MULTIPLE BINDING MODES OF THE SINGLE-STRANDED-DNA BINDING-PROTEIN FROM ESCHERICHIA-COLI AS DETECTED BY TRYPTOPHAN FLUORESCENCE AND SITE-DIRECTED MUTAGENESIS
MULTIPLE BINDING MODES OF THE SINGLE-STRANDED-DNA BINDING-PROTEIN FROM ESCHERICHIA-COLI AS DETECTED BY TRYPTOPHAN FLUORESCENCE AND SITE-DIRECTED MUTAGENESIS
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DOI：
10.1021/bi00061a016
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发表时间：
1993-03-16
期刊：
影响因子：
2.9
通讯作者：
MAASS, G
中科院分区：
文献类型：
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作者：
CURTH, U;GREIPEL, J;MAASS, G
我们系统地用极性（丝氨酸或苏氨酸）和芳香（酪氨酸或苯丙氨酸）氨基酸取代了大肠杆菌（EcoSSB）单链DNA结合蛋白的四个色氨酸残基。由此产生的突变体，无论是单氨基酸交换还是三重取代，都在ssDNA结合中具有活性，尽管在某些情况下具有降低的亲和力。突变的EcoSSBs的荧光测量表明，四种不同的色氨酸残基之间没有相互作用。我们用荧光滴定法分析突变蛋白的ssDNA结合。在0.3 M NaCl（“高盐”）下，所有单取代蛋白以与野生型EcoSSB相当的方式与poly（dT）结合，覆盖65个核苷酸和1个EcoSSB四聚体。W54S突变蛋白是一个例外，因为即使在0.3 M NaCl下，它也覆盖了大约35个核苷酸，这是盐浓度低于10 mM NaCl（“低盐”）的典型行为。根据这一观察，我们推断色氨酸-54参与了与ssDNA的直接相互作用，有利于“高盐”结合模式。所有缺乏色氨酸-54但在“低盐”浓度下具有色氨酸-88的突变蛋白在荧光滴定中表现出非单调行为。假设EcoSSB与poly（dT）具有两种不同结合位点大小（n几乎等于27和n几乎等于33）和不同结合亲和力的模型，可以解释这种行为。多齿配体与线性聚合物相互作用中多重结合模式问题的定量处理应用于这些滴定。
We have systematically substituted the four tryptophan residues of the single-stranded DNA binding protein from Escherichia coli (EcoSSB) by polar (serine or threonine) and aromatic (tyrosine or phenylalanine) amino acids. The resulting mutants with either single amino acid exchanges or triple substitutions are all active in ssDNA binding, though in some cases with reduced affinities. Measurements of the fluorescence of the mutated EcoSSBs show that there is no interaction between the four different tryptophan residues. We analyzed the ssDNA binding of the mutant proteins by fluorescence titrations. At 0.3 M NaCl (''high salt''), all singly substituted proteins bind to poly(dT) in a manner comparable to wild-type EcoSSB, covering 65 nucleotides with 1 EcoSSB tetramer. W54S mutant protein is an exception since even at 0.3 M NaCl it covers approximately 35 nucleotides, a behavior which is typical of salt concentrations below 10 mM NaCl (''low salt''). From this observation, it is inferred that tryptophan-54 is involved in a direct interaction with the ssDNA favoring the ''high-salt'' binding mode. All mutant proteins lacking tryptophan-54 but possessing tryptophan-88 at ''low-salt'' concentrations show a nonmonotonous behavior in the fluorescence titrations. This behavior can be interpreted assuming a model of cooperative binding of EcoSSB to poly(dT) with two different binding site sizes (n almost-equal-to 27 and n almost-equal-to 33) and different binding affinities. A quantitative treatment of the problem of multiple binding modes in the interaction of a multidentate ligand with a linear polymer is applied to these titrations.