ALPHA HELIX STABILIZATION--ITS ROLE IN PROTEIN FOLDING

阿尔法螺旋稳定性——它在蛋白质折叠中的作用

• 批准号: 2180567
• 负责人: NEVILLE Robert KALLENBACH
• 金额: $ 20.38万
• 依托单位: NEW YORK UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-04-01 至 1997-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2180567
• 关键词: chemical fingerprinting; circular dichroism; conformation; fluorescent dye /probe; hemoprotein structure; high performance liquid chromatography; myoglobin; nuclear magnetic resonance spectroscopy; protein folding; protein purification; protein structure; protein structure function; site directed mutagenesis; synthetic peptide; thermodynamics

The alpha helix is the most common secondary structure in the native state of proteins, involving nearly one third of the amino acids present. This project seeks to identify the interactions responsible for helix stabilization, and to determine at what stage(s) in folding a helical structure is important. The free energies of individual interactions involved in stabilizing helical structure will be determined, using synthetic model peptides and measurement of hydrogen exchange rates by 1/H NMR. The interactions to be monitored include the helical propensity of a given side chain, in various contexts; side chain-side chain interactions, including charged and hydrophobic side chains, and helix capping interactions. The role of helix stabilizing interactions in the native state and in folding intermediates of a helical protein will be investigated, using a series of mutant myoglobins and apomyoglobins. Methods include spectroscopic definition of mutant proteins in the presence of heme, using absorbance and CD. The stability of the mutant myoglobins will be determined by thermal and solvent induced unfolding. Structures of interesting proteins will be further investigated using a new surface fingerprinting technique as well as standard techniques. The structure and stability of compact, molten globular intermediate states in apomyoglobin and the protein with heme and dyes such as ANS which bind at the heme pocket will be investigated. Specific questions to be answered include: (i) What interactions control the stability of isolated alpha helical structure? (ii) Do these play a role in early intermediates in folding a protein? (iii) How is the helix content of intermediates in myoglobin folding established? (iv) Can the globin fold be redesigned using mutants at helix-helix cross-overs and interfaces? The results should provide a detailed picture of alpical structure in models and in folding of helical proteins.

α螺旋是本地状态中最常见的二级结构 蛋白质的含量，涉及近三分之一的氨基酸。 这 项目旨在确定负责螺旋的互动 稳定，并确定在折叠螺旋的哪个阶段 结构很重要。 单个互动的自由能量 将使用稳定螺旋结构参与 合成模型肽和氢汇率的测量值1/h NMR。 要监视的相互作用包括 给定的侧链，在各种情况下；侧链侧链 相互作用，包括带电和疏水的侧链以及螺旋 上限交互。 螺旋稳定相互作用在 本地状态和螺旋蛋白的折叠中间体将是 研究了一系列突变的肌红蛋白和阿霉素。 方法包括对突变蛋白的光谱定义 血红素的存在，使用吸光度和CD。 突变体的稳定性 肌球蛋白将由热和溶剂诱导的展开确定。 有趣的蛋白质结构将使用 新的表面指纹技术以及标准技术。 这 紧凑，熔融球状中间状态的结构和稳定性 apomyoglobin和带有血红素和染料（例如ANS）结合的蛋白质 血红素口袋将进行调查。 要回答的具体问题 包括：（i）哪些相互作用控制孤立α的稳定性 螺旋结构？ （ii）这些在早期中间体中发挥作用 折叠蛋白质？ （iii）中间体的螺旋含量如何 建立了肌红蛋白折叠？ （iv）可以重新设计球蛋白折 在螺旋 - 螺旋交叉和接口上使用突变体？ 结果 应该在模型和 旋转蛋白的折叠。