Preferential interactions between small solutes and the protein backbone: a computational analysis.

To improve our understanding of the effects of small solutes on protein stability, we conducted atomistic simulations to quantitatively characterize the interactions between two broadly used small solutes, urea and glycine betaine (GB), and a triglycine peptide, which is a good model for a protein backbone. Multiple solute concentrations were analyzed, and each solute-peptide-water ternary system was studied with approximately 200-300 ns of molecular dynamics simulations with the CHARMM force field. The comparison between calculated preferential interaction coefficients (Gamma(23)) and experimentally measured values suggests that semiquantitative agreement with experiments can be obtained if care is exercised to balance interactions among the solute, protein, and water. On the other hand, qualitatively incorrect (i.e., wrong sign in Gamma(23)) results can be obtained if a solute model is constructed by directly taking parameters for chemically similar groups from an existing force field. Such sensitivity suggests that small solute thermodynamic data can be valuable in the development of accurate force field models of biomolecules. Further decomposition of Gamma(23) into group contributions leads to additional insights regarding the effects of small solutes on protein stability. For example, use of the CHARMM force field predicts that urea preferentially interacts with not only amide groups in the peptide backbone but also aliphatic groups, suggesting a role for these interactions in urea-induced protein denaturation; quantitatively, however, it is likely that the CHARMM force field overestimates the interaction between urea and aliphatic groups. The results with GB support a simple thermodynamic model that assumes additivity of preferential interaction between GB and various biomolecular surfaces.

Quantifying accumulation or exclusion of H+, HO-, and Hofmeister salt ions near interfaces.

Recently, surface spectroscopies and simulations have begun to characterize the non-uniform distributions of salt ions near macroscopic and molecular surfaces. The thermodynamic consequences of these non-uniform distributions determine the often-large ion-specific effects of Hofmeister salts on a very wide range of processes in water. For uncharged surfaces, where these nonuniform ion distributions are confined to the first few layers of water at the surface, a two-state approximation to the distributions of water and ions, called the salt ion partitioning model (SPM) has both molecular and thermodynamic signiicance. Here, we summarize SPM results quantifying the local accumulation of H+, exclusion of HO-, and range of partitioning behavior of Hofmeister anions and cations near macroscopic and molecular interfaces. These results provide a database to interpret or predict Hofmeister salt effects on aqueous processes in terms of structural information regarding amount and composition of the surface exposed or buried in these processes.

Do sigma factors need help with a meltdown?

In this issue of Cell, Hsu et al. (2006) report on the binding activity of a variant of the bacterial transcriptional specificity factor sigma (sigma) to promoter DNA. This study demonstrates that the sigma variant induces a large distortion in the transcriptional start site in the absence of core RNA polymerase, raising intriguing new questions about the roles of sigma and core RNA polymerase in transcription initiation.

Thermodynamics of interactions of urea and guanidinium salts with protein surface: relationship between solute effects on protein processes and changes in water-accessible surface area.

To interpret effects of urea and guanidinium (GuH(+)) salts on processes that involve large changes in protein water-accessible surface area (ASA), and to predict these effects from structural information, a thermodynamic characterization of the interactions of these solutes with different types of protein surface is required. In the present work we quantify the interactions of urea, GuHCl, GuHSCN, and, for comparison, KCl with native bovine serum albumin (BSA) surface, using vapor pressure osmometry (VPO) to obtain preferential interaction coefficients (Gamma(mu3)) as functions of nondenaturing concentrations of these solutes (0-1 molal). From analysis of Gamma(mu3) using the local-bulk domain model, we obtain concentration-independent partition coefficients K(nat)(P) that characterize the accumulation of these solutes near native protein (BSA) surface: K(nat)(P,urea)= 1.10 +/- 0.04, K(nat)(P,SCN(-)) = 2.4 +/- 0.2, K(nat)(P,GuH(+)) = 1.60 +/- 0.08, relative to K(nat)(P,K(+)) identical with 1 and K(nat)(P,Cl(-)) = 1.0 +/- 0.08. The relative magnitudes of K(nat)(P) are consistent with the relative effectiveness of these solutes as perturbants of protein processes. From a comparison of partition coefficients for these solutes and native surface (K(nat)(P)) with those determined by us previously for unfolded protein and alanine-based peptide surface K(unf)(P), we dissect K(P) into contributions from polar peptide backbone and other types of protein surface. For globular protein-urea interactions, we find K(nat)(P,urea) = K(unf)(P,urea). We propose that this equality arises because polar peptide backbone is the same fraction (0.13) of total ASA for both classes of surface. The analysis presented here quantifies and provides a physical basis for understanding Hofmeister effects of salt ions and the effects of uncharged solutes on protein processes in terms of K(P) and the change in protein ASA.

