DETERMINANTS OF TERTIARY STRUCTURE IN MYOGLOBINS

肌红蛋白三级结构的决定因素

• 批准号: 3298626
• 负责人: NEVILLE Robert KALLENBACH
• 金额: $ 15.47万
• 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 1994-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3298626
• 关键词: X ray crystallography; circular dichroism; conformation; genetic manipulation; hemoprotein structure; high performance liquid chromatography; myoglobin; nuclear magnetic resonance spectroscopy; protein folding; protein structure function; site directed mutagenesis; synthetic peptide; thermodynamics

An experimental investigation of the stability and determinants of tertiary interactions in globular proteins is proposed. The helical protein myoglobin (Mb) is chosen for detailed study, because it crystallizes readily, its structure has been determined to high resolution, and the function, dynamics and thermodynamics of he molecule have been analyzed intensively. Variant peptides and proteins will be produced by: 1. site-directed mutagenesis of a cloned gene for sperm whale Mb in the plasmid pMB 413, in collaboration with Dr. S. Sligar and 2. solid state synthesis of fragments corresponding to the C-terminal 22 residues of the chain. The latter fragment from the H helix results from CNBr cleavage of Mb, and is helical in solution. We find it can be relegated with the residual 1-131 fragments to yield a native-like Mb containing two homoserines at sties 55 and 131. The spectroscopic, liganding, folding and NMR spectral properties of the synthetic fragments-MbC3 -and mutant Mb's will be determined. We will attempt to crystallize the mutants molecules, starting with Kendrew's standard ammonium sulfate conditions for sperm whale Mb, and compare the structure of these with the "wild-type" species by X-ray crystallographic methods. A parallel analysis of the structural differences among peptides and mutant proteins will be initiated using 2D NMR spectroscopy. In this way, we hope to answer two important questions about protein folding. First, what is the relationship, if any, between the stability of an isolated helical fragment from a protein such as myoglobin to that of the intact protein? Second, can one establish structural, dynamic and energetic requirements for packing alpha helices within a protein matrix? Specifically, the tertiary interactions controlling three of the H helix subdomain packings will be analyzed, using structural data to characterize the local folding and changes in this folding in this folding on the one hand, and spectroscopic and thermodynamic data to define the corresponding changes in the function, stability, energetics and dynamics of the proteins on the other.

对稳定性和决定因素的实验研究 提出了球状蛋白中的三级相互作用。 这 选择螺旋蛋白肌红蛋白（MB）进行详细研究， 因为它很容易结晶，所以已经确定了其结构 高分辨率以及功能，动力学和热力学 HE分子已进行了深入分析。 变体肽 蛋白质将由以下方式生产：1。 质粒PMB 413中的晶石鲸MB的克隆基因，在 与S. Sligar博士的合作和2。 对应于链的C末端22残基的片段。 H螺旋的后一个片段是由CNBR裂解引起的 MB，在溶液中是螺旋的。 我们发现它可以降级 残留的1-131片段，产生包含天然的MB 两个同层在55和131中。光谱，配体， 合成片段的折叠和NMR光谱特性MBC3 - 将确定MB的突变体。 我们将尝试 从肯德鲁的标准开始，结晶突变体分子 硫酸铵条件的肺鲸MB，并比较 X射线的“野生型”物种的结构 晶体学方法。 对结构的平行分析 肽和突变蛋白之间的差异将开始 使用2D NMR光谱法。 这样，我们希望回答两个 有关蛋白质折叠的重要问题。 首先，什么是 隔离螺旋的稳定性之间的关系（如果有的话） 从蛋白质（例如肌红蛋白）到完整的碎片 蛋白质？ 其次，一个人可以建立结构，动态和 在蛋白质中包装α螺旋的能量要求 矩阵？ 具体而言，控制三个的三级互动 使用H螺旋子域包装中的 结构数据以表征本地折叠和变化 一方面折叠的折叠，光谱和 热力学数据定义了相应的变化 蛋白质的功能，稳定性，能量和动力学 其他。