Exploring Protein Folding Landscapes by Circular Permutation

通过循环排列探索蛋白质折叠景观

• 批准号: 8650905
• 负责人: Niels Hjorth Andersen
• 金额: $ 25.98万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8650905
• 关键词: 3-Dimensional; Address; Amides; Amino Acid Sequence; Amino Acids; Biological Models; Cleaved cell; Communities; Cyclization; Data; Databases; Disease; Distal; Docking; Elements; Event; Fluorescence; Goals; Head; Helix-Turn-Helix Motifs; Human; Intervention; Knowledge; Length; Life; Location; Measures; Medical; Metal Binding Site; Modeling; Molecular Conformation; Monitor; Mutation; Parents; Pathway interactions; Peptide Sequence Determination; Peptides; Plague; Protein Engineering; Proteins; Recycling; Relaxation; Research; Resolution; Series; Simulate; Site; Structure; Surface; System; Techniques; Temperature; Testing; Time; Uncertainty; Ursidae Family; Work; chromophore; design; disulfide bond; human disease; innovation; insight; melting; molecular dynamics; movie; polypeptide; protein folding; protein misfolding; research study; simulation; therapeutic protein; tool; villin

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).

描述（适用提供）：蛋白质折叠问题仍未解决。该领域的最新努力集中在显示快速折叠的小蛋白上，也适合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似乎都不是循环置换研究的主题。为此，我们选择了TRP式式（TC），Villin头饰（HP36）和井井有条的WW域（PIN1）。我们新设计的这些蛋白质的圆形排列物保留了类似天然的结构，尽管链实际上是在新位置进行了自行车和清除的。圆形置换代表了折叠途径的强大探针，因为它可以回收可以驱动折叠并因此将折叠拓扑分开的蛋白质序列的键相互作用的接触顺序。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松弛动力学，UV-resonance-raman（UVRR）和荧光监测的T-Jumps。通过应用所有这些动力学和熔化措施，监视序列中众多站点的内源性问题，我们应该能够区分谨慎（即使多个）折叠途径和下坡折叠方案。预计可得出完整的实验折叠景观，可用于与计算折叠研究进行比较。这些模型系统可以解决的特定蛋白质折叠问题是：1）接触顺序对动态和折叠途径选择的影响（来自所有三个系统），2）串行与全面的疏水核心形成（TC和HP36），3）在多个特定螺旋/螺旋/螺旋/螺旋/螺旋中相互作用（helix/loop）的时间和helix/loop and-helix/loop protworce and-plph proteins（ - 表构成（发夹成核与效果更长的循环构象搜索时间）（WW域）。