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PubMed1975

Thermodynamic properties of superhelical DNAs.

Binding isotherms of ethidium to the superhelical DNA from phage PM2, and to PM2 DNA containing -1 single-chain scission per molecule, have been determined at six temperatures from 2.5 to 50 degrees, in 3M CsCl-0.01 M Na3EDTA. Spectrophotometric measurements in both the visible and ultraviolet (uv) regions were used to obtain the binding isotherms. The isotherm at 20 degrees was also obtained by determining the free ethidium concentrations in equilibrium with DNA-ethidium complexes in boundary sedimentation experiments. A simple thermodynamic analysis shows that for a superhelical DNA with v bound ethidium per nucleotide, the change in free energy per unit change in the number of superhelical turns T, dGT/dT, is directly related the ratio of the free ethidium concentrations, cf,v and cf,v, in equilibrium with the ethidium complexes of the superhelical and the nicked DNA, respectively, at the same values of v. The relationship is dGT/dT = (360/E)RTln (cf,v/cf,v), where e is the unwinding angle of the DNA helix per bound ethidium molecule. Experimentally, it was found that ln (cf,v/cf,v) = a1(v - vc), where a1 is a constant at a given temperature and vc is the value of v at which the originally superhelical DNA is completely relaxed, i.e., containing no superhelical turns. The equation relating dGT/dT and v, upon transformation and integration, gives finite difference GT,v=o = -a1NRTvc2/2, where finite difference GT,v=0 is the free energy of superhelix formation of the superhelical DNA in the absence of ethidium and N is the number of nucleotides per DNA molecule. This equation is independent of the unwinding angle e. The values of a1 are 11.2 plus or minus 0.9, 11.2 plus or minus 0.7, 11.2 plus or minus 0.6, 10.7 plus or minus 0.8, 10.0 pl.us or minus 1.0, and 9.8 plus or minus 1.4 at 2.5, 10, 20, 30, 40, and 50 degrees, respectively. Measurements with lambda b2b5c DNA, and monomeric and trimeric lambda dv DNA, indicate that the constant a1 is insensitive to the molecular length of DNA. The present results are compared with the previous results of Bauer and Vinograd (Bauer, W., and Vinograd, M. (1970), J. Mol. Biol. 47, 419), obtained by a statistical mechanical analysis of the ethidium binding isotherms (determined by density gradient centrifugation measurements) to two forms of simian virus 40 DNA at 25 degrees. The effects of superhelical turns on a number of processes, such as the binding of a ligand which unwinds or winds the DNA helix, the denaturation of a DNA segment, and the formation of hair-pinned structures from base sequences with a twofold rotational symmetry, are discussed from the thermodynamic point of view, based on the present results.

Bacteriophages↗
PubMed1975

The pH dependence of the thermodynamics of the interaction of 3'-cytidine monophosphate with ribonuclease A.

The apparent free energy (deltaGapp) and enthalpy changes (deltaHB) associated with the interaction of 3'-cytosine monophosphate (3'-CMP) and ribonuclease A (RNase) are reported for the pH range 4--9, T = 25 degrees, mu = 0.05. The pH dependence of deltaGapp and deltaHB has been interpreted in terms of coupled ionization of histidine residues 12, 48, and 119, assuming that only the dianionic form of the inhibitor is bound. The results of this analysis are consistent with the calorimetric and potentiometric titration results for the free enzyme and its 3'-CMP complex reported in the previous paper (M. Flogel and R. L. Biltonen ((1975), Biochemistry, preceding paper in this issue). This analysis allows the calculation of the thermodynamic quantities associated with hypothetical but clearly defined reactions (e.g., the reaction of the dianionic inhibitor with the completely protonated enzyme). It is concluded that the primary thermodynamic driving forces for the reaction are van der Waals interactions between the riboside moiety and the protein fabric and electrostatic interaction between the negatively charged phosphate group of the inhibitor and the positively charged histidine residues at the binding locus. It is also suggested that the binding reaction is weakly coupled (approximately to 0.5 kcal/mol) with a conformational change of the protein associated with protonation of residue 48. These results are consistent with the model originally proposed by G. G. Hammes ((1968), Adv. Protein Chem. 23, 1) and lend additional quantitative detail to the nature of the reaction.