Analytic binding isotherms describing competitive interactions of a protein ligand with specific and nonspecific sites on the same DNA oligomer.

Many studies of specific protein-nucleic acid binding use short oligonucleotides or restriction fragments, in part to minimize the potential for nonspecific binding of the protein. However, when the specificity ratio is low, multiple nonspecifically bound proteins may occupy the region of DNA corresponding to one specific site; this situation was encountered in our recent calorimetric study of binding of integration host factor (IHF) protein to its specific 34-bp H' DNA site. Here, beginning from the analytical McGhee and von Hippel infinite-lattice nonspecific binding isotherm, we derive a novel analytic isotherm for nonspecific binding of a ligand to a finite lattice. This isotherm is an excellent approximation to the exact factorial-based Epstein finite lattice isotherm even for short lattices and therefore is of great practical significance for analysis of experimental data and for analytic theory. Using this isotherm, we develop an analytic treatment of the competition between specific and nonspecific binding of a large ligand to the same finite lattice (i.e., DNA oligomer) containing one specific and multiple overlapping nonspecific binding sites. Analysis of calorimetric data for IHF-H' DNA binding using this treatment yields enthalpies and binding constants for both specific and nonspecific binding and the nonspecific site size. This novel analysis demonstrates the potential contribution of nonspecific binding to the observed thermodynamics of specific binding, even with very short DNA oligomers, and the need for reverse (constant protein) titrations or titrations with nonspecific DNA to resolve specific and nonspecific contributions. The competition treatment is useful in analyzing low-specificity systems, including those where specificity is weakened by mutations or the absence of specificity factors.

Specific and non-specific interactions of integration host factor with DNA: thermodynamic evidence for disruption of multiple IHF surface salt-bridges coupled to DNA binding.

Site-specific DNA binding of architectural protein integration host factor (IHF) is involved in formation of functional multiprotein-DNA assemblies in Escherichia coli, while non-specific binding of IHF and other histone-like proteins serves to structure the nucleoid. Here, we report an isothermal titration calorimetry study of the thermodynamics of binding IHF to a 34 bp fragment composed entirely of the specific H' site from lambda-phage DNA. At low to moderate [K(+)] (60-100 mM), strong competition is observed between specific and non-specific binding as a result of a low specificity ratio (approximately 10(2)) and a very small non-specific site size. In this [K(+)] range, both specific and non-specific binding are enthalpy-driven, with large negative enthalpy, entropy and heat capacity changes and binding constants that are insensitive to [K(+)]. Above 100 mM K(+), only specific binding is observed, and both the binding constant and the magnitudes of enthalpy, entropy and heat capacity changes all decrease strongly with increasing [K(+)]. When interpreted in the context of the structure of the specific complex, the thermodynamics provide compelling evidence for a previously unrecognized design principle by which proteins that form extensive binding interfaces with nucleic acids control binding constants, binding site sizes and effects of temperature and ion concentrations on stability and specificity. We propose that up to 22 of the 23 IHF cationic side-chains that are located within 6 A of DNA phosphate oxygen atoms in the complex, are masked in the absence of DNA by pairing with anionic carboxylate groups in intramolecular salt-bridges (dehydrated ion-pairs). These salt-bridges increase in stability with increasing temperature and decreasing [K(+)]. To explain the unusual thermodynamics of IHF-DNA interactions, we propose that both specific and non-specific binding at low [K(+)] require disruption of salt-bridges (as many as 18 for specific binding) whereupon many of the unmasked charged groups hydrate and the cationic groups interact with DNA. From structural or thermodynamic parallels with IHF, we propose that large-scale coupling of disruption of protein salt-bridges to DNA binding is significant for other large-interface DNA wrapping proteins including the nucleosome, lac repressor core tetramer, RNA polymerase core protein, HU and SSB.

