NMR SPECTROSCOPY OF IRON-SULFUR PROTEINS

核磁共振波谱学和计算化学的方法将 用于研究铁的结构-功能关系- 具有生物学和生物医学重要性的硫蛋白。蛋白 待研究的包括那些含有单一金属位点的化合物， 4 Cys、3 Cys/1 His和2 Cys/2 His连接（红曲霉毒素和 锌/铁指），各种类型的[2Fe-2S] 4 Cys簇 （植物，细菌和脊椎动物铁氧化还原蛋白），和两类 [2Fe-2S] 2 Cys/2 His簇（Rieske蛋白）。 的作用 各种相互作用（金属与特定氨基酸的配位 侧链，氢键和附近的电荷）将被 通过选择性蛋白质修饰--通过诱变和 化学合成或通过掺入不同的金属离子。 一 我们的方法的独特之处是我们证明了我们的能力， 联合收割机从顺磁核磁共振谱、x-射线 晶体学和量子力学计算来评估 蛋白质中金属中心的结构模型。信息 这对药物的设计至关重要 靶向金属结合位点。 预计这一 方法将产生新的见解的作用， 相互作用在稳定蛋白质中发挥作用， 氧化态 不断增长的指定超精细1H数据库， 铁硫蛋白质的H、13 C和15 N化学位移应 有助于确定新发现的 从NMR数据中提取蛋白质并用于开发氨基酸共有序列 与特定簇类型相关的序列。 小说 这些方法有望提高核磁共振结构的质量， 顺磁性蛋白质来源于NMR数据。 通常的 约束来自NOE和三键J耦合来自 反磁性区域将补充信息， 场依赖的单键偶极耦合， 磁场对蛋白质的排序， 从超精细位移共振的分析中得出， 从已知蛋白质数据库中导出的人工电位 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.