M. Thomas Record

Thermodynamic analysis of ion effects on the binding and conformational equilibria of proteins and nucleic acids: the roles of ion association or release, screening, and ion effects on water activity

The purpose of this review is to examine the various effects of low- molecular-weight electrolytes on the associations and interactions of proteins and nucleic acids. Our primary interest is in general electrostatic effects, rather than chemical effects (specific interactions) of particular ions (e.g. transition metals, protons). We consider those interactions in which a variation in salt concentration has a significant effect on the macromolecular equilibrium, and analyse the effects of salt in these situations in terms of (i) direct participation of ions in the biopolymer reaction, (ii) Debye—Huckel screening by salt ions of the charge interactions on the biopolymers, and (iii) the reduction in water activity brought about at high salt concentrations.

Ion effects on ligand-nucleic acid interactions.

We have developed a general thermodynamic analysis of monovalent ion effects on the observed association constants K obs of ligand-nucleic acid interactions. Our approach is based on the binding theory of Wyman (1964) and the polyelectrolyte theory of Manning (1969) . In the case of model ligands such as Mg 2+ or short oligolysines, where there is no anion binding by the ligand, the dependence of K obs on monovalent ion (M + ) concentration results from the release of M + counterions from the nucleic acid in the association reaction. We find that, for these systems, log K obs is a linear function of log [M + ]. The slope of such a graph yields the number of charge interactions, or ion pairs, formed between ligand and nucleic acid; the intercept of a linear extrapolation to a 1 m -M + standard state yields the non-electrostatic component of the binding free energy. From an analysis of the data of Latt & Sober (1967) on the interactions of oligolysines with polyribonucleotides, we have concluded that the dominant factor driving complex formation between these charged ligands and the nucleic acid is the entropic contribution from the release of counterions. Counterion release also appears to drive the non-specific interactions of proteins with nucleic acids.

Role of the Hydrophobic Effect in Stability of Site-specific Protein-DNA Complexes

The site-specific binding interaction of lac repressor with a symmetric operator sequence and of EcoRI endonuclease with its specific recognition site both exhibit a characteristic dependence of equilibrium binding constant (Kobs) on temperature, in which Kobs attains a relative maximum in the physiologically relevant temperature range. This behavior, which appears to be quite general for site-specific protein-DNA interactions, is indicative of a large negative standard heat capacity change (delta C0P,obs) in the association process. By analogy with model compound transfer studies and protein folding data, we propose that this delta C0P,obs results primarily from the removal of non-polar surface from water in the association process. From delta C0P,obs we obtain semiquantitative information regarding the change in water-exposed non-polar surface area (delta Anp) and the corresponding hydrophobic driving force for association (delta G0hyd): delta G0hyd approximately equal to 8(+/- 1) x 10(1) delta C0P,obs approximately equal to -22(+/- 5) delta Anp. We propose that removal of non-polar surface from water (the hydrophobic effect) and release of cations (the polyelectrolyte effect) drive the thermodynamically unfavorable process (e.g. conformational distortions) necessary to achieve mutually complementary recognition surfaces (at a steric and functional-group level) in the specific complex.

Novel computer program for fast exact calculation of accessible and molecular surface areas and average surface curvature.

New computer programs, SurfRace and FastSurf, perform fast calculations of the solvent accessible and molecular (solvent excluded) surface areas of macromolecules. Program SurfRace also calculates the areas of cavities inaccessible from the outside. We introduce the definition of average curvature of molecular surface and calculate average molecular surface curvatures for each atom in a structure. All surface area and curvature calculations are analytic and therefore yield exact values of these quantities. High calculation speed of this software is achieved primarily by avoiding computationally expensive mathematical procedures wherever possible and by efficient handling of surface data structures. The programs are written initially in the language C for PCs running Windows 2000/98/NT, but their code is portable to other platforms with only minor changes in input‐output procedures. The algorithm is robust and does not ignore either multiplicity or degeneracy of atomic overlaps. Fast, memory‐efficient and robust execution make this software attractive for applications both in computationally expensive energy minimization algorithms, such as docking or molecular dynamics simulations, and in stand‐alone surface area and curvature calculations.

