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1.
Protein Sci ; 10(12): 2485-97, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11714916

ABSTRACT

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.


Subject(s)
Guanidine/chemistry , Guanidines/chemistry , Serum Albumin/chemistry , Thiocyanates/chemistry , Urea/chemistry , Animals , Cattle , Dose-Response Relationship, Drug , Guanidine/metabolism , Guanidines/metabolism , Models, Theoretical , Osmolar Concentration , Potassium Chloride/chemistry , Protein Binding , Protein Folding , Protein Structure, Tertiary , Serum Albumin/metabolism , Thermodynamics , Thiocyanates/metabolism , Urea/metabolism , Water/chemistry , Water/metabolism
2.
Proteins ; Suppl 4: 72-85, 2000.
Article in English | MEDLINE | ID: mdl-11013402

ABSTRACT

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.


Subject(s)
Proteins/chemistry , Guanidine/chemistry , Models, Chemical , Protein Denaturation , Thermodynamics , Urea/chemistry
3.
Biochemistry ; 39(15): 4455-71, 2000 Apr 18.
Article in English | MEDLINE | ID: mdl-10757995

ABSTRACT

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.


Subject(s)
Biopolymers/metabolism , Serum Albumin/metabolism , Solutions , Water/metabolism , Animals , Betaine/metabolism , Cattle , Cell Division , Cytoplasm/metabolism , Dialysis , Escherichia coli/cytology , Escherichia coli/metabolism , Glutamates/metabolism , Glycerol/metabolism , Methylamines/metabolism , Osmolar Concentration , Pressure , Proline/metabolism , Thermodynamics , Trehalose/metabolism
4.
J Mol Biol ; 294(3): 639-55, 1999 Dec 03.
Article in English | MEDLINE | ID: mdl-10610786

ABSTRACT

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.


Subject(s)
Bacterial Proteins/metabolism , DNA/metabolism , Escherichia coli Proteins , Lac Operon , Nucleic Acid Conformation , Repressor Proteins/metabolism , Binding Sites , Lac Repressors , Models, Molecular , Potassium/metabolism , Protein Conformation , Structure-Activity Relationship , Thermodynamics
5.
Biochemistry ; 38(26): 8409-22, 1999 Jun 29.
Article in English | MEDLINE | ID: mdl-10387087

ABSTRACT

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.


Subject(s)
DNA, Single-Stranded/chemistry , Hot Temperature , Nucleic Acid Heteroduplexes/chemistry , Oligonucleotides/chemistry , Calorimetry , Calorimetry, Differential Scanning , DNA, Single-Stranded/radiation effects , Nucleic Acid Denaturation/radiation effects , Nucleic Acid Heteroduplexes/radiation effects , Oligonucleotides/radiation effects , Temperature , Thermodynamics , Ultraviolet Rays
6.
Biophys J ; 76(2): 1008-17, 1999 Feb.
Article in English | MEDLINE | ID: mdl-9916032

ABSTRACT

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.


Subject(s)
Cations/metabolism , DNA, Single-Stranded/chemistry , Oligopeptides/chemistry , Oligopeptides/metabolism , Static Electricity , Anions/metabolism , Binding Sites , Fluorescence , Poly T/metabolism , Salts/pharmacology , Thermodynamics
7.
J Mol Biol ; 283(4): 741-56, 1998 Nov 06.
Article in English | MEDLINE | ID: mdl-9790837

ABSTRACT

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 (

Subject(s)
DNA Footprinting , DNA-Directed RNA Polymerases/chemistry , Escherichia coli/enzymology , Promoter Regions, Genetic/genetics , Bacteriophage lambda/genetics , DNA, Bacterial/metabolism , DNA-Binding Proteins/chemistry , Deoxyribonuclease I/metabolism , Kinetics , Nucleic Acid Conformation , Potassium Permanganate/metabolism , Temperature , Transcription, Genetic/genetics
8.
J Mol Biol ; 267(5): 1186-206, 1997 Apr 18.
Article in English | MEDLINE | ID: mdl-9150406

ABSTRACT

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.


