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

Nina Y Sidorova,Shakir Muradymov,Donald C Rau

doi:10.1074/jbc.m608018200

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.

Highlights

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
Our goal here is 2-fold: 1) to measure directly differences in sequestered water between specific and nonspecific BamHI complexes to confirm the correspondence between waters measured by osmotic stress and structure; and 2) to demonstrate the complications connected with using osmotic stress for reactions that are accompanied by significant changes in exposed surface area as, for example, the specific binding of free protein
We demonstrate that when the same solutes are used to probe changes in hydration accompanying the binding of free BamHI to its recognition sequence, the measured difference in the number of participating water molecules is strikingly solute-dependent as expected for a reaction coupled with a significant change in exposed surface area

Summary

Introduction

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. Our goal here is 2-fold: 1) to measure directly differences in sequestered water between specific and nonspecific BamHI complexes to confirm the correspondence between waters measured by osmotic stress and structure; and 2) to demonstrate the complications connected with using osmotic stress for reactions that are accompanied by significant changes in exposed surface area as, for example, the specific binding of free protein. The osmotic pressure dependence of Knsp-sp indicates that about 120 –150 extra water molecules are retained in the nonspecific versus specific BamHI-DNA complex in very good agreement with the x-ray structural data This number is only slightly dependent on the nature of the solute used to set water activity for seven osmolytes, consistent with a steric exclusion of solutes from a cavity at the interface of the BamHI-DNA nonspecific complex. This 10-fold difference in the numbers of waters associated with specific binding should be compared with differences in the number of waters of only ϳ126 and ϳ145 for sucrose and stachyose for the difference between specific and nonspecific complexes measured by competition

Results

Discussion

Conclusion