Tsao D H, Casas-Finet J R, Maki A H, Chase J W
Department of Chemistry, University of California, Davis 95616.
Biophys J. 1989 May;55(5):927-36. doi: 10.1016/S0006-3495(89)82891-7.
Complexes of point-mutated E. coli single-stranded DNA-binding protein (Eco SSB) with homopolynucleotides have been investigated by optical detection of magnetic resonance (ODMR) of the triplet state of tryptophan (Trp) residues. Investigation of the individual sublevel kinetics of the lowest triplet state of Trp residues 40 and 54 in the poly (dT) complex of Eco SSB-W88F,W135F (a mutant protein whose Trp residues at positions 88 and 135 have been substituted by Phe) shows that Trp 54 is the most affected residue upon stacking with thymine bases, confirming previous results based on SSB mutants having single Trp----Phe substitutions. (Zang, L. H., A. H. Maki, J. B. Murphy, and J. W. Chase. 1987. Biophys. J. 52:867-872). The Tx sublevel of Trp 54 shows a fourfold increase in the decay rate constant, as well as an increase in its populating rate constant by selective spin-orbit coupling. The two nonradiative sublevels show no change in lifetime, relative to unstacked Trp. For Trp 40, a weaker perturbation of Tx by thymine results in a sublevel lifetime about one-half that of normal Trp. Trp54 displays a 2[E]transition of negative polarity in the double mutant SSB complex with Poly (dT), but gives a vanishingly weak [D] - [E] signal, thus implying that the steady-state sublevel populations of Tx and Tz are nearly equal in this residue. Poly (5-BrU) induces the largest red-shift of the Eco SSB-W88F,W135F Trp phosphorescence 0,0-band of all polynucleotides investigated. Its phosphorescence decay fits well to two exponential components of 1.02 and 0.12 s, with no contribution from long-lived Trp residues. This behavior provides convincing evidence that both Trp 40 and 54 are perturbed by stacking with brominated uridine. The observed decrease in the Trp [D] values further confirms the stacking of the Trp residues with 5-BrU. Wave-length-selected ODMR experiments conducted on the [D[ + [E] transition of Eco SSB-W88F,W135F complexed with poly(5HgU) indicate the presence of multiple heavy atom-perturbed sites. Measurements made on poly (5-HgU) which each of its 4 Trp residues has been replaced in turn by Phe demonstrate that Trp 40 and 54 are the only Trp residues undergoing stacking with nucleotide bases, as previously proposed.
通过对色氨酸(Trp)残基三重态的磁共振光学检测(ODMR),研究了点突变的大肠杆菌单链DNA结合蛋白(Eco SSB)与同聚核苷酸的复合物。对Eco SSB-W88F、W135F(一种突变蛋白,其88位和135位的Trp残基已被Phe取代)的聚(dT)复合物中Trp残基40和54最低三重态的各个子能级动力学研究表明,Trp 54是与胸腺嘧啶碱基堆积时受影响最大的残基,这证实了基于具有单个Trp→Phe取代的SSB突变体的先前结果。(Zang, L. H., A. H. Maki, J. B. Murphy, and J. W. Chase. 1987. Biophys. J. 52:867-872)。Trp 54的Tx子能级的衰变率常数增加了四倍,并且通过选择性自旋轨道耦合其填充率常数也增加。相对于未堆积的Trp,两个非辐射子能级的寿命没有变化。对于Trp 40,胸腺嘧啶对Tx的扰动较弱,导致子能级寿命约为正常Trp的一半。在与聚(dT)形成的双突变体SSB复合物中,Trp54显示出负极性的2[E]跃迁,但给出的[D] - [E]信号极其微弱,因此意味着该残基中Tx和Tz的稳态子能级分布几乎相等。在所有研究的多核苷酸中,聚(5-溴尿嘧啶)引起Eco SSB-W88F、W135F Trp磷光0,0带的最大红移。其磷光衰变很好地拟合为1.02和0.12 s的两个指数成分,没有来自长寿命Trp残基的贡献。这种行为提供了令人信服的证据,表明Trp 40和54都因与溴化尿苷堆积而受到扰动。对与聚(5-汞尿嘧啶)复合的Eco SSB-W88F、W135F的[D] + [E]跃迁进行的波长选择ODMR实验表明存在多个重原子扰动位点。对聚(5-汞尿嘧啶)进行的测量,其中其4个Trp残基依次被Phe取代,结果表明Trp 40和54是仅有的与核苷酸碱基发生堆积的Trp残基,如先前所提出的。