Michael W. Capp

Quantifying why urea is a protein denaturant, whereas glycine betaine is a protein stabilizer

To explain the large, opposite effects of urea and glycine betaine (GB) on stability of folded proteins and protein complexes, we quantify and interpret preferential interactions of urea with 45 model compounds displaying protein functional groups and compare with a previous analysis of GB. This information is needed to use urea as a probe of coupled folding in protein processes and to tune molecular dynamics force fields. Preferential interactions between urea and model compounds relative to their interactions with water are determined by osmometry or solubility and dissected using a unique coarse-grained analysis to obtain interaction potentials quantifying the interaction of urea with each significant type of protein surface (aliphatic, aromatic hydrocarbon (C); polar and charged N and O). Microscopic local-bulk partition coefficients Kp for the accumulation or exclusion of urea in the water of hydration of these surfaces relative to bulk water are obtained. Kp values reveal that urea accumulates moderately at amide O and weakly at aliphatic C, whereas GB is excluded from both. These results provide both thermodynamic and molecular explanations for the opposite effects of urea and glycine betaine on protein stability, as well as deductions about strengths of amide NH—amide O and amide NH—amide N hydrogen bonds relative to hydrogen bonds to water. Interestingly, urea, like GB, is moderately accumulated at aromatic C surface. Urea m-values for protein folding and other protein processes are quantitatively interpreted and predicted using these urea interaction potentials or Kp values.

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.

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 (Γμ3) as functions of nondenaturing concentrations of these solutes (0–1 molal). From analysis of Γμ3 using the local‐bulk domain model, we obtain concentration‐independent partition coefficients KnatP that characterize the accumulation of these solutes near native protein (BSA) surface: KnatP,urea= 1.10 ± 0.04, Knat  P,SCN − = 2.4 ± 0.2, Knat  P,GuH + = 1.60 ± 0.08, relative to Knat  P,K + ≡ 1 and Knat  P,Cl − = 1.0 ± 0.08. The relative magnitudes of KnatP 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 (KnatP) with those determined by us previously for unfolded protein and alanine‐based peptide surface KunfP, we dissect KP into contributions from polar peptide backbone and other types of protein surface. For globular protein‐urea interactions, we find KnatP,urea = KunfP,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 KP and the change in protein ASA.

Interactions of the Osmolyte Glycine Betaine with Molecular Surfaces in Water: Thermodynamics, Structural Interpretation, and Prediction of m-Values

Noncovalent self-assembly of biopolymers is driven by molecular interactions between functional groups on complementary biopolymer surfaces, replacing interactions with water. Since individually these interactions are comparable in strength to interactions with water, they have been difficult to quantify. Solutes (osmolytes, denaturants) exert often large effects on these self-assembly interactions, determined in sign and magnitude by how well the solute competes with water to interact with the relevant biopolymer surfaces. Here, an osmometric method and a water-accessible surface area (ASA) analysis are developed to quantify and interpret the interactions of the remarkable osmolyte glycine betaine (GB) with molecular surfaces in water. We find that GB, lacking hydrogen bond donors, is unable to compete with water to interact with anionic and amide oxygens; this explains its effectiveness as an osmolyte in the Escherichia coli cytoplasm. GB competes effectively with water to interact with amide and cationic nitrogens (hydrogen bonding) and especially with aromatic hydrocarbon (cation-pi). The large stabilizing effect of GB on lac repressor-lac operator binding is predicted quantitatively from ASA information and shown to result largely from dehydration of anionic DNA phosphate oxygens in the protein-DNA interface. The incorporation of these results into theoretical and computational analyses will likely improve the ability to accurately model intra- and interprotein interactions. Additionally, these results pave the way for development of solutes as kinetic/mechanistic and thermodynamic probes of conformational changes and formation/disruption of molecular interfaces that occur in the steps of biomolecular self-assembly processes.

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.

