Solution NMR Study of the Electronic and Molecular Structures of Hyperthermostable Ferredoxins

9600759 LaMar将通过NMR阐明铁氧还蛋白（Fds）的分子和电子结构以及动力学性质，所述铁氧还蛋白来自一系列超嗜热古菌，Pyroccus furiosus，Pf，Thermococcus litoralis，T1，以及尚未命名的ES-4和JDF-3。 这些小的电子转移Fd在环境温度下具有两种氧化态的顺磁性簇。 适当调整1D和2D NMR方法，如在选择的N-15标记的Fd上通过3D NMR增强，以检测和分配所有顺磁影响的共振，预期提供：还原簇中单个铁的价态的序列特异性鉴定;阐明配体突变和/或外源配体在调节整体簇氧化还原电位以及个体铁氧化态中的影响;非半胱氨酸配体的光谱特征，用于识别其他铁硫簇酶中的蛋白质配体;利用各种顺磁光谱数据作为结构约束的协议，以取代丢失的常规NMR约束附近的集群;最后，但最重要的是，这些FDS的强大的分子模型。 pf、ES-4和JDF-3 Fds还具有一对远程Cys，其作为2 e-2 H+氧化还原位点，在基本上与簇相同的电位下。 预期在环境温度下所得到的四个Fd氧化还原状态的完整时间分辨率将提供独特的洞察力的影响，每个步骤在调节蛋白质结构附近和远离集群，以及在这些Fd的集群电子转移的大重组能量的结构基础。 所得到的分子结构预计将揭示什么结构特性赋予这些蛋白质显著的热稳定性（在95 ° C（24小时内可忽略不计的变性），尽管与来自嗜温菌的稳定性低得多的Fds具有显著的序列同源性。 在本发明的Fds中鉴定“稳定化结构基序”可以允许将来通过诱变“基序”引入以增加工业生物催化剂的热稳定性。 这些铁氧化还原蛋白的表征也将有助于理解这些新的生物体（古生菌），这些生物体被认为是最古老和进化最缓慢的生命形式，可能是最接近普遍生命形式的后代。 一系列称为铁氧还蛋白的小（（60个氨基酸残基）电子转移蛋白的分子结构将通过溶液质子核磁共振测定，所得结构用于解释与其在电子转移中的作用相关的性质。 铁氧还蛋白选自生活在沸水温度下的深海火山喷口中的许多古细菌（即激烈热球菌（Pyrococcus furiosus）、滨海热球菌（Thermococcus litoralis））。 目的是确定这些铁氧还蛋白中不同残基对之间的相互作用，并将高温状态古细菌铁氧还蛋白中这些相互作用的差异与更正常真细菌的类似蛋白联系起来，这些蛋白在37 ℃下稳定，但在远高于50 ℃的温度下迅速变性。 对有助于高温稳定性的相互作用类型的理解可以为如何通过定点诱变增加生物技术工业中使用的酶的高温稳定性提供线索。 拟议的研究还将有助于了解这些相对较新发现的古细菌，这些古细菌被认为是地球上最早生命形式的最接近的活后代。 ***

9600759 LaMar The molecular and electronic structural and dynamic properties of ferredoxins (Fds), from a series of hyperthermophilic archaea, Pyroccus furiosus, Pf, Thermococcus litoralis, Tl, and the as yet unnamed ES-4 and JDF-3, will be elucidated by NMR. These small electron transfer Fd possess paramagnetic clusters at ambient temperature in both oxidation states. The appropriate tailoring of 1D and 2D NMR methods, as augmented by 3D NMR on select N-15 labeled Fd, to detect and assign all paramagnetically influenced resonances is expected to provide: the sequence-specific identification of the valence states for he individual iron in the reduced cluster; elucidation of the influence of ligand mutation and/or exogenous ligands in modulating the overall cluster redox potential as well as the individual iron oxidation states; the spectral signatures of non cysteine ligands for identifying protein ligands in other iron-sulfur cluster enzymes; a protocol for utilizing various paramagnetic spectral data for use as structural constraints to substitute for lost conventional NMR constraints near the cluster; and, lastly, but most importantly, robust molecular models for these Fds. The pf, ES-4 and JDF-3 Fds also possess a pair of remote Cys which serve as 2e- 2H+ redox sites at essentially the same potential as the cluster. The complete time resolution of the resulting four Fd redox states at ambient temperature is expected to provide unique insight into the influence of each step in modulating the protein architecture both near and remote from the cluster, as well as the structural basis of the large reorganizational energy for cluster electron transfer in these Fd. The resulting molecular structures are expected to shed light on what structural properties impart the remarkable thermostability (negligible denaturation at 95(C over 24h) to these proteins in spite of significant sequence homology to much less stable Fds from mesophiles. The identification of "stabilizing structural motifs" i n the present Fds may permit future introduction by mutagenesis of the "motifs" to increase thermostability of industrial biocatalysts. The characterization of these ferredoxins will also contribute to the understanding of these novel organisms (archaea) that are proposed to be the most ancient and slowly evolving form of life and possibly the closest living descendant to a universal life form. %%% The molecular structures of a series of small ((60 amino acid residues) electron transfer proteins called ferredoxins will be determined by solution proton nuclear magnetic resonance and the resulting structures used to interpret properties relevant to their role in electron transfer. The ferredoxins are selected from a number of archaebacteria (i.e. Pyrococcus furiosus, Thermococcus litoralis) that live in deep ocean volcano vents at temperatures of boiling water. The goal is to define the interactions among the various pairs of residues in these ferredoxins and to relate differences in these interactions in the high temperature-state archaebacteria ferredoxins with similar proteins of more normal eubacteria which are stable at 37(C, but rapidly denature at temperatures much higher than 50(C. The understanding of the types of interactions that contribute to the high temperature stability may provide clues as how to increase by site-directed mutagenesis the high temperature stability of enzymes used in the biotechnology industry. The proposed studies will also contribute to the understanding of these relatively recently discovered archaebacteria which are proposed to be the closest living descendants of the earliest forms of life on this planet. ***