Finucane Michael D, Jardetzky Oleg
Department of Molecular Pharmacology, Stanford University, Stanford, CA 94305-5174, USA.
Protein Sci. 2003 Aug;12(8):1613-20. doi: 10.1110/ps.0305703.
The interactions of wild-type (WT) and AV77 tryptophan repressor (TR) with several operators have been studied using surface plasmon resonance. The use of this real-time method has been able to settle several outstanding issues in the field, in a way that has heretofore not been possible. We resolve the issue of the super-repressor status of the AV77 aporepressor and find that in contrast to early studies, which found no significant difference in the binding constants in vitro to those of the WT, that there is indeed a clear difference in the binding constant that can simply account for the phenotype. Accordingly, there is no need for alternative proposals invoking complex equilibria with in vivo components not found in the in vitro experiments. In addition, we find that the AV77 holorepressor-DNA complex is much more stable than the equivalent WT complex, which has not been apparent from either in vitro or equilibrium binding experiments.
利用表面等离子体共振研究了野生型（WT）和AV77色氨酸阻遏物（TR）与多个操纵基因的相互作用。这种实时方法的应用能够以一种前所未有的方式解决该领域中几个悬而未决的问题。我们解决了AV77无辅基阻遏物的超阻遏物状态问题，发现与早期研究不同，早期研究发现其体外结合常数与野生型无显著差异，而实际上结合常数存在明显差异，这足以解释其表型。因此，无需提出涉及体外实验中未发现的体内成分复杂平衡的替代方案。此外，我们发现AV77全阻遏物 - DNA复合物比等效的野生型复合物稳定得多，这在体外或平衡结合实验中都不明显。