Binding Sites↗
PubMed2001

Thermodynamics of electron transfer in oxygenic photosynthetic reaction centers: volume change, enthalpy, and entropy of electron-transfer reactions in manganese-depleted photosystem II core complexes.

We have previously reported the thermodynamic data of electron transfer in photosystem I using pulsed time-resolved photoacoustics [Hou et al. (2001) Biochemistry 40, 7109-7116]. In the present work, using preparations of purified manganese-depleted photosystem II (PS II) core complexes from Synechocystis sp. PCC 6803, we have measured the DeltaV, DeltaH, and estimated TDeltaS of electron transfer on the time scale of 1 micros. At pH 6.0, the volume contraction of PS II was determined to be -9 +/- 1 A3. The thermal efficiency was found to be 52 +/- 5%, which corresponds to an enthalpy change of -0.9 +/- 0.1 eV for the formation of the state P680+Q(A-) from P680*. An unexpected volume expansion on pulse saturation of PS II was observed, which is reversible in the dark. At pH 9.0, the volume contraction, the thermal efficiency, and the enthalpy change were -3.4 +/- 0.5 A3, 37 +/- 7%, and -1.15 +/- 0.13 eV, respectively. The DeltaV of PS II, smaller than that of PS I and bacterial centers, is assigned to electrostriction and analyzed using the Drude-Nernst equation. To explain the small DeltaV for the formation of P680+Q(A-) or Y(Z*)Q(A-), we propose that fast proton transfer into a polar region is involved in this reaction. Taking the free energy of charge separation of PS II as the difference between the energy of the excited-state P680* and the difference in the redox potentials of the donor and acceptor, the apparent entropy change (TDeltaS) for charge separation of PS II is calculated to be negative, -0.1 +/- 0.1 eV at pH 6.0 (P680+Q(A-)) and -0.2 +/- 0.15 eV at pH 9.0 (Y(Z*)Q(A-)). The thermodynamic properties of electron transfer in PS II core reaction centers thus differ considerably from those of bacterial and PS I reaction centers, which have DeltaV of approximately -27 A3, DeltaH of approximately -0.4 eV, and TDeltaS of approximately +0.4 eV.

Cyanobacteria↗
PubMed2001

A molecular beacon strategy for the thermodynamic characterization of triplex DNA: triplex formation at the promoter region of cyclin D1.

We studied the formation of triplex DNA in the purine-pyrimidine-rich promoter site sequence of cyclin D1, located at -116 to -99 from the transcription initiation site, with a molecular beacon comprised of a G-rich 18-mer triplex forming oligodeoxyribonucleotide. Formation of triplex DNA was monitored by enhanced fluorescence of the beacon, due to the weakening of fluorescence energy transfer, upon its binding to the target duplex. Electrophoretic mobility shift assay confirmed triplex DNA formation by these oligonucleotides. In low salt buffer (10 mM Na(+)), triplex DNA formation was not observed in the absence of a ligand such as spermine. At room temperature (22 degrees C), the equilibrium association constant (K(a)) calculated in the presence of 1 microM spermine and 10 mM Na(+) was 3.2 x 10(8) M(-1). The K(a) value was 1.0 x 10(9) M(-1) in the presence of 150 mM Na(+), and it increased by 10-fold by the addition of 1 mM spermine. Delta H, Delta S, and Delta G of triplex DNA formation, calculated from the temperature dependence of K(a) in the range of 20--45 degrees C, were -35.9 kcal/mol, -77 cal/(mol.K), and -13 kcal/mol, respectively, in the presence of 150 mM NaCl. The corresponding values were -52.9 kcal/mol, -132.5 cal/(mol.K), and -13.4 kcal/mol in the presence of 150 mM NaCl and 1 mM spermine. Structurally related polyamines exerted different degrees of triplex DNA stabilization, as determined by binding constant measurements. Comparison of spermine versus hexamine showed a 17-fold increase in the equilibrium association constant, whereas bis(ethyl) derivatization lead to a 4-fold decrease of this value. In the absence of added duplex and polyamines, the molecular beacon dissociated with a melting temperature of 67 degrees C. Thermodynamic parameters of beacon melting were calculated from the melting curve, and the Delta H, Delta S, and Delta G values were 37.8 kcal/mol, 112 cal/(mol.K), and 4.4 kcal/mol, respectively. These results demonstrate that molecular beacons can be used for the direct determination of the equilibrium association constants and thermodynamic parameters of triplex DNA formation in the presence of ligands such as polyamines.

