The reversible two-state unfolding of a monocot mannose-binding lectin from garlic bulbs reveals the dominant role of the dimeric interface in its stabilization

Allium sativum agglutinin (ASAI) is a heterodimeric mannose-specific bulb lectin possessing two polypeptide chains of molecular mass 11.5 and 12.5 kDa. The thermal unfolding of ASAI, characterized by differential scanning calorimetry and circular dichroism, shows it to be highly reversible and can be defined as a two-state process in which the folded dimer is converted directly to the unfolded monomers (A2 if 2U). Its conformational stability has been determined as a function of temperature, GdnCl concentration, and pH using a combination of thermal and isothermal GdnCl-induced unfolding monitored by DSC, far-UV CD, and fluorescence, respectively. Analyses of these data yielded the heat capacity change upon unfolding (DeltaC(p) and also the temperature dependence of the thermodynamic parameters, namely, DeltaG, DeltaH, and DeltaS. The fit of the stability curve to the modified Gibbs-Helmholtz equation provides an estimate of the thermodynamic parameters DeltaH(g), DeltaS(g), and DeltaC(p) as 174.1 kcal x mol(-1), 0.512 kcal x mol(-1) x K(-1), and 3.41 kcal x mol(-1) x K(-1), respectively, at T(g) = 339.4 K. Also, the free energy of unfolding, DeltaG(s), at its temperature of maximum stability (T(s) = 293 K) is 13.13 kcal x mol(-1). Unlike most oligomeric proteins studied so far, the lectin shows excellent agreement between the experimentally determined DeltaC(p) (3.2 +/- 0.28 kcal x mol(-1) x K(-1)) and those evaluated from a calculation of its accessible surface area. This in turn suggests that the protein attains a completely unfolded state irrespective of the method of denaturation. The absence of any folding intermediates suggests the quaternary interactions to be the major contributor to the conformational stability of the protein, which correlates well with its X-ray structure. The small DeltaC(p) for the unfolding of ASAI reflects a relatively small, buried hydrophobic core in the folded dimeric protein.     

Denaturant mediated unfolding of both native and molten globule states of maltose binding protein are accompanied by large deltaCp's.

Maltose binding protein (MBP) is a large, monomeric two domain protein containing 370 amino acids. In the absence of denaturant at neutral pH, the protein is in the native state, while at pH 3.0 it forms a molten globule. The molten globule lacks a tertiary circular dichroism signal but has secondary structure similar to that of the native state. The molten globule binds 8-anilino-1-naphthalene sulfonate (ANS). The unfolding thermodynamics of MBP at both pHs were measured by carrying out a series of isothermal urea melts at temperatures ranging from 274-329 K. At 298 K, values of deltaGdegrees , deltaCp, and Cm were 3.1+/-0.2 kcal mol(-1), 5.9+/-0.8 kcal mol(-1) K(-1) (15.9 cal (mol-residue)(-1) K(-1)), and 0.8 M, respectively, at pH 3.0 and 14.5+/-0.4 kcal mol(-1), 8.3+/-0.7 kcal mol(-1) K(-1) (22.4 kcal (mol-residue)(-1) K(-1)), and 3.3 M, respectively, at pH 7.1. Guanidine hydrochloride denaturation at pH 7.1 gave values of deltaGdegrees and deltaCp similar to those obtained with urea. The m values for denaturation are strongly temperature dependent, in contrast to what has been previously observed for small globular proteins. The value of deltaCp per mol-residue for the molten globule is comparable to corresponding values of deltaCp for the unfolding of typical globular proteins and suggests that it is a highly ordered structure, unlike molten globules of many small proteins. The value of deltaCp per mol-residue for the unfolding of the native state is among the highest currently known for any protein.     

