NMR Study of Structural and Dynamic Properties of Paramagnetic Proteins ,

为了从超精细位移中提取与蛋白质中连接通道的形成、电子介质氧化还原电位的控制以及蛋白质展开过程中中间体的检测相关的独特电子/分子结构信息，提出了对各种顺磁性金属蛋白进行核磁共振研究。所有这些研究的核心是二维核磁共振策略的开发和实施，这将为顺磁弛豫、拓宽和移位共振提供关键任务。在氰基甲基肌红蛋白远端血红素口袋突变/化学修饰时不稳定质子交换率的变化将用于定义允许血红素口袋形成瞬时连接通道的相互作用。将阐明细胞色素b5的血红素取向和轴向组氨酸的氢键对血红素电子结构的影响，并确定调节细胞色素b562氧化还原电位的pKs的来源。结合半胱氨酸在各种铁硫簇电子介质中的接触位移共振将被分配到蛋白质序列中的特定残基上。并根据现有的x射线晶体结构解释簇中单个铁的价态，以阐明蛋白质控制局部铁氧化还原电位的机制。高自旋铁胞色素c′的血红素和轴向组氨酸共振将被分配来解释在可逆热展开过程中检测到的平衡过渡前结构变化的性质。

Nuclear magnetic resonance studies of a variety of paramagnetic metalloproteins are proposed in order to extract unique electronic/molecular structural information from the hyperfine shifts as relevant to the formation of ligation channels in proteins, the control of redox potential of electron mediators, and the detection of intermediates in the unfolding of proteins. Central to all of these studies is the development and implementation of two-dimensional NMR strategies, that will provide the critical assignments for the paramagnetically relaxed, broadened and shifted resonances. Changes in labile proton exchange rates upon mutation/chemical modification in the distal heme pocket of cyano-metmyoblobin will be used to define the interactions that allows formation of a transient ligation channel to the heme pocket. The influence of heme orientation and hydrogen bonding by an axial histidine on heme electronic structure will be elucidated for cytochrome b5, and the origins of the pKs that modulate the redox potential of cytochrome b562 will be identified. The contact shifted resonance of bound cysteines in a variety of iron-sulfur cluster electron mediators will be assigned to specific residues in the protein sequence, and the valence states of individual irons in the cluster will be interpreted on the basis of available X-ray crystal structure to elucidate the mechanism of protein control of localized iron redox potential. The heme and axial histidine resonances of high-spin ferricytochromes c' will be assigned to interpret the nature of the equilibrium pre-transition structural changes detected during reversible thermal unfolding.