Cyclin D1↗
PubMed2001

Thermodynamic analysis of the binding of aromatic hydroxamic acid analogues to ferric horseradish peroxidase.

Peroxidases typically bind their reducing substrates weakly, with K(d) values in the millimolar range. The binding of benzhydroxamic acid (BHA) to ferric horseradish peroxidase isoenzyme C (HRPC) [K(d) = 2.4 microM; Schonbaum, G. R. (1973) J. Biol. Chem. 248, 502-511] is a notable exception and has provided a useful tool for probing the environment of the peroxidase aromatic-donor-binding site and the distal heme cavity. Knowledge of the underlying thermodynamic driving forces is key to understanding the roles of the various H-bonding and hydrophobic interactions in substrate binding. The isothermal titration calorimetry results of this study on the binding of aromatic hydroxamic acid analogues to ferric HRPC under nonturnover conditions (no H(2)O(2) present) confirm the significance of H-bonding interactions in the distal heme cavity in complex stabilization. For example, the binding of BHA to HRPC is enthalpically driven at pH 7.0, with the H-bond to the distal Arg38 providing the largest contribution (6.74 kcal/mol) to the binding energy. The overall relatively weak binding of the hydroxamic acid analogues to HRPC is due to large entropic barriers (-11.3 to -37.9 eu) around neutral pH, with the distal Arg38 acting as an "entropic gate keeper". Dramatic enthalpy-entropy compensation is observed for BHA and 2-naphthohydroxamic acid binding to HRPC at pH 4.0. The enthalpic loss and entropic gain are likely due to increased flexibility of Arg38 in the complexes at low pH and greater access by water to the active site. Since the Soret absorption band of HRPC is a sensitive probe of the binding of hydroxamic acids and their analogues, it was used to investigate the binding of six donor substrates over the pH range of 4-12. The negligible pH dependence of the K(d) values corrected for substrate ionization suggests that enthalpy-entropy compensation is operative over a wide pH range. Examination of the thermodynamics of binding of ring-substituted hyrazides to HRPC reveals that the binding affinities of aromatic donors are highly sensitive to the position and nature of the ring substituent.

Benzamides↗
PubMed2001

Thermodynamic differences among homologous thermophilic and mesophilic proteins.

Here, we analyze the thermodynamic parameters and their correlations in families containing homologous thermophilic and mesophilic proteins which show reversible two-state folding <--> unfolding transitions between the native and the denatured states. For the proteins in these families, the melting temperatures correlate with the maximal protein stability change (between the native and the denatured states) as well as with the enthalpic and entropic changes at the melting temperature. In contrast, the heat capacity change is uncorrelated with the melting temperature. These and additional results illustrate that higher melting temperatures are largely obtained via an upshift and broadening of the protein stability curves. Both thermophilic and mesophilic proteins are maximally stable around room temperature. However, the maximal stabilities of thermophilic proteins are considerably greater than those of their mesophilic homologues. At the living temperatures of their respective source organisms, homologous thermophilic and mesophilic proteins have similar stabilities. The protein stability at the living temperature of the source organism does not correlate with the living temperature of the protein. We tie thermodynamic observations to microscopics via the hydrophobic effect and a two-state model of the water structure. We conclude that, to achieve higher stability and greater resistance to high and low temperatures, specific interactions, particularly electrostatic, should be engineered into the protein. The effect of these specific interactions is largely reflected in an increased enthalpy change at the melting temperature.

Acid Anhydride Hydrolases↗
PubMed2001

Thermodynamic characterization of ligand-induced conformational changes in UDP-N-acetylglucosamine enolpyruvyl transferase.

