NMR SPECTROSCOPY OF IRON-SULFUR PROTEINS

核磁共振波谱和计算化学的方法将 被用来研究铁的结构-功能关系 具有生物和生物医学重要性的硫蛋白。蛋白质 要研究的包括含有单一金属位置的那些 4个半胱氨酸，3个半胱氨酸/1个组氨酸，2个半胱氨酸/2个组氨酸结扎(Rubredoxins和 锌/铁指)，各种类型的[2Fe-2S]4-Cys簇合物 (植物、细菌和脊椎动物铁氧还蛋白)和两类 [2Fe-2S]2Cys/2His簇(Rieske蛋白)。的角色 各种相互作用(金属对特定氨基酸的配位作用 侧链、氢键和附近的电荷) 通过选择性蛋白质修饰进行评估--通过突变和 化学合成或通过加入不同的金属离子。一个 我们方法的独特之处在于我们证明有能力 结合来自顺磁核磁共振谱、x射线的信息 结晶学和量子力学计算来评估 蛋白质中金属中心的结构模型。的信息 这类药物对于药物的设计是至关重要的。 以金属结合部位为目标。预计这将是一次 方法将对各种不同的角色产生新的见解 相互作用在稳定蛋白质在其可及性中的作用 氧化态。指定的超精细1H的不断增长的数据库， 铁硫蛋白的H，13C和15N化学位移应该 对确定新发现的星团类别很有用 来自核磁共振数据的蛋白质和开发氨基酸共识 与特定集群类型相关联的序列。小说 方法有望提高核磁共振结构的质量 从核磁共振数据中提取的顺磁性蛋白质。和往常一样 NOE和三键J-联结产生的约束来自 反磁区将由来自 偏振光引起的依赖于场的单键偶极耦合 磁场对蛋白质的排序，限制条件 源自对超精细位移共振的分析，以及 从已知蛋白质数据库中提取的人工势能 结构。

The methods of NMR spectroscopy and computational chemistry will be used to investigate structure-function relationships in iron- sulfur proteins of biological and biomedical importance. Proteins to be studied include those that contain single metal sites with 4 Cys, 3 Cys/1 His, and 2 Cys/2His ligation (rubredoxins and zinc/iron fingers), various classes of [2Fe-2S] 4 Cys clusters (plant, bacterial, and vertebrate ferredoxins), and two classes of [2Fe-2S] 2 Cys/2 His clusters (Rieske proteins). The roles of various interactions (metal coordination to particular amino acid side chains, hydrogen bonds, and nearby charges) will be evaluated by selective protein modifications--by mutagenesis and chemical synthesis or by incorporating different metal ions. A unique feature of our approach is our demonstrated ability to combine information from paramagnetic NMR spectra, x-ray crystallography, and quantum mechanical calculations to evaluate structural models for metal centers in proteins. Information of this kind is of fundamental importance for the design of drugs that target metal binding sites. It is expected that this approach will yield new insights about the role that various interactions play in stabilizing the protein in its accessible oxidation states. The growing database of assigned hyperfine 1H, H, 13C, and 15N chemical shifts for iron-sulfur proteins should be useful for determining cluster categories in newly discovered proteins from NMR data and for developing amino acid consensus sequences associated with particular cluster types. Novel approaches promise to improve the quality of NMR structures of paramagnetic proteins derived from NMR data. The usual constraints derived from NOEs and three-bond J-couplings from the diamagnetic region will be supplemented by information from field-dependent one-bond dipolar couplings arising from partial ordering of the proteins by the magnetic field, constraints derived from analysis of hyperfine-shifted resonances, and artificial potentials derived from the database of known protein structures.