Binding of urate and caffeine to hemocyanin of the lobster Homarus vulgaris (E.) as studied by isothermal titration calorimetry

Hemocyanin serves as an oxygen carrier in the hemolymph of the European lobster Homarus vulgaris. The oxygen binding behavior of the pigment is modulated by metabolic effectors such as lactate and urate. Urate and caffeine binding to 12-meric hemocyanin (H. vulgaris) was studied using isothermal titration calorimetry (ITC). Binding isotherms were determined for fully oxygenated hemocyanin between pH 7.55 and 8.15. No pH dependence of the binding parameters could be found for either effector. Since the magnitude of the Bohr effect depends on the urate concentration, the absence of any pH dependence of urate and caffeine binding to oxygenated hemocyanin suggests two conformations of the pigment under deoxygenated conditions. Urate binds to two identical binding sites (n = 2) each with a microscopic binding constant K of 8500 M(-1) and an enthalpy change DeltaH degrees of -32.3 kcal mol(-1). Caffeine binds cooperatively to hemocyanin with two microscopic binding constants: K(1) = 14 100 M(-1) and K(2) = 40 400 M(-1). The corresponding enthalpy changes in binding are as follows: DeltaH degrees (1) = -23.3 kcal mol(-1) and DeltaH degrees (2) = -27.1 kcal mol(-1). The comparison of urate and caffeine binding to the oxygenated pigment indicates the existence of two protein conformations for oxygen-saturated hemocyanin. Since effector binding is not influenced by protons, four different conformations are required to create a convincing explanation for caffeine and urate binding curves. This was predicted earlier on the basis of the analysis of oxygen binding to lobster hemocyanin, employing the nesting model.     

Unfolding thermodynamics of the tetrameric chaperone, SecB

SecB is a cytosolic tetrameric chaperone in Escherichia coli, which maintains polypeptides, destined for export in a translocation competent state. The thermodynamics of unfolding of SecB was studied as a function of protein concentration, by using high sensitivity-differential scanning calorimetry and spectroscopic methods. The thermal unfolding of tetrameric SecB is reversible and can be well described as a two-state transition in which the folded tetramer is converted directly to unfolded monomers. Increasing the pH decreases the stability of the tetramer significantly, the T(m) changing from 341.3 K at pH 6.5 to 332.6 K at pH 9.5. The value of DeltaC(p) obtained from measurements of DeltaH(m) as a function of T(m) was 10.7 +/- 0.7 kcal mol(-1) K(-1). The value of DeltaC(p) is among the highest measured for a multimeric protein. At 298 K, pH 7.4, the DeltaG degrees (u) for the SecB tetramer is 27.9 +/- 2 kcal mol(-1). Denaturant-mediated unfolding of SecB was found to be irreversible. The reactivity of the four solvent-exposed free thiols in tetrameric SecB is salt dependent. The kinetics of reactivity suggests that these four cysteines are in close proximity to each other and that these residues on each monomer are in chemically identical environments. The thermodynamic data suggest that SecB is a stable, well-folded, and tightly packed tetramer and that substrate binding occurs at a surface site rather than at an interior cavity.     

The conformational stability of the Streptomyces coelicolor histidine-phosphocarrier protein. Characterization of cold denaturation and urea-protein interactions.

Thermodynamic parameters describing the conformational stability of the histidine-containing phosphocarrier protein from Streptomyces coelicolor, scHPr, have been determined by steady-state fluorescence measurements of isothermal urea-denaturations, differential scanning calorimetry at different guanidinium hydrochloride concentrations and, independently, by far-UV circular dichroism measurements of isothermal urea-denaturations, and thermal denaturations at fixed urea concentrations. The equilibrium unfolding transitions are described adequately by the two-state model and they validate the linear free-energy extrapolation model, over the large temperature range explored, and the urea concentrations used. At moderate urea concentrations (from 2 to 3 m), scHPr undergoes both high- and low-temperature unfolding. The free-energy stability curves have been obtained for the whole temperature range and values of the thermodynamic parameters governing the heat- and cold-denaturation processes have been obtained. Cold-denaturation of the protein is the result of the combination of an unusually high heat capacity change (1.4 +/- 0.3 kcal.mol(-1).K(-1), at 0 m urea, being the average of the fluorescence, circular dichroism and differential scanning calorimetry measurements) and a fairly low enthalpy change upon unfolding at the midpoint temperature of heat-denaturation (59 +/- 4 kcal.mol(-1), the average of the fluorescence, circular dichroism and differential scanning calorimetry measurements). The changes in enthalpy (m(DeltaH(i) )), entropy (m(DeltaS(i) )) and heat capacity (m(DeltaC(pi) )), which occur upon preferential urea binding to the unfolded state vs. the folded state of the protein, have also been determined. The m(DeltaH(i) ) and the m(DeltaS(i) ) are negative at low temperatures, but as the temperature is increased, m(DeltaH(i) ) makes a less favourable contribution than m(DeltaS(i) ) to the change in free energy upon urea binding. The m(DeltaC(pi) ) is larger than those observed for other proteins; however, its contribution to the global heat capacity change upon unfolding is small.     

