Toward the physical basis of thermophilic proteins: linking of enriched polar interactions and reduced heat capacity of unfolding

The enrichment of salt bridges and hydrogen bonding in thermophilic proteins has long been recognized. Another tendency, featuring lower heat capacity of unfolding (DeltaC(p)) than found in mesophilic proteins, is emerging from the recent literature. Here we present a simple electrostatic model to illustrate that formation of a salt-bridge or hydrogen-bonding network around an ionized group in the folded state leads to increased folding stability and decreased DeltaC(p). We thus suggest that the reduced DeltaC(p) of thermophilic proteins could partly be attributed to enriched polar interactions. A reduced DeltaC(p) might serve as an indicator for the contribution of polar interactions to folding stability.     

A new method for determining the constant-pressure heat capacity change associated with the protein denaturation induced by guanidinium chloride (or urea)

Differential scanning calorimetry (DSC) provides authentic and accurate value of DeltaC(p)(X), the constant-pressure heat capacity change associated with the N (native state)<-->X (heat denatured state), the heat-induced denaturation equilibrium of the protein in the absence of a chemical denaturant. If X retains native-like buried hydrophobic interaction, DeltaC(p)(X) must be less than DeltaC(p)(D), the constant-pressure heat capacity change associated with the transition, N<-->D, where the state D is not only more unfolded than X but it also has its all groups exposed to water. One problem is that for most proteins D is observed only in the presence of chemical denaturants such as guanidinium chloride (GdmCl) and urea. Another problem is that DSC cannot yield authentic DeltaC(p)(D), for its measurement invokes the existence of putative specific binding sites for the chemical denaturants on N and D. We have developed a non-calorimetric method for the measurements of DeltaC(p)(D), which uses thermodynamic data obtained from the isothermal GdmCl (or urea)-induced denaturation and heat-induced denaturation in the presence of the chemical denaturant concentration at which significant concentrations of both N and D exist. We show that for each of the proteins (ribonuclease-A, lysozyme, alpha-lactalbumin and chymotrypsinogen) DeltaC(p)(D) is significantly higher than DeltaC(p)(X). DeltaC(p)(D) of the protein is also compared with that estimated using the known heat capacities of amino acid residues and their fractional area exposed on denaturation.     

A new method for the determination of stability parameters of proteins from their heat-induced denaturation curves

A new method has been developed for determining the stability parameters of proteins from their heat-induced transition curves followed by observation of changes in the far-UV circular dichroism (CD). This method of analysis of the thermal denaturation curve of a protein gave values of stability parameters that not only are identical to those measured by the differential scanning calorimetry (DSC), but also are measured with the same error as that observed with a calorimeter. This conclusion has been reached from our studies of the reversible heat-induced denaturation of lysozyme and ribonuclease A at various pH values. For each protein, the conventional method of analysis of the conformational transition curve, which assumes a linear temperature dependence of the pre- and posttransition baselines, gave the estimate of DeltaH(van)(m) (enthalpy change on denaturation at T(m), the midpoint of denaturation) which is significantly lower than DeltaH(cal)(m), the value obtained from DSC measurements. However, if the analysis of the same denaturation curve assumes that a parabolic function describes the temperature dependence of the pre- and posttransition baselines, there exists an excellent agreement between DeltaH(van)(m) and DeltaH(cal)(m) of the protein. The latter analysis is supported by the far-UV CD measurements of the oxidized ribonuclease A as a function of temperature, for the temperature dependence of this optical property of the protein is indeed nonlinear. Furthermore, it has been observed that, for each protein, the constant-pressure heat capacity change (DeltaC(p)) determined from the plots of DeltaH(van)(m) versus T(m) is independent of the method of analysis of the transition curve.     

Conformational detours during folding of a collapsed state

The protein S6 is a useful model to probe the role of partially folded states in the folding process. In the absence of salt, S6 folds from the denatured state D to the native state N without detectable intermediates. High concentrations of sodium sulfate induce the accumulation of a collapsed state C, which is off the direct folding route. However, the mutation VA85 enables S6 to fold from C directly to N through the transition state TS(C). According to the denaturant dependence of this reaction, TS(C) and C are equally compact, but the data are difficult to deconvolute. Therefore, I have measured the heat capacities (DeltaC(p)) for the D-->C and C-->TS(C) transitions. The DeltaC(p)-values suggest that C needs to increase its surface area in order to fold directly to N. This underlines that it is a misfolded state that can only fold by at least partial unfolding. In contrast to the C-state formed by S6 wildtype, the VA85 C-state is just as compact as the native state, and this may be a prerequisite for direct folding. Individual "gatekeeper" residues may thus play a disproportionately large role in guiding proteins through different folding pathways.     

Heat capacity and thermodynamic characteristics of denaturation and glass transition of hydrated and anhydrous proteins

Calorimetric measurements of absolute heat capacity have been performed for hydrated (11)S-globulin (0 < C(H(2)O) < 25%) and for lysozyme in a concentrated solution, both in the native and denatured states. The denaturation process is observed in hydrated and completely anhydrous proteins; it is accompanied by the appearance of heat capacity increment (Delta(N)(D)C(p)), as is the case for protein solutions. It has been shown that, depending on the temperature and water content, the hydrated denatured proteins can be in a highly elastic or glassy states. Glass transition is also observed in hydrated native proteins. It is found that the denaturation increment Delta(N)(D)C(p) in native protein, like the increment DeltaC(p) in denatured protein in glass transition at low water contents, is due to additional degrees of freedom of thermal motion in the protein globule. In contrast to the conventional notion, comparison of absolute C(p) values for hydrated denatured proteins with the C(p) values for denatured proteins in solution has indicated a dominant contribution of the globule thermal motion to the denaturation increment of protein heat capacity in solutions. The concentration dependence of denaturing heat absorption (temperature at its maximum, T(D), and thermal effect, DeltaQ(D)) and that of glass transition temperature, T(g), for (11)S-globulin have been studied in a wide range of water contents. General polymeric and specific protein features of these dependencies are discussed.     