The binding of UDP-N-acetylglucosamine (UDPNAG) to the enzyme UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) was studied in the absence and presence of the antibiotic fosfomycin by isothermal titration calorimetry. Fosfomycin binds covalently to MurA in the presence of UDPNAG and also in its absence as demonstrated by MALDI mass spectrometry. The covalent attachment of fosfomycin affects the thermodynamic parameters of UDPNAG binding significantly: In the absence of fosfomycin the binding of UDPNAG is enthalpically driven (DeltaH = -35.5 kJ mol(-1) at 15 degrees C) and opposed by an unfavorable entropy change (DeltaS = -25 J mol(-1) K(-1)). In the presence of covalently attached fosfomycin the binding of UDPNAG is entropically driven (DeltaS = 187 J mol(-1)K(-1) at 15 degrees C) and associated with unfavorable changes in enthalpy (DeltaH = 28.8 kJ mol(-1)). Heat capacities for UDPNAG binding in the absence or presence of fosfomycin were -1.87 and -2.74 kJ mol(-1) K(-1), respectively, indicating that most ( approximately 70%) of the conformational changes take place upon formation of the UDPNAG-MurA binary complex. The major contribution to the heat capacity of ligand binding is thought to be due to changes in the solvent-accessible surface area. However, associated conformational changes, if any, also contribute to the experimentally measured magnitude of the heat capacity. The changes in solvent-accessible surface area were calculated from available 3D structures, yielding a DeltaC(p) of -1.3 kJ mol(-1) K(-1); i.e., the experimentally determined heat capacity exceeds the calculated one. This implies that other thermodynamic factors exert a large influence on the heat capacity of protein-ligand interactions.

Alkyl and Aryl Transferases↗
PubMed2001

Structure, dynamics, and thermodynamics of the structural domain of troponin C in complex with the regulatory peptide 1-40 of troponin I.

The structure of the calcium-saturated C-domain of skeletal troponin C (CTnC) in complex with a regulatory peptide comprising residues 1-40 (Rp40) of troponin I (TnI) was determined using nuclear magnetic resonance (NMR) spectroscopy. The solution structure determined by NMR is similar to the structure of the C-domain from intact TnC in complex with TnI(1)(-)(47) determined by X-ray crystallography [Vassylyev, D. G., Takeda, S., Wakatsuki, S., Maeda, K., and Maeda, Y. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 4847-4852]. Changes in the dynamic properties of CTnC.2Ca2+ induced by Rp40 binding were investigated using backbone amide (15)N NMR relaxation measurements. Analysis of NMR relaxation data allows for extraction of motional order parameters on a per residue basis, from which the contribution of changes in picosecond to nanosecond time scale motions to the conformational entropy associated with complex formation can be estimated. The results indicate that binding of Rp40 decreases backbone flexibility in CTnC, particularly at the end of the C-terminal helix. The backbone conformational entropy change (-TDeltaS) associated with binding of Rp40 to CTnC.2Ca2+ determined from (15)N relaxation data is 9.6 +/- 0.7 kcal mol(-1) at 30 degrees C. However, estimation of thermodynamic quantities using a structural approach [Lavigne, P., Bagu, J. R., Boyko, R., Willard, L., Holmes, C. F., and Sykes, B. D. (2000) Protein Sci. 9, 252-264] reveals that the change in solvation entropy upon complex formation is dominant and overcomes the thermodynamic "cost" associated with "stiffening" of the protein backbone upon Rp40 binding. Additionally, backbone amide (15)N relaxation data measured at different concentrations of CTnC.2Ca2+.Rp40 reveal that the complex dimerizes in solution. Fitting of the apparent global rotational correlation time as a function of concentration to a monomer-dimer equilibrium yields a dimerization constant of approximately 8.3 mM.

Amino Acid Sequence↗
PubMed2001

Insights into the stability of native and partially folded states of ubiquitin: effects of cosolvents and denaturants on the thermodynamics of protein folding.