Thermodynamic characterization of the human acidic fibroblast growth factor: evidence for cold denaturation.

The thermodynamic parameters characterizing the conformational stability of the human acidic fibroblast growth factor (hFGF-1) have been determined by isothermal urea denaturation and thermal denaturation at fixed concentrations of urea using fluorescence and far-UV CD circular dichroism (CD) spectroscopy. The equilibrium unfolding transitions at pH 7.0 are adequately described by a two-state (native <--> unfolded state) mechanism. The stability of the protein is pH-dependent, and the protein unfolds completely below pH 3.0 (at 25 degrees C). hFGF-1 is shown to undergo a two-state transition only in a narrow pH range (pH 7.0-8.0). Under acidic (pH <6.0) and basic (pH >8.0) conditions, hFGF-1 is found to unfold noncooperatively, involving the accumulation of intermediates. The average temperature of maximum stability is determined to be 295.2 K. The heat capacity change (DeltaC(p)()) for the unfolding of hFGF-1 is estimated to be 2.1 +/- 0.5 kcal.mol(-1).K(-1). Temperature denaturation experiments in the absence and presence of urea show that hFGF-1 has a tendency to undergo cold denaturation. Two-dimensional (1)H-(15)N HSQC spectra of hFGF-1 acquired at subzero temperatures clearly show that hFGF-1 unfolds under low-temperature conditions. The significance of the noncooperative unfolding under acidic conditions and the cold denaturation process observed in hFGF-1 are discussed in detail.     

Thermodynamic analysis of the structural stability of phage 434 Cro protein

Thermodynamic parameters describing the phage 434 Cro protein have been determined by calorimetry and, independently, by far-UV circular dichroism (CD) measurements of isothermal urea denaturations and thermal denaturations at fixed urea concentrations. These equilibrium unfolding transitions are adequately described by the two-state model. The far-UV CD denaturation data yield average temperature-independent values of 0.99 +/- 0.10 kcal mol(-)(1) M(-)(1) for m and 0.98 +/- 0.05 kcal mol(-)(1) K(-)(1) for DeltaC(p)()(,U), the heat capacity change accompanying unfolding. Calorimetric data yield a temperature-independent DeltaC(p)()(,U) of 0.95 +/- 0.30 kcal mol(-)(1) K(-)(1) or a temperature-dependent value of 1.00 +/- 0.10 kcal mol(-)(1) K(-)(1) at 25 degrees C. DeltaC(p)()(,U) and m determined for 434 Cro are in accord with values predicted using known empirical correlations with structure. The free energy of unfolding is pH-dependent, and the protein is completely unfolded at pH 2.0 and 25 degrees C as judged by calorimetry or CD. The stability of 434 Cro is lower than those observed for the structurally similar N-terminal domain of the repressor of phage 434 (R1-69) or of phage lambda (lambda(6)(-)(85)), but is close to the value reported for the putative monomeric lambda Cro. Since a protein's structural stability is important in determining its intracellular stability and turnover, the stability of Cro relative to the repressor could be a key component of the regulatory circuit controlling the levels and, consequently, the functions of the two proteins in vivo.     