Quantifying Additive Interactions of the Osmolyte Proline with Individual Functional Groups of Proteins: Comparisons with Urea and Glycine Betaine, Interpretation of m-Values

To quantify interactions of the osmolyte l-proline with protein functional groups and predict their effects on protein processes, we use vapor pressure osmometry to determine chemical potential derivatives dμ2/dm3 = μ23, quantifying the preferential interactions of proline (component 3) with 21 solutes (component 2) selected to display different combinations of aliphatic or aromatic C, amide, carboxylate, phosphate or hydroxyl O, and amide or cationic N surface. Solubility data yield μ23 values for four less-soluble solutes. Values of μ23 are dissected using an ASA-based analysis to test the hypothesis of additivity and obtain α-values (proline interaction potentials) for these eight surface types and three inorganic ions. Values of μ23 predicted from these α-values agree with the experiment, demonstrating additivity. Molecular interpretation of α-values using the solute partitioning model yields partition coefficients (Kp) quantifying the local accumulation or exclusion of proline in the hydration water of each functional group. Interactions of proline with native protein surfaces and effects of proline on protein unfolding are predicted from α-values and ASA information and compared with experimental data, with results for glycine betaine and urea, and with predictions from transfer free energy analysis. We conclude that proline stabilizes proteins because of its unfavorable interactions with (exclusion from) amide oxygens and aliphatic hydrocarbon surfaces exposed in unfolding and that proline is an effective in vivo osmolyte because of the osmolality increase resulting from its unfavorable interactions with anionic (carboxylate and phosphate) and amide oxygens and aliphatic hydrocarbon groups on the surface of cytoplasmic proteins and nucleic acids.

One-step DNA melting in the RNA polymerase cleft opens the initiation bubble to form an unstable open complex

Though opening of the start site (+1) region of promoter DNA is required for transcription by RNA polymerase (RNAP), surprisingly little is known about how and when this occurs in the mechanism. Early events at the λPR promoter load this region of duplex DNA into the active site cleft of Escherichia coli RNAP, forming the closed, permanganate-unreactive intermediate I1. Conversion to the subsequent intermediate I2 overcomes a large enthalpic barrier. Is I2 open? Here we create a burst of I2 by rapidly destabilizing open complexes (RPo) with 1.1 M NaCl. Fast footprinting reveals that thymines at positions from -11 to +2 in I2 are permanganate-reactive, demonstrating that RNAP opens the entire initiation bubble in the cleft in a single step. Rates of decay of all observed thymine reactivities are the same as the I2 to I1 conversion rate determined by filter binding. In I2, permanganate reactivity of the +1 thymine on the template (t) strand is the same as the RPo control, whereas nontemplate (nt) thymines are significantly less reactive than in RPo. We propose that: (i) the +1(t) thymine is in the active site in I2; (ii) conversion of I2 to RPo repositions the nt strand in the cleft; and (iii) movements of the nt strand are coupled to the assembly and DNA binding of the downstream clamp and jaw that occurs after DNA opening and stabilizes RPo. We hypothesize that unstable open intermediates at the λPR promoter resemble the unstable, transcriptionally competent open complexes formed at ribosomal promoters.

Probing DNA binding, DNA opening, and assembly of a downstream clamp/jaw in Escherichia coli RNA polymerase-lambdaP(R) promoter complexes using salt and the physiological anion glutamate.

