Elucidating Protein Folding Mechanisms

描述(申请人提供)：功能蛋白质的生产需要多肽在一个巨大的错误折叠空间中找到唯一的构象。失败的后果是严重的；错误折叠的蛋白质与迅速增长的致衰性疾病清单有关，其中包括II型糖尿病以及艾兹海默氏症和克雅氏病。部分折叠的多肽是蛋白质正确组装和形成有害的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蛋白质进化到自然状态时供体-受体距离分布的纳秒时间快照。这些实验可以揭示折叠过程中存在的多肽构象。实验旨在解决四个具体目标：(1)阐明折叠过程中快速平衡紧凑和延伸结构的相对能量学；(2)紧凑折叠中间体的结构和异质性的表征；(3)通过在折叠前形成本地和近本地接触来探索构象空间减少的影响；以及(4)确定血红素蛋白折叠过程中核心水化的程度。 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，研究细胞色素c和c‘折叠过程中的紧凑-伸展构象平衡。紧凑和延伸的多肽在酰基辅酶A结合蛋白折叠中的作用也将通过FET动力学进行研究。蛋白质正常折叠的细胞内环境是大分子密集堆积的环境：FET动力学将被用来探索高分子浓度对细胞色素c折叠过程中紧凑-伸展构象平衡的影响。紧凑折叠中间体的构象异质性将通过FET动力学测量来评估。在未折叠的血红素蛋白中，通过在突变体中引入His残基，将建立天然和近天然的接触：这些接触对折叠的影响将在Fe(111)蛋白质以及Co(111)和Zn(11)取代的衍生物中进行研究。将通过测量溶剂中的氚同位素对锌-细胞色素c‘的三重态寿命的影响来评估在折叠过程中卟啉的溶剂化作用的变化。

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.