Conformational stability and mechanism of folding of ribonuclease T1

Urea and thermal unfolding curves for ribonuclease T1 (RNase T1) were determined by measuring several different physical properties. In all cases, steep, single-step unfolding curves were observed. When these results were analyzed by assuming a two-state folding mechanism, the plots of fraction unfolded protein versus denaturant were coincident. The dependence of the free energy of unfolding, delta G (in kcal/mol), on urea concentration is given by delta G = 5.6 - 1.21 (urea). The parameters characterizing the thermodynamics of unfolding are: midpoint of the thermal unfolding curve, Tm = 48.1 degrees C, enthalpy change at Tm, delta Hm = 97 kcal/mol, and heat capacity change, delta Cp = 1650 cal/mol deg. A single kinetic phase was observed for both the folding and unfolding of RNase T1 in the transition and post-transition regions. However, two slow kinetic phases were observed during folding in the pre-transition region. These two slow phases account for about 90% of the observed amplitude, indicating that a faster kinetic phase is also present. The slow phases probably result from cis-trans isomerization at the 2 proline residues that have a cis configuration in folded RNase T1. These results suggest that RNase T1 folds by a highly cooperative mechanism with no structural intermediates once the proline residues have assumed their correct isomeric configuration. At 25 degrees C, the folded conformation is more stable than the unfolded conformations by 5.6 kcal/mol at pH 7 and by 8.9 kcal/mol at pH 5, which is the pH of maximum stability. At pH 7, the thermodynamic data indicate that the maximum conformational stability of 8.3 kcal/mol will occur at -6 degrees C.     

Energetic basis of molecular recognition in a DNA aptamer

The thermal stability and ligand binding properties of the L-argininamide-binding DNA aptamer (5'-GATCGAAACGTAGCGCCTTCGATC-3') were studied by spectroscopic and calorimetric methods. Differential calorimetric studies showed that the uncomplexed aptamer melted in a two-state reaction with a melting temperature T(m)=50.2+/-0.2 degrees C and a folding enthalpy DeltaH(0)(fold)=-49.0+/-2.1 kcal mol(-1). These values agree with values of T(m)=49.6 degrees C and DeltaH(0)(fold)=-51.2 kcal mol(-1) predicted for a simple hairpin structure. Melting of the uncomplexed aptamer was dependent upon salt concentration, but independent of strand concentration. The T(m) of aptamer melting was found to increase as L-argininamide concentrations increased. Analysis of circular dichroism titration data using a single-site binding model resulted in the determination of a binding free energy DeltaG(0)(bind)=-5.1 kcal mol(-1). Isothermal titration calorimetry studies revealed an exothermic binding reaction with DeltaH(0)(bind)=-8.7 kcal mol(-1). Combination of enthalpy and free energy produce an unfavorable entropy of -TDeltaS(0)=+3.6 kcal mol(-1). A molar heat capacity change of -116 cal mol(-1) K(-1) was determined from calorimetric measurements at four temperatures over the range of 15-40 degrees C. Molecular dynamics simulations were used to explore the structures of the unligated and ligated aptamer structures. From the calculated changes in solvent accessible surface areas of these structures a molar heat capacity change of -125 cal mol(-1) K(-1) was calculated, a value in excellent agreement with the experimental value. The thermodynamic signature, along with the coupled CD spectral changes, suggest that the binding of L-argininamide to its DNA aptamer is an induced-fit process in which the binding of the ligand is thermodynamically coupled to a conformational ordering of the nucleic acid.     

pH dependence thermal stability of a chymotrypsin inhibitor from Schizolobium parahyba seeds

The thermal stability of a Schizolobium parahyba chymotrypsin inhibitor (SPCI) as a function of pH has been investigated using fluorescence, circular dichroism, and differential scanning calorimetry (DSC). The thermodynamic parameters derived from all methods are remarkably similar and strongly suggest the high stability of SPCI under a wide range of pH. The transition temperature (T(m)) values ranging from 57 to 85.3 degrees C at acidic, neutral, and alkaline pH are in good agreement with proteins from mesophilic and thermophilic organisms and corroborate previous data regarding the thermal stability of SPCI. All methods gave transitions curves adequately fitted to a two-state model of the unfolding process as judged by the cooperative ratio between the van't Hoff and the calorimetric enthalpy energies close to unity in all of the pH conditions analyzed, except at pH 3.0. Thermodynamic analysis using all these methods reveals that SPCI is thermally a highly stable protein, over the wide range of pH from 3.0 to 8.8, exhibiting high stability in the pH region of 5.0-7.0. The corresponding maximum stabilities, DeltaG(25), were obtained at pH 7.0 with values of 15.4 kcal mol(-1) (combined fluorescence and circular dichroism data), and 15.1 kcal mol(-1) (DSC), considering a DeltaC(p) of 1.72 +/- 0.24 kcal mol(-1) K(-1). The low histidine content ( approximately 1.7%) and the high acidic residue content ( approximately 22.5%) suggests a flat pH dependence of thermal stability in the region 2.0-8.8 and that the decrease in thermal stability at low pH can be due to the differences in pK values of the acidic groups.     

