Elucidating Protein Folding Mechanisms

描述（由申请人提供）：功能性蛋白质的生产需要多肽在大量不正确折叠的空间中找到独特的构象。失败的后果是严重的;错误折叠的蛋白质与迅速增长的衰弱性疾病清单有关，包括II型糖尿病以及阿尔茨海默病和克雅氏病。部分折叠的多肽是蛋白质正确组装和形成有害错误折叠结构的关键中间体。 荧光能量转移（FET）动力学的测量可以提供染料标记的蛋白质中供体-受体距离分布的纳秒时间尺度快照，因为它们演变到天然状态。这些实验可以揭示折叠过程中存在的多肽构象。实验设计有四个具体目标：（1）阐明折叠过程中快速平衡的紧凑和伸展结构的相对能量学;（2）表征紧凑折叠中间体的结构和异质性;（3）探索在重折叠之前通过形成天然和近天然接触减少构象空间的影响;（4）血红素蛋白折叠过程中核心水合程度的测定。 天然色氨酸荧光团以及共价连接的染料将作为供体和受体的研究中的紧凑扩展的构象平衡在折叠的细胞色素c和c '。紧凑和扩展的多肽在酰基辅酶A结合蛋白的折叠中的作用也将通过FET动力学进行研究。蛋白质通常折叠的细胞内环境中密集地填充有大分子：FET动力学将用于探测细胞色素c折叠期间高大分子浓度对紧凑扩展构象平衡的影响。将使用FET动力学测量来评价紧凑折叠中间体的构象异质性。未折叠血红素蛋白中的天然和近天然接触将通过在突变体中引入His残基来产生：这些接触对折叠的影响将在Fe（III）蛋白以及Co（III）和Zn（II）取代的衍生物中进行检查。将测量溶剂氘同位素对Zn-细胞色素c'的三重态寿命的影响，以评估折叠期间卟啉的溶剂化的变化。

DESCRIPTION (provided by applicant): The production of functional proteins requires that polypeptides find a unique conformation in a vast space of incorrect folds. The consequences of failure are severe; misfolded proteins are implicated in a rapidly growing list of debilitating illnesses that includes type II diabetes as well as AIzheimer's and Creutzfeldt-Jakob diseases. Partially folded polypeptides are key intermediates in both the proper assembly of proteins and in the formation of harmful misfolded structures. Measurements of fluorescence-energy-transfer (FET) kinetics can provide nanosecond timescale snapshots of donor-acceptor distance distributions in dye-labeled proteins as they evolve to native states. These experiments can reveal the polypeptide conformations that are present during folding. Experiments have been designed to address four specific aims: (1) elucidation of the relative energetics of rapidly equilibrating compact and extended structures during folding; (2) characterization of the structures and heterogeneity of compact folding intermediates; (3) exploration of the effects of reduced conformational space by forming native and near-native contacts prior to refolding; and (4) determination of the extent of core hydration during heme protein folding. Natural tryptophan fluorophores as well as covalently attached dyes will serve as donors and acceptors in studies of compact-extended conformational equilibria during the folding of cytochromes c and c'. The role of compact and extended polypeptides in the folding of acyl-coenzyme A-binding protein also will be investigated by FET kinetics. The intracellular environment in which proteins normally fold is densely packed with macromolecules: FET kinetics will be used to probe the effects of high macromolecular concentrations on the compact-extended conformational equilibrium during folding of cytochrome c. The conformational heterogeneity of compact folding intermediates will be evaluated using FET kinetics measurements. Native and near-native contacts in unfolded heme proteins will be created by the introduction of His residues in mutants: the effects of these contacts on folding will be examined in Fe(lll) proteins as well as Co(Ill)- and Zn(ll)-substituted derivatives. Solvent deuterium isotope effects on the triplet lifetime of Zn-cytochrome c' will be measured to evaluate changes in solvation of the porphyrin during folding.