BIOPHYSICAL & MOLECULAR BIOLOGICAL STUDIES OF HEMOGLOBIN

生物物理

• 批准号: 7924974
• 负责人: CHIEN HO
• 金额: $ 5.78万
• 依托单位: CARNEGIE-MELLON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7924974
• 关键词: Accounting; Adult; Allosteric Regulation; Basic Science; Binding; Biochemistry; Biological; Biophysics; Bohr effect; Code; Computer Simulation; Conflict (Psychology); Data; Development; Distal; Enzymes; Equilibrium; Escherichia coli; Generations; Goals; Grant; Heme Iron; Hemoglobin; Human; Ions; Kinetics; Knowledge; Ligand Binding; Ligands; Medical; Medicine; Methodology; Modeling; Molecular; Molecular Biology; NMR Spectroscopy; Nuclear Magnetic Resonance; Oxygen; Patients; Phosphoric Acid Esters; Physiological; Property; Proteins; Raman Spectrum Analysis; Relative (related person); Research; Research Project Grants; Resolution; Roentgen Rays; Sickle Cell Anemia; Solutions; Structure; System; Techniques; Technology; Variant; X ray diffraction analysis; X-Ray Crystallography; X-Ray Diffraction; base; insight; mutant; novel strategies; structural biology

DESCRIPTION (provided by applicant): The goal of our proposed research is to provide a detailed structural description of how O2 binding to one subunit of the hemoglobin (Hb) molecule can alter the accessibility and reactivity of the heme-iron atoms in adjacent subunits. Even though Hb is one of the best-studied proteins, many details of its structure and function are not fully understood and several aspects are controversial. Simple two-structure allosteric models cannot account for the stereochemical mechanism for the cooperative oxygenation of Hb. For example, there are at least three quaternary structures (T, R, and R2) of Hb in crystals and the functional properties of Hb in crystals are distinctly different from those in solution. Recent multinuclear nuclear magnetic resonance (NMR) results indicate that the solution structure of human normal adult Hb in the CO form is a dynamic ensemble of the R and R2 crystal structures. In order to correlate the structure, dynamics, and function of Hb under physiological conditions and to resolve the conflicting results derived from studies obtained from Hb crystals and Hb in solution, we need to know the details of the structures and dynamics of Hb as a function of oxygenation in solution. We plan to apply techniques of biochemistry, biophysics, molecular biology, and structural biology to our Hb research and to correlate the results obtained from NMR spectroscopy, X-ray crystallography, resonance Raman spectroscopy, computer modeling, and equilibrium and kinetic studies of the binding of O2 and other ligands to Hb. With our Escherichia coli expression system for Hb, we can express any desired mutants of Hb that can aid in our study. Our specific aims are: (i) to determine the relative functional and structural importance of changes at the a^ interface vs the a$2 interface; (ii) to evaluate the effects of differential ligand binding to the a- and (3-chains on the expression of cooperative O2 binding; and (iii) to investigate the effects of allosteric effectors (H+ ions and organic phosphates) on the proximal geometry and distal accessibility of the heme-iron atoms to describe the structural basis behind the Bohr effect and the effect of organic phosphates. The knowledge that we gain can also provide insights into the development of a new generation of Hb-based oxygen carriers and of a new approach to the treatment of patients with sickle cell anemia. Hb research is a good illustration of how discoveries from basic research on proteins can make important contributions to medicine and technology.

描述（由申请人提供）：我们提出的研究的目标是提供详细的结构描述，说明 O2 与血红蛋白 (Hb) 分子的一个亚基结合如何改变相邻亚基中血红素铁原子的可及性和反应性。尽管 Hb 是研究最充分的蛋白质之一，但其结构和功能的许多细节尚未完全了解，并且在几个方面存在争议。简单的二结构变构模型无法解释 Hb 协同氧化的立体化学机制。例如，晶体中的 Hb 至少有 3 个四级结构（T、R 和 R2），晶体中 Hb 的功能特性与溶液中的 Hb 明显不同。最近的多核核磁共振（NMR）结果表明，人类正常成人血红蛋白CO形式的溶液结构是R和R2晶体结构的动态系综。为了关联生理条件下 Hb 的结构、动力学和功能，并解决从 Hb 晶体和溶液中 Hb 获得的研究中得出的相互矛盾的结果，我们需要了解 Hb 的结构和动力学细节作为溶液中氧合的函数。我们计划将生物化学、生物物理学、分子生物学和结构生物学技术应用于 Hb 研究，并将 NMR 光谱、X 射线晶体学、共振拉曼光谱、计算机建模以及 O2 和其他配体与 Hb 结合的平衡和动力学研究所获得的结果关联起来。利用我们的 Hb 大肠杆菌表达系统，我们可以表达任何所需的 Hb 突变体，以帮助我们的研究。我们的具体目标是：(i) 确定 a^ 接口与 a$2 接口变化的相对功能和结构重要性； （ii）评估不同配体结合到α-和（3-链）对协同O2结合表达的影响；（iii）研究变构效应子（H+离子和有机磷酸盐）对血红素铁原子的近端几何形状和远端可及性的影响，以描述玻尔效应和有机磷酸盐效应背后的结构基础。我们获得的知识还可以提供 深入了解新一代血红蛋白氧载体的开发以及治疗镰状细胞性贫血患者的新方法。血红蛋白研究很好地说明了蛋白质基础研究的发现如何为医学和技术做出重要贡献。