The stability, structural organization, and denaturation of pectate lyase C, a parallel beta-helix protein

Pectate lyase C (pelC) was the first protein in which the parallel beta-helix structure was recognized. The unique features of parallel beta-helix-containing proteins-a relatively simple topology and unusual interactions among side chains-make pelC an interesting protein to study with respect to protein folding. In this paper, we report studies of the unfolding equilibrium of pelC. PelC is unfolded reversibly by gdn-HCl at pH 7 and 5, as monitored by far- and near-UV CD and fluorescence. The coincidence of these spectroscopically detected transitions is consistent with a two-state transition at pH 7, but the three probes are not coincident at pH 5. No evidence was found for a loosely folded intermediate in the transition region at pH 5. At pH 7, the for unfolding is 12.2 kcal/mol, with the midpoint of the transition at 0.99 M gdn-HCl and m = 12.3 kcal/(mol.M). Thus, pelC is unusually stable and has an m value that is much larger than for typical globular proteins. Thermal denaturation of pelC has been studied by differential scanning calorimetry (DSC) and by CD. Although thermal denaturation is not reversible, valid thermodynamic data can be obtained for the unfolding transition. DeltaH(van't Hoff)/DeltaH(cal) is less than 1 for pHs between 5 and 8, with a maximum value of 0.91 at pH 7 decreasing to 0.85 at pH 8 and to 0.68 at pH 5. At all pHs studied, the excess heat capacity can be deconvoluted into two components corresponding to two-state transitions that are nearly coincident at pH 7, but deviate more at higher and lower pH. Thus, pelC appears to consist of two domains that interact strongly and unfold in a cooperative fashion at pH 7, but the cooperativity decreases at higher and lower pH. The crystal structure of pelC shows no obvious domain structure, however.     

Site-bound water and the shortcomings of a less than perfect transition state analogue

Kinetic measurements have shown that substantial enthalpy changes accompany substrate binding by cytidine deaminase, increasing markedly as the reaction proceeds from the ground state (1/K(m), DeltaH = -13 kcal/mol) to the transition state (1/K(tx), DeltaH = -20 kcal/mol) [Snider, M. J., et al. (2000) Biochemistry 39, 9746-9753]. In the present work, we determined the thermodynamic changes associated with the equilibrium binding of inhibitors by cytidine deaminase by isothermal titration calorimetry and van't Hoff analysis of the temperature dependence of their inhibition constants. The results indicate that the binding of the transition state analogue 3,4-dihydrouridine DeltaH = -21 kcal/mol), like that of the transition state itself (DeltaH = -20 kcal/mol), is associated with a large favorable change in enthalpy. The significantly smaller enthalpy change that accompanies the binding of 3,4-dihydrozebularine (DeltaH = -10 kcal/mol), an analogue of 3,4-dihydrouridine in which a hydrogen atom replaces this inhibitor's 4-OH group, is consistent with the view that polar interactions with the substrate at the site of its chemical transformation play a critical role in reducing the enthalpy of activation for substrate hydrolysis. The entropic shortcomings of 3,4-dihydrouridine, in capturing all of the free energy involved in binding the actual transition state, may arise from its inability to displace a water molecule that occupies the binding site normally occupied by product ammonia.     