Thermodynamic analysis of interactions between denaturants and protein surface exposed on unfolding: interpretation of urea and guanidinium chloride m-values and their correlation with changes in accessible surface area (ASA) using preferential interaction coefficients and the local-bulk domain model.

A denaturant m-value is the magnitude of the slope of a typically linear plot of the unfolding free energy change DeltaG degrees (obs) vs. molar concentration (C(3)) of denaturant. For a given protein, the guanidinium chloride (GuHCl) m-value is approximately twice as large as the urea m-value. Myers et al. (Protein Sci 1995;4:2138-2148) found that experimental m-values for protein unfolding in both urea and GuHCl are proportional to DeltaASA(corr)(max), the calculated maximum amount of protein surface exposed to water in unfolding, corrected empirically for the effects of disulfide crosslinks: (urea m-value/DeltaASA(corr)(max)) = 0.14+/-0.01 cal M(-1) A(-2) and (GuHCl m-value/DeltaASA(corr)(max)) = 0.28+/-0.03 cal M(-1) A(-2). The observed linearity of plots of DeltaG degrees (obs) vs. C(3) indicates that the difference in preferential interaction coefficients DeltaGamma(3) characterizing the interactions of these solutes with denatured and native protein surface is approximately proportional to denaturant concentration. The proportionality of m-values to DeltaASA(corr)(max) indicates that the corresponding DeltaGamma(3) are proportional to DeltaASA(corr)(max) at any specified solute concentration. Here we use the local-bulk domain model of solute partitioning in the protein solution (Courtenay et al., Biochemistry 2000;39:4455-4471) to obtain a novel quantitative interpretation of denaturant m-values. We deduce that the proportionality of m-value to DeltaASA(corr)(max) results from the proportionality of B(1)(0) (the amount of water in the local domain surrounding the protein surface exposed upon unfolding) to DeltaASA(corr)(max). We show that both the approximate proportionality of DeltaGamma(3) to denaturant concentration and the residual dependence of DeltaGamma(3)/m(3) (where m(3) is molal concentration) on denaturant concentration are quantitatively predicted by the local-bulk domain model if the molal-scale solute partition coefficient K(P) and water-solute exchange stoichiometry S(1,3) are independent of solute concentration. We obtain K(P,urea) = 1.12+/-0.01 and K(P,GuHCl) = 1.16+/-0.02 (or K(P,GuH+) congruent with 1.48), values which will be useful to characterize the effect of accumulation of those solutes on all processes in which the water-accessible area of unfolded protein surface changes. We demonstrate that the local-bulk domain analysis of an m-value plot justifies the use of linear extrapolation to estimate ( less, similar 5% error) the stability of the native protein in the absence of denaturant (DeltaG(o)(o)), with respect to a particular unfolded state. Our surface area calculations indicate that published m-values/DeltaASA ratios for unfolding of alanine-based alpha-helical oligopeptides by urea and GuHCl exceed the corresponding m-value/DeltaASA ratios for protein unfolding by approximately fourfold. We propose that this difference originates from the approximately fourfold difference (48% vs. 13%) in the contribution of polar backbone residues to DeltaASA of unfolding, a novel finding which supports the long-standing but not universally accepted hypothesis that urea and guanidinium cation interact primarily with backbone amide groups. We propose that proteins which exhibit significant deviations from the average m-value/DeltaASA ratio will be found to exhibit significant deviations from the expected amount and/or average composition of the surface exposed on unfolding.