The thermodynamics of the native<-->A state and native<-->unfolded transitions for ubiquitin have been characterized in detail using the denaturants methanol and guanidinium chloride (Gdn.HCl) both separately and in combination. Gdn.HCl destabilizes the partially folded alcohol-induced A state such that the effects of alcoholic solvents on the native<-->unfolded transition can be investigated directly via a two-state model. The combined denaturing effects of methanol and Gdn.HCl appear to conform to a simple additive model. We show that ubiquitin folds and unfolds cooperatively in all cases, forming the same "native" state; however, the thermodynamics of the N<-->U transition change dramatically between alcoholic and Gdn.HCl solutions, with folding in aqueous methanol associated with large negative enthalpy and entropy terms at 298 K with a gradual falloff in DeltaC(p) at higher methanol concentrations, as previously reported for the N<-->A transition (Woolfson, D. N., Cooper, A., Harding, M. M., Williams, D. H., and Evans, P. A. (1993) J. Mol. Biol. 229, 502-511.). Both the N<-->U and the N<-->A transitions are enthalpy driven to a similar extent. We conclude that under these conditions van der Waals interactions in the packing of the nonpolar protein core, which is common to both the N<-->U and the N<-->A transitions, appear to drive folding in the absence of entropic effects associated with release of ordered solvent (hydrophobic effect). Solvent transfer studies of hydrocarbons into alcoholic solvents, with and without Gdn.HCl, are consistent with a large enthalpic driving force for burial of a nonpolar surface, with a linear dependence of protein stability (DeltaG(N)(<-->)(U)) on cosolvent concentration reflected in a similar linear dependence of hydrocarbon solubility. The data demonstrate that the hydrophobic effect is not a prerequisite for specific stabilization of the native state or the A state and that van der Waals packing of the nonpolar core appears to be the dominant factor in stabilization of the native state.

Animals↗
PubMed2001

Thermodynamic studies of the core histones: stability of the octamer subunits is not altered by removal of their terminal domains.

We have investigated the role of the labile terminal domains of the core histones on the stability of the subunits of the protein core of the nucleosome by studying the thermodynamic behavior of the products of limited trypsin digestion of these subunits. The thermal stabilities of the truncated H2A-H2B dimer and the truncated (H3-H4)/(H3-H4)(2) system were studied by high-sensitivity differential scanning calorimetry and circular dichroism spectroscopy. The thermal denaturation of the truncated H2A-H2B dimer at pH 6.0 and low ionic strength is centered at 47.3 degrees C. The corresponding enthalpy change is 35 kcal/mol of 11.5 kDa monomer unit, and the heat capacity change upon unfolding is 1.2 kcal/(K mol of 11.5 kDa monomer unit). At pH 4.5 and low ionic strength, the truncated (H3-H4)/(H3-H4)(2) system, like its full-length counterpart, is quantitatively dissociated into two truncated H3-H4 dimers. The thermal denaturation of the truncated H3-H4 dimer is characterized by the presence of a single calorimetric peak centered at 60 degrees C. The enthalpy change is 25 kcal/mol of 10 kDa monomer unit, and the change in heat capacity upon unfolding is 0.5 kcal/(K mol of 10 kDa monomer unit). The thermal stabilities of both types of truncated dimers exhibit salt and pH dependencies similar to those of the full-length proteins. Finally, like their full-length counterparts, both truncated core histone dimers undergo thermal denaturation as highly cooperative units, without the involvement of any significant population of melting intermediates. Therefore, removal of the histone "tails" does not generally affect the thermodynamic behavior of the subunits of the core histone complex, indicating that the more centrally located regions of the histone fold and the extra-fold structured elements are primarily responsible for their stability and responses to parameters of their chemical microenvironment.

Animals↗
PubMed2002

Kinetic and thermodynamic basis of promoter strength: multiple steps of transcription initiation by T7 RNA polymerase are modulated by the promoter sequence.