Interaction of a designed interleukin-10 epitope mimic with an antibody studied by isothermal titration microcalorimetry

The mechanism of recognition of proteins and peptides by antibodies and the factors determining binding affinity and specificity are mediated by essentially the same features. However, additional effects of the usually unfolded and flexible solution structure of peptide ligands have to be considered. In an earlier study we designed and optimized six peptides (pepI to pepVI) mimicking the discontinuous binding site of interleukin-10 for the anti-interleukin-10 monoclonal antibody (mab) CB/RS/1. Three of them were selected for analysis of their solution conformation by circular dichroism measurements. The peptides differ in the content of alpha-helices and in the inducibility of helical secondary structures by trifluoroethanol. These properties, however, do not correlate with the binding affinity. PepVI, a 32-mer cyclic epitope mimic, has the highest affinity to mab CB/RS/1 identified to date. CD difference spectroscopy suggests an increase of the alpha-helix content of pepVI with complex formation. Binding of pepVI to mab CB/RS/1 is characterized by a large negative, favorable binding enthalpy and a smaller unfavorable loss of entropy (DeltaH degrees = -16.4 kcal x mol(-1), TDeltaS degrees = -6.9 kcal x mol(-1)) resulting in DeltaG degrees = -9.5 kcal x mol(-1) at 25 degrees C as determined by isothermal titration calorimetry. Binding of pepVI is enthalpically driven over the entire temperature range studied (10-35 degrees C). Complex formation is not accompanied by proton uptake or release. A negative heat capacity change DeltaC(p) of -0.354 kcal x mol(-1) x K(-1) was determined from the temperature dependence of DeltaH degrees. The selection of protein mimics with the observed thermodynamic properties is promoted by the applied identification and iterative optimization procedure.     

Acid-induced changes in thermal stability and fusion activity of influenza hemagglutinin

The conformational and thermal stability of full-length hemagglutinin (HA) of influenza virus (strain X31) has been investigated using a combination of differential scanning calorimetry (DSC), analytical ultracentrifugation, fluorescence, and circular dichroism (CD) spectroscopy as a function of pH. HA sediments as a rosette comprised of 5-6 trimers (31-35 S) over the pH range of 7.4-5.4. The DSC profile of HA in the native state at pH 7.4 is characterized by a single cooperative endotherm with a transition temperature (Tm) of 66 degrees C and unfolding enthalpy (DeltaH(cal)) of 800 kcal x (mol of trimer)(-1). Upon acidification to pH 5.4, there is a significant decrease in the transition temperature (from 66 to 45 degrees C), unfolding enthalpy [from 800 to 260 kcal x (mol of trimer)(-1)], and DeltaH(cal)/DeltaH(vH) ratio (from 3.0 to approximately 1.3). Whereas the far- and near-UV ellipticities are maintained over this pH range, there is an acid-induced increase in surface hydrophobicity and decrease in intrinsic tryptophanyl fluorescence. The major contribution to the DSC endotherm arises from unfolding HA1 domains. The relationship between acid-induced changes in thermal stability and the fusion activity of HA has been examined by evaluating the kinetics and extent of fusion of influenza virus with erythrocytes over the temperature and pH range of the DSC measurements. Surprisingly, X31 influenza virus retains its fusion activity at acidic pH and temperatures significantly below the unfolding transition of HA. This finding is consistent with the notion that the fusion activity of influenza virus may involve structural changes of only a small fraction of HA molecules.     

Equilibrium dissociation and unfolding of the dimeric human papillomavirus strain-16 E2 DNA-binding domain.

The equilibrium unfolding reaction of the C-terminal 80-amino-acid dimeric DNA-binding domain of human papillomavirus (HPV) strain 16 E2 protein has been investigated using fluorescence, far-UV CD, and equilibrium sedimentation. The stability of the HPV-16 E2 DNA-binding domain is concentration-dependent, and the unfolding reaction is well described as a two-state transition from folded dimer to unfolded monomer. The conformational stability of the protein, delta GH2O, was found to be 9.8 kcal/mol at pH 5.6, with the corresponding equilibrium unfolding/dissociation constant, Ku, being 6.5 x 10(-8) M. Equilibrium sedimentation experiments give a Kd of 3.0 x 10(-8) M, showing an excellent agreement between the two different techniques. Denaturation by temperature followed by the change in ellipticity also shows a concomitant disappearance of secondary and tertiary structures. The Ku changes dramatically at physiologically relevant pH's: with a change in pH from 6.1 to 7.0, it goes from 5.5 x 10(-8) M to 4.4 x 10(10) M. Our results suggest that, at the very low concentration of protein where DNA binding is normally measured (e.g., 10(-11) M), the protein is predominantly monomeric and unfolded. They also stress the importance of the coupling between folding and DNA binding.     

