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

Abstract

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.Whereas it is generally accepted that hydration water plays an important role in DNA-protein sequence-specific recognition (1) there are only few techniques available to probe its contribution reliably. We use an approach termed the osmotic stress technique (2, 3) that measures changes in hydration coupled with changes in the functional state by measuring the effect of water activity on reaction equilibria and kinetics. The osmotic stress technique has been used previously to measure the changes in hydration accompanying the DNA binding of several regulatory proteins: Escherichia coli gal (4), lac (5), tyr (6), and Cro (7) repressors, E. coli CAP protein (8), Hin recombinase (9), Ultrathorax and Deformed homeodomains (10), the restriction endonucleases , BamHI (15,16), and EcoRV (17), HhaI methyltransferase (18), Sso7d protein (19), the TATA-binding protein (20,21), and the E2C protein from papillomavirus (22).The dependence of an equilibrium constant on the bulk solution osmotic pressure that is varied by adding neutral solutes that do not bind directly to the DNA or protein gives a difference in the number of associated waters between the initial reactants and the final products. More precisely, water is considered associated with the DNA, protein, and complex if it excludes osmolyte, otherwise there is no osmotic imbalance. Obviously, water that is sterically sequestered in cavities, channels, or pockets fulfills this requirement. All solutes that are excluded from these cavities act identically on the equilibrium. Water molecules hydrating exposed macromolecular surfaces are more problematic. For the most part, solutes are excluded from these waters due to "preferential hydration," macromolecules prefer their interactions with water over...

The Glutamate Effect on DNA Binding by Pol I DNA Polymerases: Osmotic Stress and the Effective Reversal of Salt Linkage

“…Such analysis has been widely used to estimate changes in hydration associated with macromolecular processes 36,37 and has been applied to a number of protein-DNA interactions, in particular for the comparative study of hydration changes associated with sequence-specific versus non-sequence-specific DNA binding. [51][52][53][54][55][56] It is necessary to perform control experiments examining the effects of the size and chemical nature of the solutes used. 36,37 Therefore, at a constant osmotic pressure (1120 mOsm for Klenow and 320 mOsm for Klentaq), PEGs of various sizes (MW: 200 to 10,000) were used to determine the osmolyte size at which the equilibrium constant was independent of the size of the osmolyte.…”

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

“…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.…”

“…However, the slopes for the glycine betaine, Me 2 SO, and sucrose data are higher, ranging from 40 to 60. Variable slopes are common in osmolality studies, but their origin is not clear (37,38,(41)(42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54). This issue will be broached under the "Discussion.…”

Using Single-Turnover Kinetics with Osmotic Stress To Characterize the EcoRV Cleavage Reaction

“…by changing water activity. This sensitivity to osmotic pressure is a common feature of many protein-DNA complexes (15–27). Since the specific to nonspecific mode of protein binding is a necessary initial step in dissociation of the specific complex, osmotic pressure also greatly slows the dissociation rate.…”

“…In this way we specifically measure the numbers of Mg 2+ , H + , and water molecules coupled to the cleavage kinetics between the initial specific EcoRV-DNA complex with no bound Mg 2+ and the transition cleavage state. This is an extension of the self-cleavage assay we developed previously to measure specific binding of the restriction endonucleases through their cleavage with high precision (15, 27, 28). The foundation for this technique is that specifically bound enzyme in the absence of Mg 2+ can be stoichiometrically converted to the active form simply by adding Mg 2+ .…”