其由黄素氧还蛋白样折叠的趋化反应调节蛋白CheY和TIM-或（ba）8-桶折叠的组氨酸生物合成途径蛋白HisF产生。CheYHisF通过平衡中间体在氯化胍（GdmCl）中可逆地解折叠。有趣的是，当嵌合体在尿素中变性时，该中间体不被填充。通过核磁共振光谱法在天然状态下测量的骨架酰胺弛豫来研究嵌合体的动力学，将单个残基鉴定为在毫秒时间尺度上高度动态的。该残基存在于CheY和HisF片段的界面处，并且其突变显著改变了天然状态和平衡中间体的稳定性。在这里，我们的目标是合理化的稳定性和折叠突变的影响，相对于重新分配的嵌合体中继承的疏水簇从其亲本蛋白质。 此外，我们将研究两个更多的相关嵌合体的CheYHisF探测其构象动力学多个时间尺度上使用NMR光谱，以确定高灵活性的位置，然后采用设计规则，以提高界面包装。我们还将研究三种合成嵌合体的折叠途径，并将其与亲本蛋白质进行比较，以揭示这些和现存同源物之间的相似性和差异，并了解片段成功重组的规则。

Recombination of protein fragments is a plausible mechanism for the emergence of novel protein folds and the diversification of existing ones. Does Nature select fragments for propagation that minimize misfolding and aggregation in the progeny protein? Also, is the stability and folding of the new protein an equal or asymmetric "sum of parts" or is it independent of the parent proteins? Any fragment specific information encoded during recombination to minimize unproductive pathways in protein folding would likely be lost to mutational drift. However, the study of the folding mechanism and conformational dynamics of synthetic proteins obtained by chimeric fusion of existing fragments could provide clues to how Nature selects against misfolding and aggregation. In this context, we study the folding and dynamics of chimeric proteins created by fusion of subdomain-sized protein fragments. One such chimera is CheYHisF, which was generated from the chemotactic response regulator protein CheY of the flavodoxin-like fold and the histidine biosynthetic pathway protein HisF of the TIM- or (ba)8-barrel fold. CheYHisF unfolds reversibly in guanidinium chloride (GdmCl) via an equilibrium intermediate. Interestingly, this intermediate is not populated when the chimera is denatured in urea. Studying the dynamics of the chimera by backbone amide relaxation measured in the native state via Nuclear Magnetic Resonance spectroscopy identified a single residue as being highly dynamic on the millisecond time scale. This residue is present at the interface of the CheY and HisF fragments and its mutation significantly changed the stability of the native state and the equilibrium intermediate. Here we aim to rationalize the effect of mutations on the stability and folding with respect to redistribution of the hydrophobic clusters inherited in the chimera from its parent proteins. Furthermore, we will study two more related chimeras of CheYHisF probing their conformational dynamics on multiple timescales using NMR spectroscopy to determine location of high flexibility and then employ design rules to improve interfacial packing. We will also study the folding pathways of the three synthetic chimeras and compare it to the parent proteins to reveal similarities and differences among these and extant homologues and to learn about rules for successful recombination of fragments.