Vapor pressure osmometry studies of osmolyte-protein interactions: implications for the action of osmoprotectants in vivo and for the interpretation of "osmotic stress" experiments in vitro.

To interpret or to predict the responses of biopolymer processes in vivo and in vitro to changes in solute concentration and to coupled changes in water activity (osmotic stress), a quantitative understanding of the thermodynamic consequences of interactions of solutes and water with biopolymer surfaces is required. To this end, we report isoosmolal preferential interaction coefficients (Gamma(mu1) determined by vapor pressure osmometry (VPO) over a wide range of concentrations for interactions between native bovine serum albumin (BSA) and six small solutes. These include Escherichia coli cytoplasmic osmolytes [potassium glutamate (K(+)Glu(-)), trehalose], E. coli osmoprotectants (proline, glycine betaine), and also glycerol and trimethylamine N-oxide (TMAO). For all six solutes, Gamma(mu1) and the corresponding dialysis preferential interaction coefficient Gamma(mu1),(mu3) (both calculated from the VPO data) are negative; Gamma(mu1), (mu3) is proportional to bulk solute molality (m(bulk)3) at least up to 1 m (molal). Negative values of Gamma(mu1),(mu3) indicate preferential exclusion of these solutes from a BSA solution at dialysis equilibrium and correspond to local concentrations of these solutes in the vicinity of BSA which are lower than their bulk concentrations. Of the solutes investigated, betaine is the most excluded (Gamma(mu1),(mu3)/m(bulk)3 = -49 +/- 1 m(-1)); glycerol is the least excluded (Gamma(mu1),(mu3)/m(bulk)3 = -10 +/- 1 m(-1)). Between these extremes, the magnitude of Gamma(mu1),(mu3)/m(bulk)3 decreases in the order glycine betaine >> proline >TMAO > trehalose approximately K(+)Glu(-) > glycerol. The order of exclusion of E. coli osmolytes from BSA surface correlates with their effectiveness as osmoprotectants, which increase the growth rate of E. coli at high external osmolality. For the most excluded solute (betaine), Gamma(mu1),(mu3) provides a minimum estimate of the hydration of native BSA of approximately 2.8 x 10(3) H(2)O/BSA, which corresponds to slightly less than a monolayer (estimated to be approximately 3.2 x 10(3) H(2)O). Consequently, of the solutes investigated here, only betaine might be suitable for use in osmotic stress experiments in vitro as a direct probe to quantify changes in hydration of protein surface in biopolymer processes. More generally, however, our results and analysis lead to the proposal that any of these solutes can be used to quantify changes in water-accessible surface area (ASA) in biopolymer processes once preferential interactions of the solute with biopolymer surface are properly taken into account.

Biophysical characterization of changes in amounts and activity of Escherichia coli cell and compartment water and turgor pressure in response to osmotic stress.