Subject(s)
Bacterial Proteins/metabolism , Consensus Sequence , DNA-Binding Proteins/metabolism , DNA/metabolism , Escherichia coli Proteins , Operator Regions, Genetic , Repressor Proteins/metabolism , Bacterial Proteins/chemistry , Base Sequence , Binding, Competitive , DNA/chemistry , DNA-Binding Proteins/chemistry , Electrolytes , Lac Operon , Lac Repressors , Models, Chemical , Nucleoproteins/chemistry , Protein Binding , Protein Folding , Repressor Proteins/chemistry , Thermodynamics
9.
Biochemistry ; 36(17): 5193-206, 1997 Apr 29.
Article in English | MEDLINE | ID: mdl-9136881

ABSTRACT

Coulombic interactions and coupled conformational changes make important contributions to stability and specificity of many protein-nucleic acid complexes. As models of these phenomena in simpler systems, we have investigated the binding to mononucleosomal (160 base-pair) calf thymus DNA of a high charge density (compact) 5-residue (+4) oligopeptide (with 4 lysines and 1 tryptophan) and of four lower charge density (extended) 17-residue (+4) oligopeptides (each with 4 lysines, 10-12 alanines, 0-2 glycines, and 1 tryptophan). The fractional helicity (f(h)) of each oligopeptide before and after DNA binding was determined using circular dichroism. At low univalent cation concentration ([M+] = 6.4 mM), binding to DNA increases f(h) significantly for all but one of the extended oligopeptides. Oligopeptide-DNA binding constants (K(obs)) and apparent binding site sizes (n) were quantified using the noncooperative McGhee-von Hippel isotherm to fit tryptophan fluorescence quenching data. For each of the oligopeptides studied, n is found to be approximately equal to four, the number of lysine charges. In the range 6.4 mM < or = [M+] < or = 21.5 mM, power dependences of K(obs) on [M+] (SK(obs) = d log K(obs)/d log[M+]) for all 17-residue (+4) oligopeptides are similar with an average value of -3.7 +/- 0.4, which is indistinguishable (outside uncertainty) from the value obtained here for the compact (+4) oligopeptide and from values reported elsewhere for another compact tetralysine and for spermine (+4). Our results are consistent with the conclusion that the nonspecific binding to DNA of all these tetravalent ligands is driven primarily by coulombic interactions. At any [M+] investigated, values of K(obs) for the four extended (+4) oligopeptides differ by less than an order of magnitude, but all are 1-2 orders of magnitude less than values of K(obs) for two compact (+4) oligopeptides and for spermine. The differences in K(obs) for oligopeptide-DNA complexes, which all have similar n and similar SK(obs) indicate that when an extended oligopeptide binds to DNA it becomes more compact as a result of conformational changes, such as the additional alpha-helix formation detected by circular dichroism.


Subject(s)
Alanine/metabolism , DNA/metabolism , Glycine/metabolism , Oligopeptides/metabolism , Amino Acid Sequence , Circular Dichroism , Kinetics , Models, Chemical , Molecular Sequence Data , Oligopeptides/chemistry , Protein Conformation , Thermodynamics
10.
Biochemistry ; 35(32): 10506-16, 1996 Aug 13.
Article in English | MEDLINE | ID: mdl-8756707

ABSTRACT

The thermodynamic consequences of interactions of native bovine serum albumin (BSA) with two smaller solutes (glycine betaine or urea) in aqueous solution are characterized by a novel application of vapor pressure osmometry (VPO), which demonstrates the utility of this method of investigating preferential interactions involving solutes that are either accumulated or excluded near the surface of a protein. From VPO measurements of osmolality (water activity) as a function of the solute concentration in the presence and absence of BSA, we determine the dependence of the solute molarity (C3) on that of BSA (C2) at fixed temperature (37 degrees C), pressure (approximately 1 atm), and osmolality (over the range 0-1.6 molal). After some thermodynamic transformations, these results yield values of [formula: see text] which characterizes the interdependence of solute molalities when temperature, pressure, and the chemical potential of solute 3 are fixed. This form of the preferential interaction coefficient can be interpreted directly in terms of the molecular exclusion or accumulation of the solute (relative to water) near the protein surface. Within experimental uncertainty, [formula: see text] is proportional to m3 both for glycine betaine (0-0.9 m) and for urea (0-1.6 m). For glycine betaine [formula: see text] = -49 +/- 4, a value consistent with the interpretation that this solute is completely excluded from the hydrated surface of BSA, whereas for urea [formula: see text] = 6 +/- 1, which indicates a moderate extent of accumulation at the surface of native BSA. The preferential accumulation of solutes (e.g., urea) that have some binding affinity for a protein can be quantified and interpreted using the two-domain model if the extent of hydration of the protein has been determined using a completely excluded solute (e.g., glycine betaine). Complete exclusion from the local hydration domain surrounding proteins, if general, justifies the use of glycine betaine as a thermodynamic probe of the changes in hydration that accompany protein folding, protein association, and protein-ligand binding interactions.