Transcription initiation by T7 RNA polymerase (T7 RNAP) is regulated by the specific promoter DNA sequence that is classically divided into two major domains, the binding domain (-17 to -5) and the initiation domain (-4 to +6). The occurrence of non-consensus bases within these domains is responsible for the diversity of promoter strength, the basis of which was investigated by studying T7 promoters with changes in the promoter specificity region (-13 to -6) of the binding domain and/or the melting region (-4 to -1) of the initiation domain. The transient state kinetics and thermodynamic studies revealed that multiple steps in the pathway of transcription initiation are modulated by the promoter DNA sequence. Three base changes in the promoter specificity region at -11, -12, and -13, found in the natural phi 3.8 promoter, reduced the overall affinity of the T7 RNAP for the promoter DNA by 2-3-fold and decreased the rate of pppGpG synthesis, the first RNA product. Promoter opening is thermodynamically driven in T7 RNAP, and a single base change in the melting region (TATA to TAAA) decreased the extent of open complex generated at equilibrium. This base change in the melting region also increased the K(d) of (+1) GTP and the dissociation rate of pppGpG. Thus, transcription initiation at various T7 promoters is differentially regulated by initiating GTP concentration. The specificity and melting regions of T7 promoter DNA act both independently and synergistically to affect distinct steps of transcription initiation. Although each step in the initiation pathway is affected to a small degree by promoter sequence variations, the cumulative effect dictates the overall promoter strength.

DNA-Directed RNA Polymerases↗
PubMed2002

Molecular recognition in purine-rich internal loops: thermodynamic, structural, and dynamic consequences of purine for adenine substitutions in 5'(rGGCAAGCCU)2.

The contribution of amino groups to the thermodynamics, structure, and dynamics of tandem A.A mismatches is investigated by substitution of purine (P) for adenine (A) within the RNA duplex, 5'(rGGCAAGCCU)(2), to give 5'(rGGCPAGCCU)(2), 5'(rGGCAPGCCU)(2), and 5'(rGGCPPGCCU)(2). The 5'(rGGCAAGCCU)(2) duplex has sheared A(anti).A(anti) (A.A trans Hoogsteen/Sugar-edge) pairs in which the A5 amino group is involved in hydrogen bonds but the A4 amino group is not [Znosko, B. M., Burkard, M. E., Schroeder, S. J., Krugh, T. R., and Turner, D. H. (2002) Biochemistry 41, 14969-14977]. In comparison to 5'(rGGCAAGCCU)(2), replacing the amino group of A4 with a hydrogen stabilizes the duplex by 1.3 kcal/mol, replacement of the A5 amino group destabilizes the duplex by 0.6 kcal/mol, and replacement of both A4 and A5 amino groups destabilizes the duplex by 0.8 kcal/mol. In NMR structures, the P.A noncanonical pairs of the 5'(rGGCPAGCCU)(2) duplex have a sheared anti-anti structure (P.A trans Hoogsteen/Sugar-edge) with P4.A5 interstrand hydrogen bonding and A5 bases that interstrand stack, similar to the structure of 5'(rGGCAAGCCU)(2). In contrast, the A.P pairs of the 5'(rGGCAPGCCU)(2) duplex have a face-to-face conformation (A.P trans Watson-Crick/Watson-Crick) with intrastrand stacking resembling typical A-form geometry. Although the P5 bases in 5'(rGGCPPGCCU)(2) are involved in an interstrand stack, the loop region is largely undefined. The results illustrate that both hydrogen-bonded and non-hydrogen-bonded amino groups play important roles in determining the thermodynamic, structural, and dynamic characteristics of purine rich internal loops.

Adenine↗
PubMed2002

Thermodynamic parameters for an expanded nearest-neighbor model for the formation of RNA duplexes with single nucleotide bulges.

Thirty-four RNA duplexes containing single nucleotide bulges were optically melted, and the thermodynamic parameters deltaH degrees, deltaS degrees, deltaG degrees (37), and T(M) for each sequence were determined. Data from this study were combined with data from previous thermodynamic data [Longfellow, C. E., Kierzek, R., and Turner, D. H. (1990) Biochemistry 29, 278-85] to develop a model that will more accurately predict the free energy of an RNA duplex containing a single nucleotide bulge. Differences between purine and pyrimidine bulges as well as differences between Group I duplexes, those in which the bulge is not identical to either neighboring nucleotide, and Group II duplexes, those in which the bulge is identical to at least one neighboring nucleotide, were considered. The length of the duplex, non-nearest-neighbor effects, and bulge location were also examined. A model was developed which divides sequences into two groups: those with pyrimidine bulges and those with purine bulges. The proposed model for pyrimidine bulges predicts deltaG degrees (37,bulge) = 3.9 kcal/mol + 0.10deltaG degrees (37,nn) + beta, while the model for purine bulges predicts deltaG degrees (37,bulge) = 3.3 kcal/mol - 0.30deltaG degrees (37,nn) + beta, where beta has a value of 0.0 and -0.8 kcal/mol for Group I and Group II sequences, respectively, and deltaG degrees (37,nn) is the nearest-neighbor free energy of the base pairs surrounding the bulge. The conformation of bulge loops present in rRNA was examined. Three distinct families of structures were identified. The bulge loop was either extrahelical, intercalated, or in a "side-step" conformation.