To obtain turgor pressure, intracellular osmolalities, and cytoplasmic water activity of Escherichia coli as a function of osmolality of growth, we have quantified and analyzed amounts of cell, cytoplasmic, and periplasmic water as functions of osmolality of growth and osmolality of plasmolysis of nongrowing cells with NaCl. The effects are large; NaCl (plasmolysis) titrations of cells grown in minimal medium at 0.03 Osm reduce cytoplasmic and cell water to approximately 20% and approximately 50% of their original values, and increase periplasmic water by approximately 300%. Independent analysis of amounts of cytoplasmic and cell water demonstrate that turgor pressure decreases with increasing osmolality of growth, from approximately 3.1 atm at 0.03 Osm to approximately 1.5 at 0.1 Osm and to less than 0.5 atm above 0.5 Osm. Analysis of periplasmic membrane-derived oligosaccharide (MDO) concentrations as a function of osmolality, calculated from literature analytical data and measured periplasmic volumes, provides independent evidence that turgor pressure decreases with increasing osmolality, and verifies that cytoplasmic and periplasmic osmolalities are equal. We propose that MDO play a key role in periplasmic volume regulation at low-to-moderate osmolality. At high growth osmolalities, where only a small amount of cytoplasmic water is observed, the small turgor pressure of E. coli demonstrates that cytoplasmic water activity is only slightly less than extracellular water activity. From these findings, we deduce that the activity of cytoplasmic water exceeds its mole fraction at high osmolality, and, therefore, conclude that the activity coefficient of cytoplasmic water increases with increasing growth osmolality and exceeds unity at high osmolality, presumably as a consequence of macromolecular crowding. These novel findings are significant for thermodynamic analyses of effects of changes in growth osmolality on biopolymer processes in general and osmoregulatory processes in particular in the E. coli cytoplasm.

Wrapping of flanking non-operator DNA in lac repressor-operator complexes: implications for DNA looping.

In our studies of lac repressor tetramer (T)-lac operator (O) interactions, we observed that the presence of extended regions of non-operator DNA flanking a single lac operator sequence embedded in plasmid DNA produced large and unusual cooperative and anticooperative effects on binding constants (Kobs) and their salt concentration dependences for the formation of 1:1 (TO) and especially 1:2 (TO2) complexes. To explore the origin of this striking behavior we report and analyze binding data on 1:1 (TO) and 1:2 (TO2) complexes between repressor and a single O(sym) operator embedded in 40 bp, 101 bp, and 2514 bp DNA, over very wide ranges of [salt]. We find large interrelated effects of flanking DNA length and [salt] on binding constants (K(TO)obs, K(TO2)obs) and on their [salt]-derivatives, and quantify these effects in terms of the free energy contributions of two wrapping modes, designated local and global. Both local and global wrapping of flanking DNA occur to an increasing extent as [salt] decreases. Global wrapping of plasmid-length DNA is extraordinarily dependent on [salt]. We propose that global wrapping is driven at low salt concentration by the polyelectrolyte effect, and involves a very large number (>/similar 20) of coulombic interactions between DNA phosphates and positively charged groups on lac repressor. Coulombic interactions in the global wrap must involve both the core and the second DNA-binding domain of lac repressor, and result in a complex which is looped by DNA wrapping. The non-coulombic contribution to the free energy of global wrapping is highly unfavorable ( approximately +30-50 kcal mol(-1)), which presumably results from a significant extent of DNA distortion and/or entropic constraints. We propose a structural model for global wrapping, and consider its implications for looping of intervening non-operator DNA in forming a complex between a tetrameric repressor (LacI) and one multi-operator DNA molecule in vivo and in vitro. The existence of DNA wrapping in LacI-DNA interactions motivates the proposal that most if not all DNA binding proteins may have evolved the capability to wrap and thereby organize flanking regions of DNA.

Enthalpy and heat capacity changes for formation of an oligomeric DNA duplex: interpretation in terms of coupled processes of formation and association of single-stranded helices.