The contribution of heme propionate groups to the conformational dynamics associated with CO photodissociation from horse heart myoglobin

Photoacoustic calorimetry and transient absorption spectroscopy were used to study conformational dynamics associated with CO photodissociation from horse heart myoglobin (Mb) reconstituted with either Fe protoporphyrin IX dimethylester (FePPDME), Fe octaethylporphyrin (FeOEP), or with native Fe protoporphyrin IX (FePPIX). The volume and enthalpy changes associated with the Fe-CO bond dissociation and formation of a transient deoxyMb intermediate for the reconstituted Mbs were found to be similar to those determined for native Mb (DeltaV1 = -2.5+/-0.6 ml mol(-1) and DeltaH1 = 8.1+/-3.0 kcal mol(-1)). The replacement of FePPIX by FeOEP significantly alters the conformational dynamics associated with CO release from protein. Ligand escape from FeOEP reconstituted Mb was determined to be roughly a factor of two faster (tau=330 ns) relative to native protein (tau=700 ns) and accompanying reaction volume and enthalpy changes were also found to be smaller (DeltaV2 = 5.4+/-2.5 ml mol(-1) and DeltaH2 = 0.7+/-2.2 kcal mol(-1)) than those for native Mb (DeltaV2 = 14.3+/-0.8 ml mol(-1) and DeltaH2 = 7.8+/-3.5 kcal mol(-1)). On the other hand, volume and enthalpy changes for CO release from FePPIX or FePPDME reconstituted Mb were nearly identical to those of the native protein. These results suggest that the hydrogen bonding network between heme propionate groups and nearby amino acid residues likely play an important role in regulating ligand diffusion through protein matrix. Disruption of this network leads to a partially open conformation of protein with less restricted ligand access to the heme binding pocket.     

Energetics of a strongly pH dependent RNA tertiary structure in a frameshifting pseudoknot

Retroviruses employ -1 translational frameshifting to regulate the relative concentrations of structural and non-structural proteins critical to the viral life cycle. The 1.6 A crystal structure of the -1 frameshifting pseudoknot from beet western yellows virus reveals, in addition to Watson-Crick base-pairing, many loop-stem RNA tertiary structural interactions and a bound Na(+). Investigation of the thermodynamics of unfolding of the beet western yellows virus pseudoknot reveals strongly pH-dependent loop-stem tertiary structural interactions which stabilize the molecule, contributing a net of DeltaH approximately -30 kcal mol(-1) and DeltaG degrees (37) of -3.3 kcal mol(-1) to a total DeltaH and DeltaG degrees (37) of -121 and -16 kcal mol(-1), respectively, at pH 6.0, 0.5 M K(+) by DSC. Characterization of mutant RNAs supports the presence of a C8(+).G12-C26 loop 1-stem 2 base-triple (pK(a)=6.8), protonation of which contributes nearly -3.5 kcal mol(-1) in net stability in the presence of a wild-type loop 2. Substitution of the nucleotides in loop 2 with uridine bases, which would eliminate the minor groove triplex, destroys pseudoknot formation. An examination of the dependence of the monovalent ion and type on melting profiles suggests that tertiary structure unfolding occurs in a manner quantitatively consistent with previous studies on the stabilizing effects of K(+), NH(4)(+) and Na(+) on other simple duplex and pseudoknotted RNAs.     

Thermodynamics of ribonuclease T1 denaturation.