Adenosine↗
PubMed2002

Selection for thermodynamically stable DNA tetraloops using temperature gradient gel electrophoresis reveals four motifs: d(cGNNAg), d(cGNABg),d(cCNNGg), and d(gCNNGc).

Hairpins play important roles in the function of DNA, forming cruciforms and affecting processes such as replication and recombination. Temperature gradient gel electrophoresis (TGGE) and in vitro selection have been used to isolate thermodynamically stable DNA hairpins from a six-nucleotide random library. The TGGE-selection process was optimized such that known stable DNA tetraloops were recovered, and the selection appears to be exhaustive. In the selection, four families of exceptionally stable DNA loops were identified: d(cGNNAg), d(cGNABg), d(cCNNGg), and d(gCNNGc). (Lowercase denotes the closing base pair; N = A, C, G, or T; and B = C, G, or T.) It appears that the known stable d(cGNAg) triloop motif can be embedded into a tetraloop, with the extra nucleotide inserted into either the middle of the loop, d(cGNNAg), or at the 3'-end of the loop, d(cGNABg). For d(cGNNAg) and d(cGNABg), a CG closing base pair was strongly preferred over a GC, with DeltaDeltaG degrees (37) approximately 2 kcal/mol. Members of the two families, d(cCNNGg) and d(gCNNGc), are similar in stability. The loop sequences and closing base pairs identified for exceptionally stable DNA tetraloops show many similarities to those known for exceptionally stable RNA tetraloops. These data provide an expanded set of thermodynamic rules for the formation of tetraloops in DNA.

Base Pairing↗
PubMed2002

Thermodynamics of the acid transition in blue copper proteins.

The thermodynamic parameters of the conformational transition occurring at low pH (acid transition, AT) in blue copper proteins, involving protonation and detachment from the Cu(I) ion of one histidine ligand, have been determined electrochemically for spinach and cucumber plastocyanins, Rhus vernicifera stellacyanin, cucumber basic protein (CBP), and Paracoccus versutus amicyanin. These data were obtained from direct protein electrochemistry experiments carried out at varying pH and temperature. For all species but CBP, the overall conformational change turns out to be exothermic. The entropy change is remarkably species-dependent. In particular, we found that (i) the balance of bond breaking/formation favors the acid transition in plastocyanins, which show remarkably negative DeltaH degrees '(AT) values, and (ii) the transition enthalpy turns out to be much less negative (or even positive) for the two phytocyanins (stellacyanin and CBP): for these species, the transition turns out to be observable thanks to the favorable (positive) entropy change. Thus, it is apparent that the thermodynamic "driving force" for this transition is enthalpic for the plastocyanins and entropic for the phytocyanins. Amicyanin is an intermediate case in which both enthalpic and entropic terms favor the transition. Under the assumption that the transition entropy originates from solvent reorganization effects, which are known to involve compensative enthalpy and entropy changes, the free energy change of the transition would also correspond to the enthalpy change due to bond breaking/formation in the first coordination sphere of the metal and in its immediate environment. Indeed, this term turns out to be very similar for the proteins investigated, in line with the conservation of the Cu(I)-His bond strengths in these species, except for amicyanin, for which the greater exothermicity of the transition can be ascribed to peculiar features of the active site.

Acids↗
PubMed2002

Structure-based thermodynamic analysis of a coupled metal binding-protein folding reaction involving a zinc finger peptide.

