Folding and dynamics of protein chimeras build from two different folds

由两种不同折叠构建的蛋白质嵌合体的折叠和动力学

• 批准号: 445695864
• 负责人: Professorin Dr. Birte Höcker
• 金额: --
• 依托单位: Arbeitsgruppe Biochemie III - Protein Design
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/445695864?language=en
• 关键词: Folding; dynamics; protein; chimeras; build

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.

蛋白质片段的重组是出现新的蛋白质折叠和现有蛋白质折叠多样化的一种可能的机制。大自然是否选择将后代蛋白质的错误折叠和聚集降至最低的片段进行繁殖？另外，新蛋白质的稳定性和折叠是相等的还是不对称的“部分之和”，或者它独立于亲本蛋白质？在重组过程中编码的任何片段特异性信息都可能会丢失，从而将蛋白质折叠中的无效路径减少到突变漂移。然而，对通过嵌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防止错误折叠和聚集提供线索。在此背景下，我们研究了亚域大小的蛋白质片段融合所产生的嵌合蛋白质的折叠和动力学。其中一个嵌合体是CheYHisF，它是由黄毒素样折叠的趋化反应调节蛋白Chey和Tim-or(Ba)8桶折叠的组氨酸生物合成途径蛋白HISF产生的。CheYHisF在氯化胍(GdmCl)中通过平衡中间体可逆展开。有趣的是，当嵌合体在尿素中变性时，这种中间体不会被填充。通过核磁共振光谱研究嵌合体在天然状态下测量的主链酰胺松弛的动力学，发现单个残基在毫秒时间尺度上是高度动态的。这种残基存在于Chey和HISF片段的交界处，它的突变显著改变了自然态和平衡中间体的稳定性。在这里，我们的目的是使突变对嵌合体中从其亲本蛋白继承的疏水簇的重新分布的稳定性和折叠的影响合理化。此外，我们还将研究另外两个相关的CheYHisF嵌合体，利用核磁共振光谱探测它们在多个时间尺度上的构象动力学，以确定高柔性的位置，然后使用设计规则来改进界面填充。我们还将研究三个合成嵌合体的折叠路径，并将其与亲本蛋白质进行比较，以揭示这些同源物和现有同源物之间的异同，并了解成功重组片段的规则。