Differential scanning calorimetry has been used to investigate the thermodynamics of denaturation of ribonuclease T1 as a function of pH over the pH range 2-10, and as a function of NaCl and MgCl2 concentration. At pH 7 in 30 mM PIPES buffer, the thermodynamic parameters are as follows: melting temperature, T1/2 = 48.9 +/- 0.1 degrees C; enthalpy change, delta H = 95.5 +/- 0.9 kcal mol-1; heat capacity change, delta Cp = 1.59 kcal mol-1 K-1; free energy change at 25 degrees C, delta G degrees (25 degrees C) = 5.6 kcal mol-1. Both T1/2 = 56.5 degrees C and delta H = 106.1 kcal mol-1 are maximal near pH 5. The conformational stability of ribonuclease T1 is increased by 3.0 kcal/mol in the presence of 0.6 M NaCl or 0.3 M MgCl2. This stabilization results mainly from the preferential binding of cations to the folded conformation of the protein. The estimates of the conformational stability of ribonuclease T1 from differential scanning calorimetry are shown to be in remarkably good agreement with estimates derived from an analysis of urea denaturation curves.     

Unfolding of rabbit muscle creatine kinase induced by acid. A study using electrospray ionization mass spectrometry,isothermal titration calorimetry,and fluorescence spectroscopy

Electrospray ionization mass spectrometry, isothermal titration calorimetry (ITC), fluorescence spectroscopy, and glutaraldehyde cross-linking SDS-PAGE have been used to study the unfolding of rabbit muscle creatine kinase (MM-CK) induced by acid. The mass spectrometric experiments show that MM-CK is unfolded gradually when titrated with acid. MM-CK is a dimer (the native state) at pH 7.0 and becomes an equilibrium mixture of the dimer and a partially folded monomer (the intermediate) between pH 6.7 and 5.0. The dimeric protein becomes an equilibrium mixture of the intermediate and an unfolded monomer (the unfolded state) between pH 5.0 and 3.0 and is almost fully unfolded at pH 3.0 reached. The results from a "phase diagram" method of fluorescence show that the conformational transition between the native state and the intermediate of MM-CK occurs in the pH range of 7.0-5.2, and the transition between the intermediate and the unfolded state of the protein occurs between pH 5.2 and 3.0. The intrinsic molar enthalpy changes for formation of the unfolded state of MM-CK induced by acid at 15.0, 25.0, 30.0, and 37.0 degrees C have been determined by ITC. A large positive molar heat capacity change of the unfolding, 8.78 kcal mol-1 K-1, at all temperatures examined indicates that hydrophobic interaction is the dominant driving force stabilizing the native structure of MM-CK. Combining the results from these four methods, we conclude that the acid-induced unfolding of MM-CK follows a "three-state" model and that the intermediate state of the protein is a partially folded monomer.     

Thermodynamic analysis of binding between mouse major urinary protein-I and the pheromone 2-sec-butyl-4,5-dihydrothiazole

The mouse pheromone 2-sec-butyl-4,5-dihydrothiazole (SBT) binds to an occluded, nonpolar cavity in the mouse major urinary protein-I (MUP-I). The thermodynamics of this interaction have been characterized using isothermal titration calorimetry (ITC). MUP-I-SBT binding is accompanied by a large favorable enthalpy change (DeltaH = -11.2 kcal/mol at 25 degrees C), an unfavorable entropy change (-TDeltaS = 2.8 kcal/mol at 25 degrees C), and a negative heat capacity change [DeltaC(p)() = -165 cal/(mol K)]. Thermodynamic analysis of binding between MUP-I and several 2-alkyl-4,5-dihydrothiazole ligands indicated that the alkyl chain contributes more favorably to the enthalpy and less favorably to the entropy of binding than would be expected on the basis of the hydrophobic desolvation of short-chain alcohols. However, solvent transfer experiments indicated that desolvation of SBT is accompanied by a net unfavorable change in enthalpy (DeltaH = +1.0 kcal/mol) and favorable change in entropy (-TDeltaS = -1.8 kcal/mol). These results are discussed in terms of the possible physical origins of the binding thermodynamics, including (1) hydrophobic desolvation of both the protein and the ligand, (2) formation of a buried water-mediated hydrogen bond network between the protein and ligand, (3) formation of strong van der Waals interactions, and (4) changes in the structure, dynamics, and/or hydration of the protein upon binding.