Subject(s)
Betaine/chemistry , Serum Albumin, Bovine/chemistry , Models, Chemical , Osmolar Concentration , Osmotic Pressure , Thermodynamics , Urea/chemistry
11.
J Mol Biol ; 258(1): 25-36, 1996 Apr 26.
Article in English | MEDLINE | ID: mdl-8613989

ABSTRACT

Ion concentrations (K+, Glu-) in the cytoplasm of growing Escherichia coli cells increase strongly with increases in the osmolarity of a defined growth medium. While in vitro experiments demonstrate that the extent of protein-nucleic acid interactions (PNAI) depends critically on salt concentration, in vivo measurements indicate that cells maintain a relatively constant extent of PNAI independent of the osmolarity of growth. How do cells buffer PNAI against changes in the cytoplasmic environment? At high osmolarity, the increase in macromolecular crowding which accompanies the reduction in amount of cytoplasmic water in growing cells appears quantitatively sufficient to compensate for the increase in [K+]. At low osmolarity, however, changes in crowding appear to be insufficient to compensate for changes in [K+], and additional mechanisms must be involved. Here we report quantitative determinations of in vivo total concentrations of polyamines (putrescine(2+), spermidine(3+)) as a function of osmolarity (OsM) of growth, and in vitro binding data on the effects of putrescine concentration on a specific PNAI (lac repressor-lac operator) as a function of [K+]. The total concentration of putrescine in cytoplasmic water decreases at least eightfold from low osmolarity (approximately 64 mmol (l H2O)-1 at 0.03 OsM) to high osmolarity (approximately 8 mmol (l H2O)-1 at 1.02 OsM). Over this osmotic range the total [K+] increases from approximately 0.2 mol (l H2O)-1 to approximately 0.8 mol (lH2O)-1. We find that the effect of putrescine concentration on the repressor-operator interaction in vitro is purely competitive and is quantitatively described by a simple competition formalism in which lac repressor behaves a a specific-binding oligocation (ZR = 8+/-3). We demonstrate that this thermodynamic result is consistent with a structural analysis of the number of positively charged side-chains on two DNA binding domains of repressor which interact with the phosphodiester backbone of the operator site. Since this oligocation character of the binding surface of DNA-binding proteins appears to be general, we propose the competitive effects of putrescine and K+ concentrations on the strength of specific binding are general. At low osmolarity, compensating changes in putrescine and K+ concentration in response to changes in external osmolarity provide a general mechanism for E. coli to vary cytoplasmic osmolarity while maintaining a constant extent of PNAI.


Subject(s)
DNA, Bacterial/metabolism , Operator Regions, Genetic/genetics , Potassium/physiology , Putrescine/physiology , Repressor Proteins/metabolism , Base Sequence , Cations , Culture Media , Cytoplasm/chemistry , DNA, Bacterial/chemistry , Escherichia coli/chemistry , Helix-Turn-Helix Motifs , Magnetic Resonance Spectroscopy , Molecular Sequence Data , Nucleic Acid Conformation , Osmolar Concentration , Potassium/analysis , Protein Binding , Putrescine/analysis , Repressor Proteins/chemistry , Spermidine/analysis , Thermodynamics
12.
Biophys J ; 68(3): 1063-72, 1995 Mar.
Article in English | MEDLINE | ID: mdl-7756526

