A major contribution to the binding free energy associated with most protein-nucleic acid complexes is the increase in entropy due to counterion release from the nucleic acid that results from electrostatic interactions. To examine this quantitatively, we have measured the thermodynamic extent of counterion release that results from the interaction between single-stranded homopolynucleotides and a series of oligolysines, possessing net charges z = 2-6, 8, and 10. This was accomplished by measuring the salt dependence of the intrinsic equilibrium binding constants-i.e., (olog Kw/ 8log [KJ])-over the range from 6 mM to 0.5 M potassium acetate. These data provide a rigorous test of linear polyelectrolyte theories that have been used to interpret the effects of changes in bulk salt concentration on protein-DNA binding equilibria, since single-stranded nucleic acids have a lower axial charge density than duplex DNA. Upon binding to poly(U), the thermodynamic extent of counterion release per oligolysine charge, z, is 0.71 ± 0.03, which is signfilcandy less than unity and less than that measured upon binding duplex DNA. These results are most simply interpreted using the limiting law predictions of counterion condensation and cylindrical Poisson-Boltzmann theories, even at the high salt concentrations used in our experiments. Accurate estimates of the thermodynamic extent of counterion binding and release for model systems such as these facilitate our understanding of the energetics of protein-nucleic acid interactions. These data indicate that for simple oligovalent cations, the number of ionic interactions formed in a complex with a linear nucleic acid can be accurately estimated from a measure of the salt dependence of the equilibrium binding constant, if the thermodynamic extent of ion release is known.The interactions of proteins with nucleic acids are central to the control of gene expression and nucleic acid metabolism. A detailed understanding of how these processes are regulated requires information about the equilibrium affinity and pathways of association and dissociation of the proteinnucleic acid complexes involved. Structural data can provide information concerning the contacts made within proteinnucleic acid complexes; however, thermodynamic information is necessary to understand the stability of these complexes. One A number of theoretical studies have sought to obtain a quantitative molecular interpretation of these dramatic salt effects (1,(3)(4)(5)(6)(7). Two of these were based on counterion condensation (CC) models that describe the electrostatic interaction of counterions with linear nucleic acids (1,5). The CC models (5,8) treat the nucleic acid as a uniform line charge and predict that a constant fraction of the phosphate charges is neutralized by the delocalized binding (condensation) of counterions. The extent of counterion condensation per phosphate, which is predicted by CC theory, is a function only of the linear charge density of the nucleic acid, the counterion charge, and the d...

No abstract

The equilibrium binding to the synthetic RNA poly(U) of a series of oligolysines containing one, two, or three tryptophans has been examined as a function of pH, monovalent salt concentration (MX), temperature, and Mg2+. Oligopeptides containing lysine (K) and tryptophan (W) of the type KWKp-NH2 and KWKp-CO2 (p = 1-8), as well as peptides containing additional tryptophans or glycines, were studied by monitoring the quenching of the peptide tryptophan fluorescence upon binding poly(U). Equilibrium association constants, K(obs), and the thermodynamic quantities delta G(o)obs, delta H(o)obs, and delta S(o)obs describing peptide-poly(U) binding were measured as well as their dependences on monovalent salt concentration, temperature, and pH. In all cases, K(obs) decreases significantly with increasing monovalent salt concentration, with (delta log K(obs)/delta log [K+]) = -0.74 (+/- 0.04)z, independent of temperature and salt concentration, where z is the net positive charge on the peptide. The origin of these salt effects is entropic, consistent with the release of counterions from the poly(U) upon formation of the complex. Upon extrapolation to 1 M K+, the value of delta G(o)obs is observed to be near zero for all oligolysines binding to poly(U), supporting the conclusion that these complexes are stabilized at lower salt concentrations due to the increase in entropy accompanying the release of monovalent counterions from the poly(U). Only the net peptide charge appears to influence the thermodynamics of these interactions, since no effects of peptide charge distribution were observed. The binding of poly(U) to the monotryptophan peptides displays interesting behavior as a function of the peptide charge. The extent of tryptophan fluorescence quenching, Qmax, is dependent upon the peptide charge for z less than or equal to +4, and the value of Qmax correlates with z-dependent changes in delta H(o)obs and delta S(o)obs(1 M K+), whereas for z greater than or equal to +4, Qmax, delta H(o)obs, and delta S(o)obs (1 M K+) are constant. The correlation between Qmax and delta H(o)obs and delta S(o)obs(1 M K+) suggests a context (peptide charge)-dependence of the interaction of the peptide tryptophan with poly(U). However the interaction of the peptide tryptophan does not contribute substantially to delta G(o)obs for any of the peptides, independent of z, due to enthalpy-entropy compensations. Each of the tryptophans in multiple Trp-containing peptides appear to bind to poly(U) independently, with delta H(o)Trp = -2.9 +/- 0.7, although delta G(o)Trp is near zero due to enthalpy-entropy compensations.(ABSTRACT TRUNCATED AT 400 WORDS)