Transcription by all RNA polymerases (RNAPs) requires a series of large-scale conformational changes to form the transcriptionally competent open complex RP(o). At the lambdaP(R) promoter, Escherichia coli sigma(70) RNAP first forms a wrapped, closed 100 bp complex I(1). The subsequent step opens the entire DNA bubble, creating the relatively unstable (open) complex I(2). Additional conformational changes convert I(2) to the stable RP(o). Here we probe these events by dissecting the effects of Na(+) salts of Glu(-), F(-), and Cl(-) on each step in this critical process. Rapid mixing and nitrocellulose filter binding reveal that the binding constant for I(1) at 25 degrees C is approximately 30-fold larger in Glu(-) than in Cl(-) at the same Na(+) concentration, with the same log-log salt concentration dependence for both anions. In contrast, both the rate constant and equilibrium constant for DNA opening (I(1) to I(2)) are only weakly dependent on salt concentration, and the opening rate constant is insensitive to replacement of Cl(-) with Glu(-). These very small effects of salt concentration on a process (DNA opening) that is strongly dependent on salt concentration in solution may indicate that the backbones of both DNA strands interact with polymerase throughout the process and/or that compensation is present between ion uptake and release. Replacement of Cl(-) with Glu(-) or F(-) at 25 degrees C greatly increases the lifetime of RP(o) and greatly reduces its salt concentration dependence. By analogy to Hofmeister salt effects on protein folding, we propose that the excluded anions Glu(-) and F(-) drive the folding and assembly of the RNAP clamp/jaw domains in the conversion of I(2) to RP(o), while Cl(-) does not. Because the Hofmeister effect of Glu(-) or F(-) largely compensates for the destabilizing Coulombic effect of any salt on the binding of this assembly to downstream promoter DNA, RP(o) remains long-lived even at 0.5 M Na(+) in Glu(-) or F(-) salts. The observation that Esigma(70) RP(o) complexes are exceedingly long-lived at moderate to high Glu(-) concentrations argues that Esigma(70) RNAP does not dissociate from strong promoters in vivo when the cytoplasmic glutamate concentration increases during osmotic stress.

Preferential interactions in aqueous solutions of urea and KCl.

A quantitative characterization of the thermodynamic effects due to interactions of salt ions and urea in aqueous solution is needed for rigorous analyses of the effects of changing urea concentration on biopolymer processes in solutions that also contain salt. Therefore, we investigate preferential interactions in aqueous solutions containing KCl and urea by using vapor pressure osmometry (VPO) to measure osmolality as a function of the molality of urea (component 3) over the range 0.09<or=m(3)<or=1.65 m at two fixed molalities of KCl (component 2) (m(2)=0.212 and 0.427 m). With this experimental input and corresponding VPO measurements on solutions that contain only urea or KCl, we evaluate approximately the chemical potential derivative micro(23)=( partial differential micro(KCl)/ partial differential m(urea))(T,P,m(KCl))=( partial differential micro(urea)/ partial differential m(KCl))(T,P,m(urea))= micro(32) and hence the preferential interaction coefficients Gammamicro(3) and Gammamicro(1),micro(3). These results show that for water-KCl-urea solutions neither of these coefficients is determined primarily by contributions from thermodynamic nonideality to micro(23). In aqueous solutions containing a biopolymer and a small solute, the contribution of ideal mixing entropy to micro(23) is negligible in comparison with the experimental uncertainty, whereas in KCl-urea solutions the contribution due to ideal mixing entropy accounts for at least half of the magnitude of micro(23). For comparison, we analyze literature data for NaCl-urea interactions and find again that nonideality makes a smaller contribution to micro(23) than does ideal mixing entropy. In contrast, for aqueous solutions of urea and the protein bovine serum albumin, the experimentally determined contribution of nonideality to micro(23) exceeds the contribution of ideal mixing by a factor of approximately 2 x 10(2).

Rapid quench mixing to quantify kinetics of steps in association of Escherichia coli RNA polymerase with promoter DNA.

Publisher Summary This chapter explores the kinetic–mechanistic studies that have characterized various steps in the process of forming the transcriptionally competent open complex between RNA polymerase (R) and promoter DNA (P). It discusses the rapid quench mixing of radiolabeled λP R promoter DNA with E. coli RNA polymerase in a commercially available apparatus providing an accurate and efficient method of investigating the kinetics of association, and subsequent conformational changes involved in forming long-lived complexes. Promoter binding as a function of time is assayed by nitrocellulose filter binding after quenching with a competitor. Under all conditions examined, the kinetics of formation of competitor-resistant complexes at the λPR promoter are single exponential with first-order rate constant β CR . Interpretation of the polymerase concentration dependence of β CR in terms of the three-step mechanism of open complex formation yields the equilibrium constant K 1 for formation of the first kinetically significant intermediate (I 1 ) and the forward rate constant (k 2 ) for the conformational change that converts I1 to the second kinetically significant intermediate I 2. This method should be applicable to kinetic studies of polymerase–promoter interactions and thus should extend biophysical characterizations of transcription initiation.