ABSTRACT

The local cation concentration at the surface of oligomeric or polymeric B-DNA is expected, on the basis of MC simulations (Olmsted, M. C., C. F. Anderson, and M. T. Record, Jr. 1989. Proc. Natl. Acad. Sci. USA. 86:7766-7770), to decrease sharply as either end of the molecule is approached. In this paper we report 23Na NMR measurements indicating the importance of this "coulombic" end effect on the average extent of association of Na+ with oligomeric duplex DNA. In solutions containing either 20-bp synthetic DNA or 160-bp mononucleosomal calf thymus DNA at phosphate monomer concentrations [P] of 4-10 mM, measurements were made over the range of ratios 1 < or = [Na]/[LP] < or = 20, corresponding to Na+ concentrations of 4-200 nM. The longitudinal 23Na NMR relaxation rates measured in these NaDNA solutions, Robs, are interpreted as population-weighted averages of contributions from "bound" (RB) and "free" (RF) 23Na relaxation rates. The observed enhancements of Robs indicate that RB significantly exceeds RF, which is approximately equal to the 23Na relaxation rate in an aqueous solution containing only NaCl. Under salt-fre-tconditions ([Na]/[P] = 1), where the enhancement in Robs is maximal, we find that Robs--RF in the solution containing 160-bp DNA is approximately 1.8 times that observed for the 20-bp DNA. For the 160-bp oligomer (which theoretical calculations predict to be effectively polyion-like), we find that a plot of Robs v. [P]/[Na] is linear, as observed previously for sonicated (approximately 700 bp) DNA samples. For the 20-bp oligonucleotide this plot exhibits a marked departure from linearity that can be fitted to a quadratic function of [P]/[Na]. Monte Carlo simulations based on a simplified model are capable of reproducing the qualitative trends in the 23Na NMR measurements analyzed here. In particular, the dependences of Robs--RF on DNA charge magnitude of Z(320 vs. 38 phosphates) and (for the 20-bp oligomer) on [Na]/[P] are well correlated with the calculated average surface concentration of Na+. Thus, effects of sodium concentration on RB appear to be of secondary importance. We conclude that 23Na NMR relaxation measurements are a sensitive probe of the effects of oligomer charge on the extent of ion accumulation near B-DNA oligonucleotides, as a function of [Na] and [P].


Subject(s)
DNA/chemistry , Animals , Base Sequence , Biophysical Phenomena , Biophysics , Cations, Monovalent , Cattle , DNA/isolation & purification , Electrolytes/chemistry , In Vitro Techniques , Magnetic Resonance Spectroscopy , Models, Chemical , Molecular Sequence Data , Monte Carlo Method , Sodium/chemistry
13.
Biotechniques ; 18(1): 94, 96, 98, 100, 1995 Jan.
Article in English | MEDLINE | ID: mdl-7702861

ABSTRACT

A method is described for large-scale (10-100 micrograms) purification of individual DNA fragment lengths from a mixture of fragment lengths using a continuous-flow, toroidal gel electrophoresis system that is commercially available. Preparative-scale separations of fragments up to approximately 1000 bp differing in length by as little as 100 bp are obtained. Typical recoveries of purified DNA fragments range from 25% to 40% of starting material.


Subject(s)
DNA/isolation & purification , Electrophoresis, Polyacrylamide Gel
14.
J Mol Biol ; 245(4): 331-50, 1995 Jan 27.
Article in English | MEDLINE | ID: mdl-7837267

ABSTRACT

Initiation of transcription of the lac operon by RNA polymerase (R) is inhibited by binding of lac repressor (L) to an operator site which overlaps the lac promoter (P). We have investigated repression of the lac UV5 promoter in vitro for a choice of the repressor--operator binding constant and ranges of thermodynamic activities of L and R which appear to be relevant in vivo. Effects of [L] on the extent of formation and the kinetics of association and dissociation of abortively-initiating open complexes (RPinit) were examined using fluorescence detected abortive initiation and KMnO4 chemical probing. The nitrocellulose filter assay was used to measure the dissociation rate constant and the equilibrium constant for binding for L to its operator site in the absence of R. For the chosen solution conditions, we find that both the observed velocity of abortive RNA oligomer synthesis and the KMnO4 reactivities of bases in the open region are functions of [L] and [R], demonstrating that formation of both RPinit and the repressor-operator complex (PL) are reversible processes under these conditions, and requiring the use of a relaxation-to-equilibrium analysis to interpret the kinetics. The agreement between dissociation rate constants of RPinit when challenged with either lac repressor or heparin, and the dependences on [L] and [R] of abortive synthesis velocities at binding equilibrium and of relaxation rate constants for reversible formation of RPinit from PL, all provide evidence for a simple competition mechanism. In this mechanism, and in contrast to recent proposals from other laboratories, lac repressor inhibits formation of RPinit and hence the observed rate of abortive product synthesis by reducing the equilibrium extent of formation of the first closed complex (RPc1), without affecting either the nature of RPinit or steps in formation of RPinit from RPc1.


Subject(s)
Lac Operon , Repressor Proteins/metabolism , Transcription, Genetic , DNA, Superhelical/chemistry , DNA-Directed RNA Polymerases/metabolism , Heparin/chemistry , Kinetics , Potassium Permanganate/pharmacology , Promoter Regions, Genetic , Thermodynamics
15.
J Mol Biol ; 228(1): 252-64, 1992 Nov 05.
Article in English | MEDLINE | ID: mdl-1447786

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 (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.