The thermodynamics of self-assembly of a 14 base pair DNA double helix from complementary strands have been investigated by titration (ITC) and differential scanning (DSC) calorimetry, in conjunction with van't Hoff analysis of UV thermal scans of individual strands. These studies demonstrate that thermodynamic characterization of the temperature-dependent contributions of coupled conformational equilibria in the individual "denatured" strands and in the duplex is essential to understand the origins of duplex stability and to derive stability prediction schemes of general applicability. ITC studies of strand association at 293 K and 120 mM Na+ yield an enthalpy change of -73 +/- 2 kcal (mol of duplex)-1. ITC studies between 282 and 312 K at 20, 50, and 120 mM Na+ show that the enthalpy of duplex formation is only weakly salt concentration-dependent but is very strongly temperature-dependent, decreasing approximately linearly with increasing temperature with a heat capacity change (282-312 K) of -1.3 +/- 0.1 kcal K-1 (mol of duplex)-1. From DSC denaturation studies in 120 mM Na+, we obtain an enthalpy of duplex formation of -120 +/- 5 kcal (mol of duplex)-1 and an estimate of the corresponding heat capacity change of -0.8 +/- 0.4 kcal K-1 (mol of duplex)-1 at the Tm of 339 K. van't Hoff analysis of UV thermal scans on the individual strands indicates that single helix formation is noncooperative with a temperature-independent enthalpy change of -5.5 +/- 0.5 kcal at 120 mM Na+. From these observed enthalpy and heat capacity changes, we obtain the corresponding thermodynamic quantities for two fundamental processes: (i) formation of single helices from disordered strands, involving only intrastrand (vertical) interactions between neighboring bases; and (ii) formation of double helices by association (docking) of single helical strands, involving interstrand (horizontal and vertical) interactions. At 293 K and 120 mM Na+, we calculate that the enthalpy change for association of single helical strands is approximately -64 kcal (mol of duplex)-1 as compared to -210 kcal (mol of duplex)-1 calculated for duplex formation from completely unstructured single strands and to the experimental ITC value of -73 kcal (mol of duplex)-1. The intrinsic heat capacity change for association of single helical strands to form the duplex is found to be small and positive [ approximately 0.1 kcal K-1 (mol of duplex)-1], in agreement with the result of a surface area analysis, which also predicts an undetectably small heat capacity change for single helix formation.

General method of analysis of kinetic equations for multistep reversible mechanisms in the single-exponential regime: application to kinetics of open complex formation between Esigma70 RNA polymerase and lambdaP(R) promoter DNA.

A novel analytical method based on the exact solution of equations of kinetics of unbranched first- and pseudofirst-order mechanisms is developed for application to the process of Esigma70 RNA polymerase (R)-lambdaPR promoter (P) open complex formation, which is described by the minimal three-step mechanism with two kinetically significant intermediates (I1, I2), [equation: see text], where the final product is an open complex RPo. The kinetics of reversible and irreversible association (pseudofirst order, [R] >> [P]) to form long-lived complexes (RPo and I2) and the kinetics of dissociation of long-lived complexes both exhibit single exponential behavior. In this situation, the analytical method provides explicit expressions relating observed rate constants to the microscopic rate constants of mechanism steps without use of rapid equilibrium or steady-state approximations, and thereby provides a basis for interpreting the composite rate constants of association (ka), isomerization (ki), and dissociation (kd) obtained from experiment for this or any other sequential mechanism of any number of steps. In subsequent papers, we apply this formalism to analyze kinetic data obtained in the reversible and irreversible binding regimes of Esigma70 RNA polymerase (R)-lambdaP(R) promoter (P) open complex formation.

The importance of coulombic end effects: experimental characterization of the effects of oligonucleotide flanking charges on the strength and salt dependence of oligocation (L8+) binding to single-stranded DNA oligomers.

