Jana R, Hazbun T R, Mollah A K, Mossing M C
Department of Biological Sciences, University of Notre Dame, Notre Dame, IN, 46556, USA.
J Mol Biol. 1997 Oct 24;273(2):402-16. doi: 10.1006/jmbi.1997.1256.
The folding, dimerization and DNA binding equilibria of the bacteriophage lambda Cro repressor have been characterized. Comparison with four engineered variants shows that a folded monomeric species is substantially populated under conditions used for the formation of dimer-DNA complexes. Although Cro dimers are the only DNA-bound species observed in electrophoretic mobility shift assays, cooperativity in Cro-DNA binding isotherms shows that the predominant free protein species is monomeric at nanomolar concentrations. Micromolar dissociation constants for Cro dimers have been measured in the absence of DNA by sedimentation equilibrium and gel filtration chromatography. Denaturation of Cro dimers in the 10 to 100 micromolar concentration range by guanidine hydrochloride (GdnHCl) is well modeled as a two-state process, with folded dimers and unfolded monomers as the only significantly populated species. However, linear extrapolation of this composite unfolding and dimer dissociation free energy predicts a nanomolar dissociation constant in the absence of denaturant. This extrapolation is clearly inconsistent with the DNA binding and hydrodynamic measurements. Our interpretation of these results is that the monomeric species detected in DNA binding and hydrodynamic experiments is predominantly folded. The stability of the folded monomeric species can be calculated as the difference between the dimerization free energy determined from hydrodynamic measurements and the folding free energy extrapolated from GdnHCl denaturation. The calculated stability of the Cro F58W monomer is greater than that of the wild-type Cro monomer. Thus, residue 58, which makes critical intermolecular contacts across the dimer interface, is also involved in intramolecular stabilization of the monomeric intermediate.
噬菌体λ Cro阻遏蛋白的折叠、二聚化及DNA结合平衡已得到表征。与四种工程变体的比较表明，在用于形成二聚体-DNA复合物的条件下，折叠的单体物种大量存在。尽管在电泳迁移率变动分析中观察到Cro二聚体是唯一与DNA结合的物种，但Cro-DNA结合等温线中的协同性表明，在纳摩尔浓度下，主要的游离蛋白物种是单体。通过沉降平衡和凝胶过滤色谱法在无DNA的情况下测量了Cro二聚体的微摩尔解离常数。盐酸胍（GdnHCl）在10至100微摩尔浓度范围内使Cro二聚体变性，很好地模拟为一个两态过程，折叠的二聚体和未折叠的单体是唯一大量存在的物种。然而，这种复合展开和二聚体解离自由能的线性外推预测在没有变性剂的情况下解离常数为纳摩尔。这种外推显然与DNA结合和流体动力学测量结果不一致。我们对这些结果的解释是，在DNA结合和流体动力学实验中检测到的单体物种主要是折叠的。折叠单体物种的稳定性可以通过流体动力学测量确定的二聚化自由能与从GdnHCl变性外推的折叠自由能之间的差值来计算。计算得出的Cro F58W单体的稳定性大于野生型Cro单体。因此，在二聚体界面形成关键分子间接触的第58位残基也参与了单体中间体的分子内稳定。