The thermodynamics of metal binding by the prototypical Cys(2)His(2) zinc finger peptide CP-1 has been examined through the use of isothermal titration calorimetry. In cholamine buffer at pH 7.0, the binding of zinc(II) to CP-1 shows an enthalpy change of DeltaH degrees (obs) = -33.7 +/- 0.8 kcal/mol. Between one and two protons appear to be released accompanying the metal binding process. The heat of protonation of the cholamine buffer used is quite large (-11.5 kcal/mol), indicating that a portion of the observed metal binding enthalpy is due to buffer protonation. Structure-based thermodynamic analysis including the effect of water release from zinc(II) appears to account for the entropy associated with the coupled metal binding-protein folding process semiquantitatively. The strongest driving force for the reaction is the enthalpy associated with the four bonds from zinc(II) to cysteinate and histidine residues, compared with the bonds from zinc(II) to water. The binding of cobalt(II) to CP-1 is less enthalpically driven than the binding of zinc(II) by -7.6 kcal/mol. This value is approximately equal to, but slightly larger than, the expectation based on considerations of ligand field stabilization energy.

Calorimetry↗
PubMed2003

Thermodynamic consequences of bipartite immunity protein binding to the ribosomal ribonuclease colicin E3.

Colicin E3 is a 60 kDa, multidomain protein antibiotic that targets its ribonuclease activity to an essential region of the 16S ribosomal RNA of Escherichia coli. To prevent suicide of the producing cell, synthesis of the toxin is accompanied by the production of a 10 kDa immunity protein (Im3) that binds strongly to the toxin and abolishes its enzymatic activity. In the present work, we study the interaction of Im3 with the isolated cytotoxic domain (E3 rRNase) and intact colicin E3 through presteady-state kinetics and thermodynamic measurements. The isolated E3 rRNase domain forms a high affinity complex with Im3 (K(d) = 10(-12) M, in 200 mM NaCl at pH 7.0 and 25 degrees C). The interaction of Im3 with full-length colicin E3 under the same conditions is however significantly stronger (K(d) = 10(-14) M). The difference in affinity arises almost wholly from a marked decrease in the dissociation rate constant for the full-length complex (8 x 10(-7) s(-1)) relative to the E3 rRNase-Im3 complex (1 x 10(-4) s(-1)), with their association rates comparable ( approximately 10(8) M(-1) s(-1)). Thermodynamic measurements show that complex formation is largely enthalpy driven. In light of the recently published crystal structure of the colicin E3-Im3 complex, the additional stabilization of the wild-type complex can be ascribed to the interaction of Im3 with the N-terminal translocation domain of the toxin. These observations suggest a mechanism whereby dissociation of the immunity protein prior to translocation into the target cell is facilitated by the loss of the Im3-translocation domain interaction.

Bacterial Proteins↗
PubMed2003

Thermodynamics of heme binding to the HasA(SM) hemophore: effect of mutations at three key residues for heme uptake.

HasA(SM) secreted by the Gram-negative bacterium Serratia marcescens belongs to the hemophore family. Its role is to take up heme from host heme carriers and to shuttle it to specific receptors. Heme is linked to the HasA(SM) protein by an unusual axial ligand pair: His32 and Tyr75. The nucleophilic nature of the tyrosine is enhanced by the hydrogen bonding of the tyrosinate to a neighboring histidine in the binding site: His83. We used isothermal titration microcalorimetry to examine the thermodynamics of heme binding to HasA(SM) and showed that binding is strongly exothermic and enthalpy driven: DeltaH = -105.4 kJ x mol(-1) and TDeltaS = -44.3 kJ x mol(-1). We used displacement experiments to determine the affinity constant of HasA(SM) for heme (K(a) = 5.3 x 10(10) M(-1)). This is the first time that this has been reported for a hemophore. We also analyzed the thermodynamics of the interaction between heme and a panel of single, double, and triple mutants of the two axial ligands His32 and Tyr75 and of His83 to assess the implication of each of these three residues in heme binding. We demonstrated that, in contrast to His32, His83 is essential for the binding of heme to HasA(SM), even though it is not directly coordinated to iron, and that the Tyr75/His83 pair plays a key role in the interaction.

Amino Acid Substitution↗