Binding constants Kobs, expressed per site and evaluated in the limit of zero binding density, are quantified as functions of salt (sodium acetate) concentration for the interactions of the oligopeptide ligand KWK6NH2 (designated L8+, with ZL = 8 charges) with three single-stranded DNA oligomers (ss dT-mers, with |ZD| = 15, 39, and 69 charges). These results provide the first systematic experimental information about the effect of changing |ZD| on the strength and salt dependence of oligocation-oligonucleotide binding interactions. In a comparative study of L8+ binding to poly dT and to a short dT oligomer (|ZD| = 10),. Proc. Natl. Acad. Sci. USA. 93:2511-2516) demonstrated the profound thermodynamic effects of phosphate charges that flank isolated nonspecific L8+ binding sites on DNA. Here we find that both Kobs and the magnitude of its power dependence on salt activity (|SaKobs|) increase monotonically with increasing |ZD|. The dependences of Kobs and SaKobs on |ZD| are interpreted by introducing a simple two-state thermodynamic model for Coulombic end effects, which accounts for our finding that when L8+ binds to sufficiently long dT-mers, both DeltaGobso = -RT ln Kobs and SaKobs approach the values characteristic of binding to poly-dT as linear functions of the reciprocal of the number of potential oligocation binding sites on the DNA lattice. Analysis of our L8+-dT-mer binding data in terms of this model indicates that the axial range of the Coulombic end effect for ss DNA extends over approximately 10 phosphate charges. We conclude that Coulombic interactions cause an oligocation (with ZL < |ZD|) to bind preferentially to interior rather than terminal binding sites on oligoanionic or polyanionic DNA, and we quantify the strong increase of this preference with decreasing salt concentration. Coulombic end effects must be considered when oligonucleotides are used as models for polyanionic DNA in thermodynamic studies of the binding of charged ligands, including proteins.

DNA footprints of the two kinetically significant intermediates in formation of an RNA polymerase-promoter open complex: evidence that interactions with start site and downstream DNA induce sequential conformational changes in polymerase and DNA.

Kinetic studies of formation and dissociation of open-promoter complexes (RPo) involving Esigma70 RNA polymerase (R) and the lambdaPR promoter (P) demonstrate the existence of two kinetically significant intermediates, designated I1 and I2, and facilitate the choice of conditions under which each accumulates. For such conditions, we report the results of equilibrium and transient DNase I and KMnO4 footprinting studies which characterize I1 and I2. At 0 degreesC, where extrapolation of equilibrium data indicates I1 is the dominant complex, DNA bases in the vicinity of the transcription start site (+1) do not react with KMnO4, indicating that this region is closed in I1. However, the DNA backbone in I1 is extensively protected from DNase I cleavage; the DNase I footprint extends approximately 30 bases downstream and at least approximately 40 bases upstream from the start site. I1 has a short lifetime (

Quantitative analysis of multiple-hit footprinting studies to characterize DNA conformational changes in protein-DNA complexes: application to DNA opening by Esigma70 RNA polymerase.

Formation of many site-specific protein-nucleic acid complexes involves sequential conformational changes subsequent to initial binding which create functionally active assemblies. Characterization of population distributions and structural characteristics of intermediate and product conformations is necessary to understand both the mechanisms and the thermodynamics of these processes. For these purposes, here we develop the quantitative method of multiple hit footprinting (MHF), where chemical or enzymatic probing is performed as a function of either concentrations of the footprinting agent and/or time of exposure to it, in the multiple hit regime where many of the population or subpopulation of reactive DNA molecules are modified at more than one site. Properly controlled MHF experiments yield both the population distribution of different conformers and reactivity rate constants of the footprinting agent at all reactive positions in each conformer, which may be interpreted in terms of the accessibility of the site or the local concentration of the reagent. MHF experiments are particularly well-suited for dissecting effects at sites where unbound DNA is non-reactive and bound DNA is reactive with base-specific probes (e.g. KMnO4, DMS). We suggest that this method will also be applicable to analysis of enhancements in reactivity of other footprinting agents (e.g. DNase I, HO.). To demonstrate the utility of the MHF analysis, we quantify fragment distributions and individual site reactivities from multiple-hit KMnO4 footprinting of the non-template strand of Esigma70 RNA polymerase-lambdaPR promoter DNA complexes populated at binding equilibrium at 37 degreesC and transiently populated at a fixed time after a temperature downshift from 37 degreesC to 0 degreesC. For this system, a MHF analysis directly addresses the following questions: (i) what fraction of the population of promoter DNA molecules is open in the vicinity of the transcription start site (RPo) both at 37 degreesC and (transiently) after a downshift to 0 degreesC; (ii) does opening of the start site region in RPo occur entirely in one mechanistic step at the lambdaPR promoter and (iii) does the structure of RPo vary with temperature? In addition, we use the MHF-determined population distribution of KMnO4-reactive (RPo) and non-reactive promoter DNA to normalize the biphasic kinetics of decay of RPo to free promoter DNA after a 37 degrees to 0 degreesC temperature downshift, and thereby characterize the kinetics of the conformational changes involved in forming RPo.

