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J. Biol. Chem., Vol. 281, Issue 47, 35656-35666, November 24, 2006

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Differences in Hydration Coupled to Specific and Nonspecific Competitive Binding and to Specific DNA Binding of the Restriction Endonuclease BamHI^*

From the Laboratory of Physical and Structural Biology, NICHD, National Institutes of Health, Bethesda, Maryland 20892

Using the osmotic stress technique together with a self-cleavage assay we measure directly differences in sequestered water between specific and nonspecific DNA-BamHI complexes as well as the numbers of water molecules released coupled to specific complex formation. The difference between specific and nonspecific binding free energy of the BamHI scales linearly with solute osmolal concentration for seven neutral solutes used to set water activity. The observed osmotic dependence indicates that the nonspecific DNA-BamHI complex sequesters some 120–150 more water molecules than the specific complex. The weak sensitivity of the difference in number of waters to the solute identity suggests that these waters are sterically inaccessible to solutes. This result is in close agreement with differences in the structures determined by x-ray crystallography. We demonstrate additionally that when the same solutes that were used in competition experiments are used to probe changes accompanying the binding of free BamHI to its specific DNA sequence, the measured number of water molecules released in the binding process is strikingly solute-dependent (with up to 10-fold difference between solutes). This result is expected for reactions resulting in a large change in a surface exposed area.

Received for publication, August 21, 2006

^* This work was supported by the Intramural Research Program of the NICHD, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Bldg. 9, Rm. 1E-108, Bethesda, MD 20892. Tel.: 301-402-4698; Fax: 301-496-2172; E-mail: sidorova{at}mail.nih.gov.

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