BIOPHYSICAL & MOLECULAR BIOLOGICAL STUDIES OF HEMOGLOBIN

描述(申请人提供)：我们提议的研究的目标是提供详细的结构描述，说明O2如何结合到血红蛋白(Hb)分子的一个亚基上，从而改变相邻亚基中的血红素-铁原子的可及性和反应性。尽管Hb是研究得最多的蛋白质之一，但其结构和功能的许多细节还没有完全了解，几个方面也存在争议。简单的二结构变构模型不能解释Hb协同氧化的立体化学机制。例如，Hb在晶体中至少有三种四元结构(T、R和R2)，并且Hb在晶体中的功能性质与在溶液中的功能性质明显不同。最近的多核核磁共振结果表明，人正常成人Hb的CO形式的溶液结构是R和R2晶体结构的动态系综。为了将Hb在生理条件下的结构、动力学和功能联系起来，并解决从Hb晶体和Hb在溶液中的研究得出的相互矛盾的结果，我们需要知道Hb的结构和动力学作为溶液中氧化的函数的细节。我们计划将生物化学、生物物理、分子生物学和结构生物学的技术应用到我们的Hb研究中，并将从核磁共振光谱、X射线结晶学、共振拉曼光谱、计算机模拟以及O2和其他配体与Hb结合的平衡和动力学研究中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，这有助于我们的研究。我们的具体目标是：(I)确定a^界面与a$2界面变化的相对功能和结构重要性；(Ii)评估差异配体与a-和(3-链)结合对协同O2结合表达的影响；以及(Iii)研究变构效应(H+离子和有机磷酸盐)对血红素-铁原子近端几何结构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。我们所获得的知识还可以为开发新一代基于Hb的氧气载体和治疗镰状细胞性贫血患者的新方法提供见解。HB的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。

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.