Karkaria C E, Steiner R F, Rosen B P
Department of Biochemistry, Wayne State University, School of Medicine, Detroit, Michigan 48201.
Biochemistry. 1991 Mar 12;30(10):2625-8. doi: 10.1021/bi00224a009.
The ars operon of the conjugative R-factor R773 produces resistance to arsenicals in cells of Escherichia coli. The operon encodes an oxyanion pump which is composed of a membrane subunit, the 45.5-kDa ArsB protein, and a catalytic subunit, the 63-kDa ArsA protein. Purified ArsA protein is an arsenite(antimonite)-stimulated ATPase. From its amino acid sequence, as deduced from the nucleotide sequence, the ArsA protein has four tryptophanyl residues which could serve as intrinsic fluorescent probes for the study of substrate-induced conformational changes. Both static and dynamic measurements of tryptophan fluorescence were performed with the ArsA protein. Results from static anisotropy measurements indicated differences in molecular motion with addition of ATP, SbO2-, or Mg2+. These results were supported by time decay measurements of fluorescence anisotropy. The results of time decay measurements indicated a shorter correlation time, reflecting localized motion in the vicinity of the probe, and a longer correlation time, which could have arisen from rotation of the major portion of the molecule. The longer correlation time changed with addition of the various effectors, especially MgCl2, suggesting that binding of Mg2+ decreases probe mobility.
接合性R因子R773的ars操纵子可使大肠杆菌细胞对砷化物产生抗性。该操纵子编码一种氧阴离子泵，它由一个膜亚基（45.5 kDa的ArsB蛋白）和一个催化亚基（63 kDa的ArsA蛋白）组成。纯化的ArsA蛋白是一种亚砷酸盐（亚锑酸盐）刺激的ATP酶。根据核苷酸序列推导的氨基酸序列，ArsA蛋白有四个色氨酸残基，可作为研究底物诱导的构象变化的内在荧光探针。对ArsA蛋白进行了色氨酸荧光的静态和动态测量。静态各向异性测量结果表明，添加ATP、SbO2-或Mg2+时分子运动存在差异。这些结果得到了荧光各向异性时间衰减测量的支持。时间衰减测量结果表明，存在较短的相关时间，反映了探针附近的局部运动，以及较长的相关时间，这可能是由于分子大部分的旋转引起的。较长的相关时间会随着各种效应物的添加而改变，尤其是MgCl2，这表明Mg2+的结合会降低探针的流动性。
