Differences in water release for the binding of EcoRI to specific and nonspecific DNA sequences.

Abstract

The free energy difference between complexes of the restriction nuclease EcoRI with nonspecific DNA and with the enzyme's recognition sequence is linearly dependent on the water chemical potential of the solution, set using several very different solutes, ranging from glycine and glycerol to triethylene glycol and sucrose. This osmotic dependence indicates that the nonspecific complex sequesters some 110 waters more than the specific complex with the recognition sequence. The insensitivity of the difference in number of waters released to the solute identity further indicates that this water is sequestered in a space that is sterically inaccessible to solutes, most likely at the protein-DNA interface of the nonspecific complex. Calculations based on the structure of the specific complex suggest that the apposing DNA and protein surfaces in the nonspecific complex retain approximately a full hydration layer of water.The correct functioning of transcription factors and other regulatory proteins that recognize specific DNA sequences requires not only binding with high affinity to the proper sequence but also the ability to distinguish effectively the recognition sequence from all others, including ones that differ by only 1 or 2 bp. A key problem in biophysics is understanding how such binding strength and specificity are so tightly linked in recognition reactions. As yet, there is no simple way to connect structure, derived from x-ray crystallography or NMR, with thermodynamics and with the physics of molecular interactions between individual groups on apposing surfaces. Direct measurements of forces between macromolecules in condensed arrays (reviewed in ref. 1) have been interpreted as showing the dominance of water structuring on interaction energies at close surface separation (<10-15 A). These measurements suggest that differences in binding free energies for the association of proteins to different DNA sequences in dilute solution should correlate with differences in the number of water molecules retained in the complexes. Crystal structures of many specific DNA-protein complexes show that direct DNA-protein contacts mostly replace DNA-water and protein-water interactions with little or no water left at the interface (2, 3). Comparison of the crystal structure of the estrogen receptor-like DNA binding domain with a noncognate DNA sequence that has a binding constant about an order of magnitude smaller than the recognition sequence shows several additional waters incorporated at the interface between protein and DNA that are not seen in the specific complex (4). Just as differences in proton or salt binding accompanying macromolecular reactions can be thermodynamically probed by the sensitivity of the reaction to pH or salt activity, respectively, differences in the number of water molecules retained by different complexes can be determined from the dependence of binding equilibrium constants on the bulk water activity, controlled by the concentration of added solutes (5).A paradigm for specific recog...

Sequence Discrimination by DNA-binding Domain of ETS Family Transcription Factor PU.1 Is Linked to Specific Hydration of Protein-DNA Interface

“…Because macromolecular surfaces in general preferentially exclude solutes (or, equivalently, are preferentially hydrated) (34 -36), osmolytes drive complex formation to minimize these soluteexposed surfaces. Moreover, high affinity interactions that appose highly complementary surfaces are expected to displace more water than low affinity and nonspecific complexes (18). Our observation that PU.1 ETS binding to the high affinity [Ϫ]GC site is destabilized by osmotic stress is therefore highly unusual.…”

“…After electrophoresis, the gel was stained with SYBR Gold (Invitrogen). No significant changes in quantum yield due to protein binding were reported for the SYBR series of dyes (18). Fractional bound fragment (f b ) as a function of total DNA oligonucleotide concentration ([D 1 ] t ) was fitted to the positive, real root of the following cubic equation,…”

Probing the role of water in protein conformation and function

“…Even the specificity of the restriction enzyme EcoR1 is dependent on its osmotic environment (Robinson & Sligar 1993;Sidorova & Rau 1996). Contacts within the tryptophan repressor-DNA operator complex are largely water mediated (Brown et al 1999).…”

Crystal structure of an engineered cro monomer bound nonspecifically to DNA: Possible implications for nonspecific binding by the wild‐type protein

“…The proposed model encompasses many of the features anticipated for proteins bound nonspecifically to DNA (e.g., see Berg et al, 1982;Pendergrast et al, 1994;Sidorova & Rau, 1996). In particular, the protein is separated from the DNA, relative to the specific complex, allowing water to intervene (Fig.…”