We have examined the equilibrium binding of a series of synthetic oligoarginines (net charge z = +2 to +6) containing tryptophan to poly(U), poly(A), poly(C), poly(I), and double-stranded (ds) DNA. Equilibrium association constants, K(obs), measured by monitoring tryptophan fluorescence quenching, were examined as functions of monovalent salt (MX) concentration and type, as well as temperature, from which deltaG(standard)obs, deltaH(obs), and deltaS(standard)obs were determined. For each peptide, K(obs) decreases with increasing [K+], and the magnitude of the dependence of K(obs) on [K+], delta log K(obs)/delta log[K+], increases with increasing net peptide charge. In fact, the values of delta log K(obs)/delta log[K+] are equivalent for oligolysines and oligoarginines possessing the same net positive charge. However, the values of K(obs) are systematically greater for oligoarginines binding to all polynucleotides, when compared to oligolysines with the same net charge. The origin of this difference is entirely enthalpic, with deltaH(obs), determined from van't Hoff analysis, being more exothermic for oligoarginine binding. The values of deltaH(obs) are also independent of [K+]; therefore, the salt concentration dependence of deltaG(standard)obs is entirely entropic in origin, reflecting the release of cations from the nucleic acid upon complex formation. These results suggest that hydrogen bonding of arginine to the phosphate backbone of the nucleic acids contributes to the increased stability of these complexes.

We have examined the thermodynamics of binding of a series of oligolysines (net charge z = +2 to +10) containing one, two, or three tryptophans to several single-stranded (ss) homo-polynucleotides [poly(A), poly(C), poly(I), poly(dU), poly(dT)] and duplex (ds) DNA in order to investigate the effects of peptide charge, tryptophan content, and polynucleotide base and sugar type. Equilibrium association constants, Kobs, were measured as a function of monovalent salt concentration (KCH3CO2) and temperature by monitoring the quenching of the peptide tryptophan fluorescence upon interaction with the polynucleotides, from which the dependence of delta G(o)obs, delta H(o)obs, and delta S(o)obs on [KCH3CO2] was obtained. As observed previously with poly(U) [Mascotti, D.P., & Lohman, T.M. (1992) Biochemistry 31, 8932], the dependence of delta G(o)obs on [K+] for peptide binding to each polynucleotide is entirely entropic in origin (i.e., delta H(o)obs is independent of [K+]), consistent with the conclusion that Kobs increases with decreasing salt concentration due to the favorable increase in entropy resulting from the displacement of bound cations (K+) from the nucleic acid upon formation of the complex. For each ss polynucleotide, we find that significantly less than one potassium ion is released thermodynamically per net positive peptide charge, as determined from the value of delta log Kobs/delta log [K+]. Interestingly, (-delta log Kobs/delta log [K+])/z decreases with increasing peptide charge for poly(A), poly(C), and poly(dT), contrary to the behavior observed with poly(U) and ds-DNA, which may reflect a significant release of bound water upon formation of peptide complexes with these ss homo-polynucleotides or an increased binding of K+ to the ss polynucleotide with increasing [K+]. Alternatively, there may be conformational differences between the bound states of oligolysines of low charge, relative to oligolysines of higher charge. However, in all cases, peptides with z < +4 display different thermodynamics of binding than peptides with z > +4. The presence of tryptophan (Trp) within these peptides does not influence the salt dependence of Kobs for binding to poly(A), poly(C), or poly(dT). However, the Trp content of the peptide does contribute significantly to the thermodynamics of these interactions: Trp interactions result in a favorable contribution to delta H(o)obs, but an unfavorable contribution to delta S(o)obs, with little effect on delta G(o)obs due to entropy-enthalpy compensations. Oligolysines containing Trp also display a small, but significant, dependence of Kobs on base composition, with Kobs decreasing in the order poly(I) >> poly(dT) approximately poly(U) approximately poly(A) >> poly(C).