Subject(s)
DNA/metabolism , Repressor Proteins/chemistry , Water/chemistry , Anions , Cations , Chlorides/metabolism , Potassium/metabolism , Repressor Proteins/metabolism , Salts , Thermodynamics
16.
In Vitro Cell Dev Biol ; 23(1): 57-62, 1987 Jan.
Article in English | MEDLINE | ID: mdl-3804939

ABSTRACT

The human fetal adrenal cortex is one of the largest fetal organs and synthesizes precursors for placental estrogen production as part of the feto-placental unit. The factors controlling the rapid growth of the human fetal adrenal cortex during the second and third trimesters are not known. Placental regulation of the growth of human fetal adrenocortical cell cultures from second trimester fetuses was studied. A placental-derived mitogenic factor (PDMF) was detected in tissue homogenates of 14 to 22 week human placentas and stimulated adrenocortical cell number and [3H]thymidine incorporation into DNA 5-8 fold. PDMF has been partially purified by ammonium sulfate precipitation and anion exchange chromatography. PDMF is a heat sensitive protein with disulfide bonds required for activity. The growth stimulation by PDMF was significantly greater than that for basic or acidic fibroblast growth factor by 25-50% and epidermal growth factor by 3-4 fold. The placental hormones, progesterone, estriol, estradiol, placental lactogen and chorionic gonadotropin, either alone or in combination did not stimulate fetal adrenocortical cell growth, except for a 41% cell number increase by progesterone. Platelet-derived growth factor and insulin-like growth factors I and II were not mitogenic for these cells. These results show that the placenta contains a potent growth factor for human fetal adrenocortical cell cultures. This implies a direct role for the placenta in control of this fetal organ's growth, which would make the human feto-placental unit a bi-directional relationship.


Subject(s)
Adrenal Cortex/embryology , Mitogens/pharmacology , Pregnancy Proteins/pharmacology , Adrenal Cortex/cytology , Adrenal Cortex/drug effects , Cell Division/drug effects , Cells, Cultured , Chromatography, DEAE-Cellulose , Female , Fetus , Growth Substances/pharmacology , Humans , Maternal-Fetal Exchange , Placenta/analysis , Placental Hormones/pharmacology , Pregnancy
17.
Anal Biochem ; 147(2): 374-81, 1985 Jun.
Article in English | MEDLINE | ID: mdl-2990253

ABSTRACT

The major adrenal steroids were separated by multistep gradient elution with a reversed-phase high-performance liquid chromatography system, employing water and 1-propanol as solvents. With this solvent system, a wide range of 21 5-ene-3 beta-ol and 4-ene-3-one steroids can be resolved in a single chromatogram, which was not possible with previously published gradient solvent systems. In particular, intermediate steroids of the biosynthetic pathway, 17 alpha-hydroxyprogesterone, 17 alpha-hydroxypregnenolone, and dehydroepiandrosterone, were separated with baseline or sufficient resolution to allow accurate quantitation. Using the 1-propanol-water gradient, the separations of 5-ene and 4-ene steroids were compared on different octadecylsilyl packings. Optimum resolution was obtained with a fully covered, spherical particle. The 1-propanol-water gradient was compared to a previously published methanol-water gradient in the analysis of steroidogenesis by adrenocortical cell cultures. HPLC analysis of the steroid production was quantitatively the same with both gradient solvent systems. However, qualitatively, the methanol-water gradient system did not resolve the above-mentioned intermediate steroids.


Subject(s)
Adrenal Glands/analysis , Chromatography, High Pressure Liquid/methods , Steroids/analysis , Adrenocorticotropic Hormone/pharmacology , Cells, Cultured , Humans
18.
J Clin Endocrinol Metab ; 59(4): 643-51, 1984 Oct.
Article in English | MEDLINE | ID: mdl-6090492

