Probing the role of water in the tryptophan repressor-operator complex

The Escherichia coli tryptophan repressor protein (TR) represses the transc ription of several genes in response to the concentration of tryptophan in the environment. In the co-crystal structure of TR bound to a DNA fragment containing its target very few direct contacts between TR and the DNA were observed. In contrast, a number of solvent mediated contacts were apparent. NMR solution structures, however, did not resolve any solvent mediated bon ds at the complex interface. To probe for the rule of water in TR operator recognition, the effect of osmolytes on the interactions between TR and a t arget oligonucleotide bearing the operator site was examined. In the absenc e of specific solvent mediated hydrogen bonding interactions between the pr otein and the DNA, increasing osmolyte concentration is expected to strongl y stabilize the TR operator interaction due to the large amount of macromol ecular surface area buried upon complexation. The results of our studies in dicate that xylose did not alter the binding affinity significantly, while glycerol and PEG had a small stabilizing effect. A study of binding as a fu nction of betaine concentration revealed that this osmolyte at low concentr ation results in a stabilization of the 1:1 TR/operator complex, but at hig her concentrations leads to a switching between binding modes to favor tand em binding. Analysis of the effects of betaine on the 1:1 complex suggest t hat this osmolyte has about 78% of the expected effect. If one accepts the analysis in terms of the number of water molecules excluded upon complexati on. these results suggest that about 75 water molecules remain at the inter face of the 1:1 dimer/DNA complex. This value is consistent with the number of water molecules found at the interface in the crystallographically dete rmined structure and supports the notion that interfacial waters play an im portant thermodynamic role in the specific complexation of one TR dimer wit h its target DNA. However, the complexity of the effects of betaine and the small or negligible effects of the other osmolytes could also arise from o smolyte induced competition between antagonistic coupled reactions.