PROTEIN FOLDING PATHWAYS

本文件中描述的研究的广泛、长期目标 建议的目的是阐明生物多样性的机制途径。 小球状蛋白从变性状态到天然状态的折叠 构象。次要和补充目标是确定 精确的氨基酸序列对这些基因的特异性影响 折叠的小路。分析最初将重点放在折叠 核糖核酸酶T1，是一种由104个氨基酸残基组成的小酶。 在天然构象核糖核酸酶T1中包含两个二硫键， α-螺旋和β-折叠二级结构和两个酰胺键 顺式构型中的脯氨酸残基。蛋白质折叠 将通过测量时间进程来探索路径 在转变过程中形成单独的氢键 展开到自然构象的状态。的时间进程 单个氢键的形成将使用二维 核磁共振光谱学。在这种方法下， 蛋白质将在D2O中展开，以标记所有的酰胺氮原子 用重氢。变性剂将被稀释到临界温度以下 浓度和允许折叠的蛋白质特定的(和 可变)时间长度(5毫秒-10 S)。然后将酸碱度提高到 在H2O存在下的短时间段(50ms)，用氢标记， 所有在反应中没有形成氢键的酰胺基团 折叠期。然后用酸降低pH值以熄灭任何 进一步的氢/重胺交换和蛋白质被允许 完成折叠过程。个人入住率(H与D) 随后将通过2D-COSY核磁共振分析确定酰胺位置 500兆赫。蛋白质未折叠状态对时间的影响 氢键形成的课程将由特定的 初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状态上 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构建 不能形成二硫键或存在于顺式- 构象。天然蛋白质的循环置换变体 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 路径。这些循环排列的蛋白质实际上将具有 原始的氨基和羧基末端通过酰胺键共价闭合 形成和在各种环路结构中创建的新终点站 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.