Exploring Protein Folding Landscapes by Circular Permutation

描述（申请人提供）：蛋白质折叠问题仍未解决。最近在这一领域的努力集中在显示快速折叠的小蛋白质，也服从于计算分子动力学模拟折叠。令人惊讶的是，这些蛋白质折叠模式似乎都不是循环排列研究的主题。我们已经选择了Trp笼（TC），绒毛蛋白的头部（HP 36），和一个良好的研究WW域（Pin 1）为此目的。我们新设计的这些蛋白质的环状排列体保留了天然的结构，尽管这些链实际上在新的位置被环化和切割。循环排列是研究折叠途径的有力工具，因为循环排列揭示了驱动折叠的关键相互作用的接触顺序，从而将折叠拓扑结构与蛋白质序列分开。我们的循环置换模型系统将用于解决有关蛋白质折叠动力学和折叠途径选择的主要问题。将采用三种不同的光谱技术：NMR弛豫动力学，紫外-共振-拉曼（UVRR）和荧光监测的T-跳跃。通过应用所有这些动力学和解链措施，监测序列中许多位点的内源性探针，我们应该能够区分离散（即使是多个）折叠途径和下坡折叠方案。我们预计得出完整的实验折叠景观，可用于与计算折叠研究的比较。可以用这些模型系统解决的特定蛋白质折叠问题是：1）接触顺序对动力学和折叠途径选择的影响（来自所有三个系统），2）连续相对于一次全部疏水核形成（TC和HP 36），3）在全α蛋白框架（HP 36）中形成多个特异性螺旋/螺旋和螺旋/环相互作用的时间，和4）形成U-折叠的途径（发夹形核与较长的环构象搜索时间的影响）（WW结构域）。 公共卫生相关性：蛋白质错误折叠与40多种人类疾病和病症有关;这些医疗状况导致人类痛苦并造成巨大的社会负担。拟议的研究将产生一个更好的理解蛋白质序列和拓扑结构的特点，导致折叠动力学阻滞或折叠陷阱，可以提高错误折叠。这将是一个有价值的补充知识，可以提高蛋白质折叠预测从序列，提供基本的见解错误折叠途径，并可以提供治疗和蛋白质工程干预的目标。

DESCRIPTION (provided by applicant): The protein folding problem remains unsolved. Recent efforts in this field have focused on small proteins that display fast folding and are also amenable to computational molecular dynamics simulated folding. Surprisingly none of these protein folding paradigms appear to have been the subject of circular permutation studies. We have selected the Trp-cage (TC), the villin headpiece (HP36), and a well studied WW domain (Pin1) for this purpose. Our newly designed circular permutants of these proteins retain native-like structure, though the chains were, in effect, cyclized and cleaved at new locations. Circular permutation represents a powerful probe of folding pathways since it recycles the contact order of key interactions that can drive folding and thus separates fold topology from protein sequence. Our circular permutant model systems will be used to address major questions concerning protein folding dynamics and folding pathway selection. Three distinct spectroscopic techniques will be employed: NMR relaxation dynamics, and UV-resonance-Raman (UVRR) & fluorescence monitored T-jumps. By applying all of these dynamics and melting measures, monitoring the endogenous probes at numerous sites in the sequences, we should be able to distinguish between discreet (even if multiple) folding pathways and downhill folding scenarios. We anticipate deriving complete experimental folding landscapes that can be used for comparisons with computational folding studies. The specific protein folding questions that can be addressed with these model systems are: 1) contact order effects on dynamics and folding pathway selection (from all three systems), 2) serial versus all-at-once hydrophobic core formation (TC and HP36), 3) the timing of the formation of multiple specific helix/helix and helix/loop interactions in an all-alpha protein framework (HP36), and 4) pathways of ¿-sheet formation (hairpin nucleation versus the effects longer loop conformation search times) (WW domains). PUBLIC HEALTH RELEVANCE: Protein misfolding is associated with more than 40 human diseases and conditions; these medical conditions cause human suffering and exact a tremendous societal burden. The proposed studies will yield a greater understanding of protein sequence and topology features that result in folding dynamics retardation or folding traps that can enhance misfolding. This would be a valuable addition to knowledge that could enhance both protein fold prediction from sequence, providing fundamental insights into misfolding pathways and could provide targets for therapeutic and protein engineering interventions.