Polypeptides containing highly conserved regions of transcription initiation factor σ70 exhibit specificity of binding to promoter DNA

The sigma 70 subunit of E. coli RNA polymerase is required for sequence-specific recognition of promoter DNA. Genetic studies and sequence analysis have indicated that sigma 70 contains two specific DNA-binding domains that recognize the two conserved portions of the prokaryotic promoter. However, intact sigma 70 does not bind to DNA. Using C-terminal and internal polypeptides of sigma 70, carrying one or both putative DNA-binding domains, we demonstrate that sigma 70 does contain two DNA-binding domains, but that N-terminal sequences inhibit the ability of intact sigma 70 to bind to DNA. Thus, we propose that sigma 70 is a sequence-specific DNA-binding protein that normally functions through an allosteric interaction with the core subunits of RNA polymerase.

Responses of E. coli to osmotic stress: large changes in amounts of cytoplasmic solutes and water

Escherichia coli is capable of growing in environments ranging from very dilute aqueous solutions of essential nutrients to media containing molar concentrations of salts or nonelectrolyte solutes. Growth in environments with such a wide range (at least 100-fold) of osmolarities poses significant physiological challenges for cells. To meet these challenges, E. coli adjusts a wide range of cytoplasmic solution variables, including the cytoplasmic amounts both of water and of charged and uncharged solutes.

[16] Analysis of equilibrium and kinetic measurements to determine thermodynamic origins of stability and specificity and mechanism of formation of site-specific complexes between proteins and helical DNA

The concentration and nature of the electrolyte are key factors determining (1) the equilibrium extent of binding of oligocations or proteins to DNA, (2) the distribution of bound protein between specific and nonspecific sites, and (3) the kinetics of association and dissociation of both specific and nonspecific complexes. Salt concentration may therefore be used to great advantage to probe the thermodynamic basis of stability and specificity of protein-DNA complexes, and the mechanisms of association and dissociation. Cation concentration serves as a thermodynamic probe of the contributions to stability and specificity from neutralization of DNA phosphate charges and/or reduction in phosphate charge density. Cation concentration also serves as a mechanistic probe of the kinetically significant steps in association and dissociation that involve cation uptake. In general, effects of electrolyte concentration on equilibrium constants (quantified by SKobs) and rate constants (quantified by Skobs) are primarily cation effects that result from the cation-exchange character of the interactions of proteins and oligocations with polyanionic DNA. The competitive effects of Mg2+ or polyamines on the equilibria and kinetics of protein-DNA interactions are interpretable in the context of the cation-exchange model. The nature of the anion often has a major effect on the magnitude of the equilibrium constant (Kobs) and rate constant (kobs) of protein-DNA interactions, but a minor effect on SKobs and Skobs, which are dominated by the cation stoichiometry. The order of effects of different anions generally follows the Hofmeister series and presumably reflects the relative extent of preferential accumulation or exclusion of these anions from the relevant surface regions of DNA-binding proteins. The question of which anion is most inert (i.e., neither accumulated nor excluded from the relevant regions of these proteins) remains unanswered. The characteristic effects of temperature on equilibrium constants and rate constants for protein-DNA interactions also serve as diagnostic probes of the thermodynamic origins of stability and specificity and of the mechanism of the interaction, since large changes in thermodynamic and activation heat capacities accompany processes with large changes in the amount of water-accessible nonpolar surface area.(ABSTRACT TRUNCATED AT 400 WORDS)

Thermodynamic origin of hofmeister ion effects.

Quantitative interpretation and prediction of Hofmeister ion effects on protein processes, including folding and crystallization, have been elusive goals of a century of research. Here, a quantitative thermodynamic analysis, developed to treat noncoulombic interactions of solutes with biopolymer surface and recently extended to analyze the effects of Hofmeister salts on the surface tension of water, is applied to literature solubility data for small hydrocarbons and model peptides. This analysis allows us to obtain a minimum estimate of the hydration b1 (H2O A(-2)), of hydrocarbon surface and partition coefficients Kp, characterizing the distribution of salts and salt ions between this hydration water and bulk water. Assuming that Na+ and SO4(2-) ions of Na2SO4 (the salt giving the largest reduction in hydrocarbon solubility as well as the largest increase in surface tension) are fully excluded from the hydration water at hydrocarbon surface, we obtain the same b1 as for air-water surface (approximately 0.18 H2O A(-2)). Rank orders of cation and anion partition coefficients for nonpolar surface follow the Hofmeister series for protein processes, but are strongly offset for cations in the direction of exclusion (preferential hydration). By applying a coarse-grained decomposition of water accessible surface area (ASA) into nonpolar, polar amide, and other polar surface and the same hydration b1 to interpret peptide solubility increments, we determine salt partition coefficients for amide surface. These partition coefficients are separated into single-ion contributions based on the observation that both Cl- and Na+ (also K+) occupy neutral positions in the middle of the anion and cation Hofmeister series for protein folding. Independent of this assignment, we find that all cations investigated are strongly accumulated at amide surface while most anions are excluded. Cation and anion effects are independent and additive, allowing successful prediction of Hofmeister salt effects on micelle formation and other processes from structural information (ASA).