ABSTRACT

The steroidogenic capacity of cells cultured from the definitive zone and fetal zone of the human fetal adrenal cortex was compared, using a serum-free medium without lipoproteins. Comparison of [3H]pregnenolone and [3H]progesterone metabolism in cultures from each zone incubated without ACTH indicated that only 3 beta-hydroxysteroid dehydrogenase, delta 4,5-isomerase (3 beta-HSD) activity was deficient in fetal zone cultures. Basal 3 beta-HSD activity was 3- to 5-fold lower in fetal zone cultures than in definitive zone cultures assayed after 3 or 10 days in culture. Although basal hydroxysteroid sulfotransferase activity was 2- to 4-fold greater in 3-day-old fetal zone cultures than in definitive zone cultures, this difference was not found in 10-day-old cultures due to a 3-fold decrease in fetal zone basal hydroxysteroid sulfotransferase activity. However, older cultures of fetal zone cells did maintain the characteristic high delta 5-steroid sulfate and low delta 4,3-ketosteroid basal production from [3H] pregnenolone as compared to definitive zone cultures. ACTH treatment for 48 h in serum-free medium increased the steroidogenic capacity of cell cultures from both zones and stimulated delta 4,3-ketosteroid production from [3H]pregnenolone and 3 beta-HSD activity in fetal zone cultures to levels characteristic of the definitive zone. These studies show that in the absence of ACTH the difference in steroidogenic capacity between the fetal zone and the definitive zone (due to the lower 3 beta-HSD activity in fetal zone cells) was maintained in cell cultures for a period up to 10 days.


Subject(s)
Adrenal Cortex Hormones/biosynthesis , Adrenal Cortex/embryology , Sulfotransferases , 3-Hydroxysteroid Dehydrogenases/metabolism , Adrenal Cortex/anatomy & histology , Adrenal Cortex/metabolism , Adrenocorticotropic Hormone/pharmacology , Cells, Cultured , Chromatography, High Pressure Liquid , Fetus/anatomy & histology , Fetus/metabolism , Gestational Age , Humans , Pregnenolone/metabolism , Progesterone/metabolism , Sulfurtransferases/metabolism
19.
J Chromatogr ; 287(1): 97-104, 1984 Mar 30.
Article in English | MEDLINE | ID: mdl-6725459

ABSTRACT

Simultaneous separation of steroid 3-sulfates and the corresponding free steroids has been accomplished by reversed-phase high-performance liquid chromatography (RP-HPLC). Addition of ammonium sulfate to the aqueous component of the mobile phase results in retention of the steroid sulfates to octadecyl silica packings by an apparent ion-pairing mechanism without affecting the retention of the free steroids. The retention time of steroid sulfates was dependent on the ammonium sulfate concentration in the mobile phase. Biologically important C19 delta 5, 3beta-hydroxysteroids and corresponding 3-sulfate conjugates or estrogen and estrogen 3-sulfates have been resolved with this RP-HPLC system.


Subject(s)
Steroids/analysis , Ammonium Sulfate , Chromatography, High Pressure Liquid/methods , Osmolar Concentration , Sulfates/analysis
20.
J Chromatogr ; 276(1): 55-68, 1983 Aug 12.
Article in English | MEDLINE | ID: mdl-6672025

ABSTRACT

Reversed-phase high-performance liquid chromatography (RP-HPLC) has been used to fractionate human parathyroid hormone (hPTH) from a variety of natural sources and to compare it with synthetic hPTH and hPTH fragments. Multiple radioimmunoassay systems for amino, mid and carboxyl regions of hPTH were used to monitor various preparations of hPTH previously prepared by conventional methods and ampouled in nanogram amounts for reference standard and reagent purposes. Results confirmed that they were free of detectable cleavage products, but showed that the intact hPTH comprised three or four closely associated components. A similar pattern of heterogeneity was obtained when hPTH was extracted from stored human parathyroid adenomata by a simple rapid HPLC bulk fractionation method. Comparison with synthetic 1-84 hPTH and modification of sample handling to minimize oxidative conditions, indicate that some of these components are probably intermediate oxidation products. A number of less hydrophobic components, with carboxyl region immunoreactivities, were obtained from the individual adenoma samples, human parathyroid cyst fluid, ampouled samples of human adenoma tissue culture medium, and secondary hyperparathyroid plasma ultrafiltrate when they were fractionated by RP-HPLC. The results strongly suggest that the biological degradation of hPTH is more complex than generally believed, and that RP-HPLC offers a new dimension in its analysis.


Subject(s)
Body Fluids/analysis , Parathyroid Glands/analysis , Parathyroid Hormone/analysis , Adenoma/metabolism , Chromatography, High Pressure Liquid/methods , Culture Techniques , Humans , Hyperparathyroidism/metabolism , Parathyroid Neoplasms/metabolism , Radioimmunoassay , Reference Standards
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