Exploring Protein Folding Landscapes by Circular Permutation

描述（由申请人提供）：蛋白质折叠问题仍未解决。该领域最近的研究集中在显示快速折叠的小蛋白质上，并且也适用于计算分子动力学模拟折叠。令人惊讶的是，这些蛋白质折叠范式中似乎没有一个是循环排列研究的主题。为此，我们选择了色氨酸笼（TC）、绒毛头套（HP36）和一个经过充分研究的www结构域（Pin1）。我们新设计的这些蛋白质的圆形置换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。圆形排列代表了折叠途径的强大探针，因为它循环了可以驱动折叠的关键相互作用的接触顺序，从而将折叠拓扑从蛋白质序列中分离出来。我们的循环排列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：核磁共振弛豫动力学，紫外共振拉曼（UVRR）和荧光监测t跳。通过应用所有这些动态和融化措施，监测序列中众多位点的内源性探针，我们应该能够区分离散（即使是多重）折叠途径和下坡折叠情况。我们期望得到完整的实验折叠景观，可用于与计算折叠研究的比较。这些模型系统可以解决的具体蛋白质折叠问题有：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)串联与一次性疏水性核心形成（TC和HP36）， 3)全α蛋白框架中多个特定螺旋/螺旋和螺旋/环相互作用形成的时间（HP36），以及4)¿-片形成途径（发夹成核与更长的环构象搜索时间的影响）（WW结构域）。

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.