Role of Hydration in the Binding of lac Repressor to DNA

Abstract

The osmotic stress technique was used to measure changes in macromolecular hydration that accompany binding of wild-type Escherichia coli lactose (lac) repressor to its regulatory site (operator O1) in the lac promoter and its transfer from site O1 to nonspecific DNA. Binding at O1 is accompanied by the net release of 260 ؎ 32 water molecules. If all are released from macromolecular surfaces, this result is consistent with a net reduction of solvent-accessible surface area of 2370 ؎ 550 Å 2 . This area is only slightly smaller than the macromolecular interface calculated for a crystalline repressor dimer-O1 complex but is significantly smaller than that for the corresponding complex with the symmetrical optimized O sym operator. The transfer of repressor from site O1 to nonspecific DNA is accompanied by the net uptake of 93 ؎ 10 water molecules. Together these results imply that formation of a nonspecific complex is accompanied by the net release of 165 ؎ 43 water molecules. The enhanced stabilities of repressor-DNA complexes with increasing osmolality may contribute to the ability of Escherichia coli cells to tolerate dehydration and/or high external salt concentrations.The control of transcription initiation involves the binding of gene regulatory proteins to regulatory and competing genomic DNA sequences. The stability and specificity of these interactions depend on their solution environment. Important variables include salt concentration and identity (1-3), pH (4, 5), pressure (6 -8), and accessible volume (9, 10). In addition, changes in hydration accompany macromolecular interactions (reviewed in Refs. 8, 11, and 12). For those interactions in which the hydration change is large, the free energies of interaction depend sensitively on the activity of water (a H 2 O ).The intimate association of protein and DNA surfaces is accompanied by the displacement of water molecules associated with those surfaces. In addition, allosteric changes that extend beyond the contact surfaces can alter the solvent-accessible surface areas of protein and DNA and, thus, the numbers of associated water molecules. Any water molecules bound or released in these transactions are reactants or products, respectively, in the binding reaction. Changes in the number of thermodynamically associated water molecules can be detected and quantitated by the osmotic stress technique (12-14), using small, neutral solutes (osmolytes) that are typically excluded from the volumes immediately adjacent to macromolecular surfaces (11, 15). With three caveats, the dependence of the affinity of protein for DNA (K obs ) on water activity is a measure of the net change in the number of water molecules that are associated with the participating macromolecules. These are as follows: (i) that K obs should not be significantly perturbed by the differential interaction of osmolytes with reactants and products; (ii) that volume exclusion by osmolytes should not significantly alter K obs ; and (iii) that changes in solvent properties that indirectly affect bin...

A Balancing Act between Net Uptake of Water during Dihydrofolate Binding and Net Release of Water upon NADPH Binding in R67 Dihydrofolate Reductase

“…To determine whether the previously observed effect of sucrose on DHF binding (10) arises solely from perturbations on water activity, osmolytes with different characteristics were used. For example, addition of glycine betaine or sucrose can both affect water activity, yet these molecules either increase or decrease the dielectric constant of the solution, respectively (30,35,36). If both compounds show similar results, then effects on the dielectric constant are not involved.…”

“…Here, the osmotically active volume depends on the size of the osmolyte, with larger osmolytes detecting changes in larger volumes. Osmotic stress occurs as the solution must compensate for this exclusion (30,31). A last possibility is the direct interaction of the osmolyte with either the ligand or protein (37,38).…”

“…A possible decrease in K m for NADPH is also observed. If a linear relationship is observed in plots of either osmolality or ln water activity versus ln k cat /K m (DHF) , then effects on water are involved (30,38,40,41). Fig.…”

Binding properties of peptidic affinity ligands for plasmid DNA capture and detection

“…211 M in the presence of the same saline buffer but with 5% ethylene glycol. 24 More significantly, the dissociation constant was weaker than for a dsDNA fragment containing the pUC19 lac binding region (186 bp) interacting with a 64mer peptide which contained helices 1-4 (see Introduction) of 5.7 6 0.3 3 10 211 M 25 . Hence, it may be concluded that although the absence of helix III (residues 32-45) has a negligible effect on the overall dissociation constant, its absence appears to increase both the association and dissociation rates.…”

“…22 The in vivo dissociation constant of the Lac repressor and the complete lac operator (lacO 1 , lacO 2 , and lacO 3 ) has been calculated as 1 3 10 213 M. 23 The in vivo dissociation constant of the Lac repressor and lacO 1 alone has been estimated as 5 3 10 210 to 1 3 10 29 M 13 and 1-1.2 3 10 210 M in the presence of a saline buffer and 2.9-3.9 3 10 211 M in the presence of the same saline buffer but with 5% ethylene glycol. 24 In a previous study, a 186 bp double stranded DNA fragment created from a pUC19 template and a 64mer peptide ligand containing the complete LacI DNA binding domain (helix I-helix IV) displayed a dissociation constant (K D ) of 5.7 3 10 211 M. 25,26 As the optimal dissociation constant for an affinity binding mechanism for use in a chromatographic system is 10 26 M-10 28 M (Natural Toxins Research Centre, 2001), all of these systems display a K D too low for a chromatographic or adsorption/desorption system such as would be employed in a biosensor. If the dissociation constant is greater than this value, nontarget molecules can be co-purified as the mechanism is normally not strong or selective enough for the target.…”