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

金属硫蛋白(Metals thioneins，MTS)是一种富含硫醇的多肽，在金属离子的刺激下表达，可以隔离金属离子，从而使金属离子解毒。这些多肽在细胞暴露于活性氧物种后也有高水平的表达。最近的工作主要集中在研究MT在急性应激反应中的表达和功能，而对正常细胞代谢过程中潜在的更微妙的作用关注较少。观察到这一蛋白质类成员的差异表达表明，需要这些更微妙的作用，每个成员对不同的金属离子(例如。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于氨基酸侧链在金属离子结合部位内的位置和类型造成的。虽然人们对真核生物的I类MTS(类型1和类型2)的结构和氨基酸组成知道得很多，但对细菌的II类蛋白质的了解较少，因为只有三个例子的氨基酸序列(都来自聚球藻属)。已经出版了。我们根据已发表的序列开发了聚合酶链式反应引物，并发现第二类MTS(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)等方法研究第二类金属硫蛋白的结构/功能关系。我们假设，硫醇以外的残基，如适当定位的羟基和羧酸盐，对金属离子结合是重要的，这些残基的位置和性质是这些多肽对金属离子亲和力差异的原因。对每一个新发现的MT的金属结合性质以及在金属离子结合部位定位不同的氨基酸官能团所产生的影响的彻底表征将有助于我们对这些多肽在急性和正常条件下的功能的基本理解。

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.