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）是一种富含巯基的多肽，对金属离子的攻击有反应，它能螯合金属离子，从而使金属离子解毒。这些多肽在细胞暴露于活性氧物质后也以高水平表达。最近的工作主要集中在研究MT的表达和功能，以响应急性应激，较少关注在正常细胞代谢过程中潜在的更微妙的作用。需要这些更微妙的作用是由观察到的这种蛋白质类的成员的差异表达，每一个可能具有不同的亲和力，为各种金属离子（如。铜与锌），在不同的生理或发育条件下。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定位和类型引起的。虽然对真核I类MT（1型和2型）的结构和氨基酸组成了解很多，但对细菌II类蛋白了解较少，因为仅三个实例（均来自聚球藻属）的衍生氨基酸序列。已出版。我们开发了PCR引物的基础上公布的序列，并发现，基因的II类MT（SMT A）是广泛存在于蓝藻。拟议的研究的主要目的是调查的结构/功能关系的第二类金属硫蛋白使用分子生物学（定点诱变），蛋白质生物化学（金属结合研究）和物理生物化学（扩展的X射线吸收精细结构）的方法。我们假设，除了硫醇，如适当定位的羟基和羧酸盐，残基是重要的金属离子结合，和定位和字符的这些残基是负责对这些多肽所表现出的金属离子的亲和力的差异。一个彻底的表征的金属结合性能的每一个新确定的MT，并通过定位不同的氨基酸官能团内的金属离子结合位点所产生的影响，将有助于我们基本了解这些多肽在急性和正常条件下的功能。