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.

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 (Osym) DNA site. To this end, binding constants Kobs and their power dependences on univalent salt (MX) concentration (SKobs = d log Kobs/d log[MX]) have been determined for the interactions of lac R with Osym operator and with non-operator DNA using filter binding and DNA cellulose chromatography, respectively. For both specific and non-specific binding of lac R, Kobs 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 Kobs for Osym is observed to be approximately 40-fold, whereas for non-operator DNA the increase in Kobs is estimated by extrapolation to be approximately 300-fold. For non-operator DNA, SKobsRD is independent of salt concentration within experimental uncertainty, and is similar in KCl (SKobs,RDKCl = -9.8(+/- 1.0) between 0.13 M and 0.18 M-KCl) and KGlu (SKobs,RDKGlu = -9.3(+/- 0.7) between 0.23 M and 0.36 M-KGlu). For Osym DNA, SKobsRO varies significantly with the nature of the anion, and, at least in KGlu appears to decrease in magnitude with increasing [KGlu]. Average magnitudes of SKobsRO are less than SKobsRD, and, for specific binding decrease in the order [SKobsRO,KCl[>[SKobsRO,KAc[>[SKobsRO,KGlu[ . Neither KobsRO nor SKobsRO is affected by the choice of univalent cation M+ (Na+, K+, NH4+, or mixtures thereof, all as the chloride salt), and SKobsRO is independent of [MCl] in the range examined (0.125 to 0.3 M). This behavior of SKobsRO 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 KobsRO at a fixed [MX] is somewhat unexpected, in view of the order of preference of cations for the immediate vicinity of DNA (NH4+ > 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 SKobs. 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 Osym operator (210(+/- 30) H2O) is determined from analysis of the apparently significant reduction in [SKobsRO,KGlu[ with increasing [KGlu] in the range 0.25 to 0.9 M. According to this analysis, SKobs values of specific and 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 Kobs and SKobs to an extent which differs for different anions and for the different binding modes.

Crowding and Confinement Effects on Protein Diffusion In Vivo

The first in vivo measurements of a protein diffusion coefficient versus cytoplasmic biopolymer volume fraction are presented. Fluorescence recovery after photobleaching yields the effective diffusion coefficient on a 1-mum-length scale of green fluorescent protein within the cytoplasm of Escherichia coli grown in rich medium. Resuspension into hyperosmotic buffer lacking K+ and nutrients extracts cytoplasmic water, systematically increasing mean biopolymer volume fraction, , and thus the severity of possible crowding, binding, and confinement effects. For resuspension in isosmotic buffer (osmotic upshift, or Delta, of 0), the mean diffusion coefficient, , in cytoplasm (6.1 +/- 2.4 microm2 s(-1)) is only 0.07 of the in vitro value (87 microm2 s(-1)); the relative dispersion among cells, sigmaD/ (standard deviation, sigma(D), relative to the mean), is 0.39. Both and sigmaD/ remain remarkably constant over the range of Delta values of 0 to 0.28 osmolal. For a Delta value of > or =0.28 osmolal, formation of visible plasmolysis spaces (VPSs) coincides with the onset of a rapid decrease in by a factor of 380 over the range of Delta values of 0.28 to 0.70 osmolal and a substantial increase in sigmaD/. Individual values of D vary by a factor of 9 x 10(4) but correlate well with f(VPS), the fractional change in cytoplasmic volume on VPS formation. The analysis reveals two levels of dispersion in D among cells: moderate dispersion at low Delta values for cells lacking a VPS, perhaps related to variation in phi or biopolymer organization during the cell cycle, and stronger dispersion at high Delta values related to variation in f(VPS). Crowding effects alone cannot explain the data, nor do these data alone distinguish crowding from possible binding or confinement effects within a cytoplasmic meshwork.