AMINOACID RESIDUE INVOLVED IN METAL ION BINDING BY CLASS II METALLOTHIONEINS (78)

参与 II 类金属硫蛋白金属离子结合的氨基酸残基 (78)

• 批准号: 6325825
• 负责人: ROBERT WEBB
• 金额: $ 4.04万
• 依托单位: UNIVERSITY OF TEXAS EL PASO
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-06-01 至 2001-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6325825
• 关键词: SDS polyacrylamide gel electrophoresis; X ray spectrometry; affinity chromatography; aminoacid; bacterial proteins; binding sites; chemical substitution; copper; ionic bond; lead; metallothionein; nucleic acid sequence; photosynthetic bacteria; polymerase chain reaction; protein binding; protein isoforms; protein structure function; site directed mutagenesis; zinc

Metallothioneins (MTs) are thiol-rich polypeptides, expressed in response to metal ion challenges, which sequester, and thus detoxify, metal ions. These polypeptides are also expressed at high levels after cellular exposure to reactive oxygen species. The primary focus of recent work has been to study MT expression and function in response to acute stresses with less attention to potentially more subtle roles during normal cellular metabolism. A need for these more subtle roles is suggested by the observed differential expression of members of this protein class, each presumably with different affinities for various metal ions (eg. copper versus zinc), under different physiological or developmental conditions. Differing affinities likely result from the positioning and type of amino acid side chains within the metal ion binding sites. While much is known about the structure and amino acid composition of the eukaryotic, class I MTs (types 1 and 2), less is known about the bacterial, class II proteins, since the derived amino acid sequences of only three examples (all from the genus Synechococcus spp.) have been published. We developed PCR primers based on the published sequences and have found that genes for the class II MTs (smt A) are widespread among cyanobacteria. The principal aim of the proposed study is to investigate the structure/function relationships of the class II metallothioneins using molecular biology (site-directed mutagenesis), protein biochemistry (metal binding studies) and physical biochemistry (extended x-ray absorption fine structure) approaches. We hypothesize that residues other than thiols, such as appropriately positioned hydroxyls and carboxylates, are important for metal ion binding, and that the positioning and character of such residues is responsible for the differences in affinity toward metal ions exhibited by these polypeptides. A thorough characterization of the metal binding properties of each of the newly identified MTs, and of the effects generated by positioning differing amino acid functional groups within the metal ion binding sites will aid our basic understanding of the functions of these polypeptides under acute and normal conditions.

金属硫蛋白 (MT) 是富含硫醇的多肽，在响应金属离子挑战时表达，可隔离金属离子，从而解毒。这些多肽在细胞暴露于活性氧后也以高水平表达。最近工作的主要焦点是研究 MT 响应急性应激的表达和功能，而很少关注正常细胞代谢过程中潜在的更微妙的作用。观察到的此类蛋白质成员的差异表达表明需要这些更微妙的作用，在不同的生理或发育条件下，每种蛋白质可能对各种金属离子（例如铜与锌）具有不同的亲和力。不同的亲和力可能是由金属离子结合位点内氨基酸侧链的位置和类型造成的。虽然人们对真核 I 类 MT（1 型和 2 型）的结构和氨基酸组成了解甚多，但对细菌 II 类蛋白质知之甚少，因为只有三个例子的衍生氨基酸序列（全部来自聚球藻属 (Synechococcus spp.) 已发表。我们根据已发表的序列开发了 PCR 引物，并发现 II 类 MT (smt A) 的基因在蓝藻中广泛存在。本研究的主要目的是利用分子生物学（定点突变）、蛋白质生物化学（金属结合研究）和物理生物化学（扩展 X 射线吸收精细结构）方法研究 II 类金属硫蛋白的结构/功能关系。我们假设除硫醇之外的残基，例如适当位置的羟基和羧酸盐，对于金属离子结合很重要，并且此类残基的位置和特征是造成这些多肽表现出的对金属离子的亲和力差异的原因。全面表征每个新鉴定的 MT 的金属结合特性，以及通过在金属离子结合位点内定位不同的氨基酸官能团所产生的效应，将有助于我们对这些多肽在急性和正常条件下的功能有基本的了解。