Thermodynamic stoichiometries of participation of water, cations and anions in specific and non-specific binding of lac repressor to DNA : Possible thermodynamic origins of the “glutamate effect” on protein-DNA interactions

Abstract

The objective of this study is to quantify the contributions of cations, anions and water to stability and specificity of the interaction of lac repressor ( lac R) protein with the strong-binding symmetric lac, operator (O sym) DNA site. To this end, binding constants K obs and their power dependences on univalent salt (MX) concentration ( SK obs ≡ d log K obs d log[MX] ) have been determined for the interactions of lac R with O sym operator and with non-operator DNA using filter binding and DNA cellulose chromatography, respectively. For both specific and non-specific binding of lac R, K obs at fixed salt concentration [KX] increases when chloride (Cl −) is replaced by the physiological anion glutamate (Glu −). At 0.25 m-KX. the increase in K obs for O sym is observed to be $ ̃ 40-fold, whereas for non-operator DNA the increase in K obs is estimated by extrapolation to be 3 ̃ 00-fold. For non-operator DNA, SK obs RD is independent of salt concentration within experimental uncertainty, and is similar in KCl ( SK obs,KCl RD = −9.8(±1.0) between 0.13 m and 0.18 m-KCl) and KGlu ( SK obs,KGlu RD = −9.3(±0.7) between 0.23 m and 0.36 m-KGlu). For O sym DNA, SK obs RO varies significantly with the nature of the anion, and, at least in KGlu appears to decrease in magnitude with increasing [KGlu]. Average magnitudes of SK obs RO are less than SK obs RD, and, for specific binding decrease in the order ¦ SK obs,KCl RO¦ > ¦ SK obs,KAc RO¦ > SK obs,KGlu RO¦. Neither K obs RO nor SK obs RO is affected by the choice of univalent cation M + (Na +, K +, NH + 4. or mixtures thereof, all as the chloride salt), and SK obs RO is independent of [MCl] in the range examined (0.125 to 0.3 m). This behavior of SK obs RO is consistent with that expected for a binding process with a large contribution from the polyelectrolyte effect. However, the lack of an effect of the nature of the cation on the magnitude of SK obs RO at a fixed [MX] is somewhat unexpected, in view of the order of preference of cations for the immediate vicinity of DNA (NH 4 + > K + > Na +) observed by 23Na nuclear magnetic resonance. For both specific and non-specific binding, the large stoichiometry of cation release from the DNA polyelectrolyte is the dominant contribution to SK obs RO. To interpret these data, we propose that Glu − is an inert anion, whereas Ac and Cl compete with DNA phosphate groups in binding to lac repressor. A thermodynamic estimate of the minimum stoichiometry of water release from lac repressor and O sym operator (210(±30) H 2O) is determined from analysis of the apparently significant reduction in ¦ SK obs,KGlu RO¦ with increasing [KGlu] in the range 0.25 to 0.9 m. According to this analysis, SK obs values of specific arid non-specific binding in KGlu differ primarily because of the release of water in specific binding. In KAc and KCl, we deduce that anion competition affects K obs and SK obs to an extent which differs for different anions and for the different binding modes.