Predki P F, Sarkar B
Department of Biochemistry, University of Toronto, Ontario, Canada.
Environ Health Perspect. 1994 Sep;102 Suppl 3(Suppl 3):195-8. doi: 10.1289/ehp.94102s3195.
Metal replacement studies were used to investigate the metal requirement of a bacterially expressed polypeptide encoding the zinc finger DNA binding domain of the estrogen receptor. Apopolypeptide was generated by dialysis of native polypeptide against low-pH buffer under reducing conditions. Specific DNA binding can be restored by refolding the apopolypeptide in the presence of ionic zinc, cadmium, or cobalt. However, refolding in the presence of copper or nickel fails to regenerate DNA binding activity. While cobalt-reconstituted polypeptide has a reduced affinity for its AGGTCA-binding site compared to zinc- or cadmium-polypeptide, it has the surprising property of increased cooperative DNA binding. Our work indicates that metal substitution results in a range of effects upon DNA binding in vitro. The potential biological significance of metal substitution in vivo is discussed.
金属置换研究被用于调查一种细菌表达的编码雌激素受体锌指DNA结合结构域的多肽的金属需求。在还原条件下，通过将天然多肽对低pH缓冲液进行透析来产生脱辅基多肽。在离子锌、镉或钴存在的情况下，通过使脱辅基多肽重新折叠，可以恢复特异性DNA结合。然而，在铜或镍存在的情况下重新折叠无法再生DNA结合活性。虽然与锌或镉多肽相比，钴重构的多肽对其AGGTCA结合位点的亲和力降低，但其具有协同DNA结合增加的惊人特性。我们的工作表明，金属置换在体外对DNA结合会产生一系列影响。讨论了体内金属置换的潜在生物学意义。
