PROTEIN FOLDING PATHWAYS

本文所述研究的广泛、长期目标 建议的目的是阐明的机制途径， 小球状蛋白质从变性状态到天然状态的折叠 构象 第二个补充目标是确定 精确的氨基酸序列对这些特定的影响， 折叠路径。 分析将首先集中在折叠 核糖核酸酶T1，一种由104个氨基酸残基组成的小酶。 在天然构象中，核糖核酸酶T1含有两个二硫键， α-螺旋和β-折叠二级结构和酰胺键， 顺式构型的脯氨酸残基。 蛋白质折叠 途径将通过测量的时间过程进行探索， 在从分子中分离的过程中， 将未折叠状态转化为天然构象。 时间进程为 单个H键形成将使用二维 核磁共振波谱学 通过这种方法， 蛋白质将在D2O中展开以标记所有酰胺氮原子 氘。 变性剂将被稀释到临界浓度以下。 浓度和蛋白质允许重新折叠特定（和 可变）时间长度（5 ms-10 s）。 然后将pH升高， 在H2O存在下短时间（50 ms）标记，用氢， 所有的酰胺基团在反应过程中没有形成氢键， 再折叠期 然后用酸降低pH以淬灭任何 进一步的氢/氘酰胺交换，使蛋白质 完成折叠过程。 个体的占用率（H与D） 随后通过2D-COSY NMR分析确定酰胺位点， 500兆赫。 未折叠蛋白质的状态对时间的影响 氢键形成的课程将通过具体的 一级序列的改变和各种折叠条件。 这些研究将集中在四种半胱氨酸的氧化态上， 参与二硫键形成的残基和两个脯氨酸 已知为顺式构象的残基。 定点突变体 蛋白质将在这些位点上用特定的氨基酸 不能形成二硫键或以顺式存在的残基 构象 天然蛋白质的环状排列变体 将构建核糖核酸酶T1的序列以确定 肽段转位对特异性折叠的影响 通路 这些环状排列的蛋白质实际上具有 通过酰胺键共价闭合的原始氨基和羧基末端 形成和新的终端内创建的各种环结构， 野生型蛋白质的天然结构。

The broad, long-term objectives for the research described in this proposal are aimed at the elucidation of the mechanistic pathways for the folding of small globular proteins from the denatured state to the native conformation. The secondary and complementary objective is to determine the specific influence of the precise amino acid sequence on these folding pathways. The analysis will focus initially on the folding of ribonuclease T1, a small enzyme consisting of 104 amino acid residues. In the native conformation ribonuclease T1 contains two disulfide bonds, alpha-helix and beta-sheet secondary structures and amide bonds to two proline residues that are in the cis-configuration. The protein folding pathways will be probed by measurement of the time courses for the formation of individual hydrogen bonds during the transformation from the unfolded state to the native conformation. The time courses for individual H-bond formation will be measured using two-dimensional nuclear magnetic resonance spectroscopy. With this methodology the protein will be unfolded in D2O to label all of the amide nitrogen atoms with deuterium. The denaturant will be diluted below the critical concentration and the protein allowed to refold for specific (and variable) lengths (5 ms - 10 s) of time. the pH will then be raised for a short period (50 ms) in the presence of H2O to label, with hydrogen, all of the amide groups that have not formed hydrogen bonds during the refolding period. The pH will then be lowered with acid to quench any further hydrogen/deuterium amide exchange and the protein allowed to complete the folding process. The occupancy (H vs. D) at individual amide sites will be subsequently determined by 2D-COSY NMR analysis at 500 MHz. The influence of the state of the unfolded protein on the time courses for hydrogen bond formation will be addressed by specific alterations in the primary sequence and various folding conditions. these studies will focus on the oxidation state of the four cysteine residues involved in disulfide bond formation and the two proline residues known to be in the cis-conformation. Site-directed mutant proteins will be constructed at these sites with specific amino acid residues that cannot form disulfide bonds or exist in the cis- conformation. Circularly permutized variants of the native protein sequence of ribonuclease T1 will be constructed in order to determine the effects of transposition of peptide segments on the specific folding pathway. These circularly permuted proteins will, in effect, have the original amino- and carboxy-terminal ends covalently closed by amide bond formation and new termini created within the various loop structures in the native structure of the wild-type protein.