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...

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The equilibrium association constant observed for many DNAIprotein interactions in vitro (Kc,h,) is strongly dependent on the salt concentration of the reaction buffer ([MX]). This dependence is often used to estimate the number of ionic contacts between protein and DNA by assuming that displacement of cations from the DNA is the predominant form of the involvement of ions in the binding reaction. With this assumption, the graph of log Koh\ versus log [MX] is predicted to have a constant slope proportional to the number of ions displaced from the DNA upon protein binding Mol. Bid. 107, 145-1581. Experimental data often deviate from linearity, however, at lower salt concentrations. Such deviations can be due to differential cation binding, anion binding or changes in macromolecular hydration, or differential screening effects of the electrolyte on protein and/or DNA charges. Here the theoretical effects on K,,h, of a simple form of ion-protein interaction are examined. A model for binding interactions is used that includes a mass balance of ions bound to both protein and DNA as the protein is transferred from the salt concentration of bulk solvent to the typically higher cation and lower anion concentrations characteristic of the volume adjacent to the DNA. We show that models in which the cation and anion stoichiometries of a protein change as it associates with DNA are consistent with the curvature of plots of log KCjh, versus log [MX]. Such mechanisms could reduce the sensitivity of gene-regulatory interactions to changes in environmental salt concentration.At the molecular level, gene expression is controlled by the interactions of gene regulatory proteins and RNA polymerase with specific and nonspecific sites on DNA. These interactions are often found to be highly sensitive to the salt concentration of the buffers in which they have been measured. For example, the observed equilibrium constants (KO,,,) Several possible mechanisms have previously been examined. Calculations based on a thermodynamic model of lac repressor-operator interactions in E. coli show that the combination of a large number of nonspecific repressor binding sites and a salt sensitivity for nonspecific binding that is greater than, or equal to, that for specific binding, can produce a buffering effect that reduces the salt sensitivity of binding at the operator site [3]. However, the nonspecific DNA buffering model does not reduce the salt sensitivity to the degree observed in vivo 131. Some protein-DNA complexes have been found to be more stable in solutions in which glutamate is the principal anion than in solutions in which other anions dominate [4]. Glutamate has been shown to be an important osmolyte in E. coli cytoplasm, especially in cells that have been allowed to adapt to high extracellular salt concentrations [ 5 ] . Finally, the volume of cytoplasmic water has been shown to decrease with increasing extracellular osmolarity, resulting in increased macromolecular concentrations [3, 61. Calculations using scaled particle theory...

The polyol 1,5-anhydroglucitol (AG) present in human plasma is derived largely from ingestion and is excreted unmetabolized. Reduction of plasma [AG] has been noted in diabetics and is due to accelerated excretion of AG during hyperglycemia. Plasma [AG] has therefore been proposed as a marker for glycemic control. A precise understanding of its utility relies on a quantitative understanding of the mass balance for AG. In this study, non-steady-state data from the literature were analyzed to develop a dynamic mass balance model for AG that is based on the two-compartment model proposed by Yamanouchi et al. [T. Yamanouchi, Y. Tachibana, H. Akanuma, S. Minoda, T. Shinohara, H. Moromizato, H. Miyashita, and I. Akaoka. Am. J. Physiol. 263 ( Endocrinol. Metab. 26): E268—E273, 1992]. The data are consistent with a model in which exchange between tissue and plasma pools is rapid and in which the tissue compartment mass is two to three times the mass of the plasma compartment. According to model estimates, accelerated excretion of AG due to hyperglycemia can cause marked net depletion of total AG over a time scale of days. Recovery from a depleted state is slow because the total body capacity represents >5 wk of normal intake. Accordingly, AG monitoring should be able to indicate the presence of past glucosuric hyperglycemic episodes during a period of days to weeks, as well as provide information on the extent to which high deviations from the average plasma glucose concentration are operative.