STRUCTURAL AND DYNAMIC STUDY OF MODEL HEME COMPLEXES

将探索和扩展特别是O2结合的肌红蛋白和血红蛋白的顺磁性衍生物的核磁共振波谱的实验方法和应用，以阐明它们的连接和自氧化的电子和分子结构控制机制。最终目的是为了了解氧结合的分子机制，以帮助设计作为血液替代品的基因工程血红蛋白，并了解天然突变血红蛋白的病理性质。该计划继续开发有效的方法来确定血红素空腔的归属和分子结构，并将重点发展强大的基础，以根据功能相关的结构决定因素解释每一种珠蛋白氧化/自旋状态的超精细位移。后一个目标将依赖于对一系列不同的、具有结构特征的参考珠蛋白的详细研究，这些参考珠蛋白的一系列点突变体旨在扰乱活性部位的选定结构/动力学性质，以及参考和点突变珠蛋白，这些珠蛋白由修饰的血红素重组，旨在将乙烯基的菱形影响与珠蛋白空腔的这一特征分离。从His轴向、His-Fe键强度以及与结合配体的远端残基氢键和空间相互作用的角度来解释花环球蛋白的超精细位移。将建立珠蛋白诱导的超精细位移变化与巨球蛋白轴向His取向之间的关系，完善定量部分水连接的探针，并表征这种水连接的热力学和动力学。决定脱氧珠蛋白中主磁轴异常取向的远端相互作用的性质，以及血红素接触位移模式与其取向和非连接远端水的存在之间的关联将被建立。所开发的方法将被用于解决摇蚊Hb远端His的溶液与晶体取向的差异，表征Aplysia Mb热变性的平衡中间体，表征序列比对，折叠拓扑结构并鉴定一系列“迷你”或“致密”(109-118残基)珠蛋白的远端残基，表征极端O2-Avid，有时极耐自氧化的吸虫珠蛋白的远端氢键网络，以及表征伴随连接HBA变构转变的远端相互作用和近端His-Fe键应变的变化。

The experimental approaches to, and application of, NMR spectroscopy of particularly paramagnetic derivatives of O2 binding myoglobin and hemoglobin will be explored and expanded to elucidate electronic and molecular structural control mechanisms of their ligation and autoxidizability. The ultimate aim is to understand the molecular mechanism of oxygen binding so as to aid in the design of genetically engineered hemoglobin as a blood substitute and to understand the pathological properties of natural mutant hemoglobins. The program continues to develop effective methods for definitive assignments and molecular structural determination of the heme cavity, and will emphasize the development of robust bases for interpreting hyperfine shifts for each of the globin oxidation/spin states in terms of functionally relevant structural determinants. This latter goal will rely on detailed studies on a series of diverse, structurally characterized reference globins, a series of point mutants of these reference globin designed to perturb selected structurally/dynamic properties of the active site, as well as both reference and point mutated globins reconstituted with modified hemes designed to separate the rhombic influences of the vinyls from that characteristic of the globin cavity. The hyperfine shifts in cyanomet globins will be interpreted in terms of the axial His orientation, the His-Fe bond strength as well as distal residue hydrogen bonding to, and steric interaction with, the bound ligand. The correlation between globin induced changes in hyperfine shift and axial His orientation in metglobins will be established, probes to quantitate partial water ligation perfected, and the thermodynamics and dynamics of such water ligation characterized. The nature of the distal interactions that determine the unusual orientation of the major magnetic axis in deoxy globins, as well as the correlation between heme contact shift pattern and both His orientation and the presence of non-ligated distal water will be established. The developed approaches will be used to resolve differences in the solution versus crystal orientation of the distal His in Chironomus Hb, structurally characterize equilibrium intermediates in the thermal denaturation of Aplysia Mb, characterize the sequence alignment, folding topology and identify distal residues in a series of "mini" or "compact" (109-118 residue) globins, characterize the distal H- bonding network in the extremely O2-avid, and on occasion, extremely autoxidative-resistant, trematode globins, and characterize the changes in distal interactions and proximal His-Fe bond strain that accompanies the allosteric transition in ligated HbA.