Biophysical compensation mechanisms buffering E. coli protein-nucleic acid interactions against changing environments.

Escherichia coli adapts to changes in growth osmolarity of at least 100-fold by making large changes in the amounts of intracellular water and solutes, including cytoplasmic K+. A wide range of in vitro salt, solute and biopolymer concentrations should therefore be considered 'physiological'. Paradoxically, these large, osmotically induced changes in cytoplasmic K+ concentration do not greatly affect the equilibria and kinetics of cytoplasmic protein-nucleic acid interactions. Biophysical effects resulting from changes in the amount of cytoplasmic water (such as macromolecular crowding) and in the concentrations of other cytoplasmic solutes appear to compensate for the effects of changes in cytoplasmic K+ concentration and thereby maintain protein-nucleic acid equilibria and kinetics in the range required for in vivo function.

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.

Thermodynamics of the interactions of lac repressor with variants of the symmetric lac operator: effects of converting a consensus site to a non-specific site.

What are the thermodynamic consequences of the stepwise conversion of a highly specific (consensus) protein-DNA interface to one that is nonspecific? How do the magnitudes of key favorable contributions to complex stability (burial of hydrophobic surfaces and reduction of DNA phosphate charge density) change as the DNA sequence of the specific site is detuned? To address these questions we investigated the binding of lac repressor (LacI) to a series of 40 bp fragments carrying symmetric (consensus) and variant operator sequences over a range of temperatures and salt concentrations. Variant DNA sites contained symmetrical single and double base-pair substitutions at positions 4 and/or 5 [sequence: see text] in each 10 bp half site of the symmetric lac operator (Osym). Non-specific interactions were examined using a 40 bp non-operator DNA fragment. Disruption of the consensus interface by a single symmetrical substitution reduces the observed equilibrium association constant (K(obs)) for Osym by three to four orders of magnitude; double symmetrical substitutions approach the six orders in magnitude difference between specific and non-specific binding to a 40 bp fragment. At these adjacent positions in the consensus site, the free energy effects of multiple substitutions are non-additive: the first reduces /deltaG(obs)o/ by 3 to 5 kcal mol(-1), approximately halfway to the non-specific level, whereas the second is less deleterious, reducing /deltaG(obs)o/ by less than 3 kcal mol(-1). Variant-specific dependences of K(obs) on temperature and salt concentration characterize these LacI-operator interactions. In general, binding constants and standard free energies of binding both exhibit characteristic extrema near 290 K. As a consequence, both the enthalpic and entropic contributions to stability of Osym and variant complexes change from positive (i.e. entropy driven) at lower temperatures to negative (i.e. enthalpy driven) at higher temperatures, indicating that the heat capacity change upon binding, deltaC(obs)o, is large and negative. In general, /deltaC(obs)o/ decreases as the specificity and stability of the variant complex decreases. Stabilities of complexes of LacI with Osym and all variant operators are strongly [salt]-dependent. Binding constants for the variant complexes exhibit a power-dependence on [salt] that is larger in magnitude (i.e. more negative) than for Osym, but no obvious trend relates changes in contributions from the polyelectrolyte effect and the observed reductions in stability (delta deltaG(obs)o). These variant-specific thermodynamic signatures provide novel insights into the consequences of converting a consensus interface to a less specific one; such insights are not obtained from comparisons at the level of delta deltaG(obs)o. We propose that this variant-specific behavior arises from a strong effect of operator sequence on the extent of induced conformational changes in the protein (and possibly also in the DNA site) which accompany binding.