Heat capacity effects in protein folding and ligand binding: a re-evaluation of the role of water in biomolecular thermodynamics

Large "anomalous" heat capacity (DeltaC(p)) effects are a common feature of the thermodynamics of biomolecular interactions in aqueous solution and, as a result of the improved facility for direct calorimetric measurements, there is a growing body of experimental data for such effects in protein folding, protein-protein and protein-ligand interactions. Conventionally such heat capacity effects have been ascribed to hydrophobic interactions, and there are some remarkably convincing demonstrations of the usefulness of this concept. Nonetheless, there is also increasing evidence that hydrophobic interactions are not the only possible source of such effects. Here we re-evaluate the possible contributions of other interactions to the heat capacity changes to be expected for cooperative biomolecular folding and binding processes, with particular reference to the role of hydrogen bonding and solvent water interactions. Simple models based on the hydrogen-bonding propensity of water as a function of temperature give quantitative estimates of DeltaC(p) that compare well with experimental observations for both protein folding and ligand binding. The thermodynamic contribution of bound waters in protein complexes is also estimated. The prediction from simple lattice models is that trapping of water in a complex should give more exothermic binding (DeltaDeltaH-6 to -12 kJ mol(-1)) with lower entropy (DeltaDeltaS(0) approximately -11 J mol(-1) K(-1)) and more negative DeltaC(p) (by about -75 J mol(-1) K(-1)) per water molecule. More generally, it is clear that significant DeltaC(p) effects are to be expected for any macromolecular process involving a multiplicity of cooperative weak interactions of whatever kind.     

Temperature range of thermodynamic stability for the native state of reversible two-state proteins

The difference between the heat (T(G)) and the cold (T(G)') denaturation temperatures defines the temperature range (T(Range)) over which the native state of a reversible two-state protein is thermodynamically stable. We have performed a correlation analysis for thermodynamic parameters in a selected data set of structurally nonhomologous single-domain reversible two-state proteins. We find that the temperature range is negatively correlated with the protein size and with the heat capacity change (DeltaC(p)) but is positively correlated with the maximal protein stability [DeltaG(T(S))]. The correlation between the temperature range and maximal protein stability becomes highly significant upon normalization of the maximal protein stability with protein size. The melting temperature (T(G)) also shows a negative correlation with protein size. Consistently, T(G) and T(G)' show opposite correlations with DeltaC(p), indicating a dependence of the T(Range) on the curvature of the protein stability curve. Substitution of proteins in our data set with their homologues and arbitrary addition or removal of a protein in the data set do not affect the outcome of our analysis. Simulations of the thermodynamic data further indicate that T(Range) is more sensitive to variations in curvature than to the slope of the protein stability curve. The hydrophobic effect in single domains is the principal reason for these observations. Our results imply that larger proteins may be stable over narrower temperature ranges and that smaller proteins may have higher melting temperatures, suggesting why protein structures often differentiate into multiple substructures with different hydrophobic cores. Our results have interesting implications for protein thermostability.     

Heat capacity and conformation of proteins in the denatured state

Heat capacity, intrinsic viscosity and ellipticity of a number of globular proteins (pancreatic ribonuclease A, staphylococcal nuclease, hen egg-white lysozyme, myoglobin and cytochrome c) and a fibrillar protein (collagen) in various states (native, denatured, with and without disulfide crosslinks or a heme) have been studied experimentally over a broad range of temperatures. It is shown that the partial heat capacity of denatured protein significantly exceeds the heat capacity of native protein, especially in the case of globular proteins, and is close to the value calculated for an extended polypeptide chain from the known heat capacities of individual amino acid residues. The significant residual structure that appears at room temperature in the denatured states of some globular proteins (e.g. myoglobin and lysozyme) at neutral pH results in a slight decrease of the heat capacity, probably due to partial screening of the protein non-polar groups from water. The heat capacity of the unfolded state increases asymptotically, approaching a constant value at about 100 degrees C. The temperature dependence of the heat capacity of the native state, which can be determined over a much shorter range of temperature than that of the denatured state and, correspondingly, is less certain, appears to be linear up to 80 degrees C. Therefore, the denaturational heat capacity increment seems to be temperature-dependent and is likely to decrease to zero at about 140 degrees C.     

Compatibility of osmolytes with Gibbs energy of stabilization of proteins

This study led to the conclusion that naturally occurring osmolytes which are known to protect proteins against denaturing stresses, do not perturb the Gibbs energy of stabilization of proteins at 25 degrees C (DeltaG(D) degrees ) which has been shown to control the in vivo rate of degradative protein turnover (Pace et al., Acta Biol. Med. Germ 40 (1981) 1385-1392). This conclusion has been reached from our studies of heat-induced denaturation of lysozyme, ribonuclease A, cytochrome c and myoglobin in the presence of different concentrations of osmolytes, namely, glycine, proline, sarcosine and glycine-betaine. At a fixed concentration of osmolyte a heat-induced denaturation curve measured by following changes in the molar absorption coefficient of the protein, was analyzed for T(m), the midpoint of the denaturation and DeltaH(m), the enthalpy change of denaturation at T(m). Values of DeltaG(D) degrees were determined with Gibbs-Helmoltz equation using known values of T(m), DeltaH(m) and DeltaC(p), the constant-pressure heat capacity change. It has been observed that T(m) increases with the osmolyte concentration, whereas DeltaG(D) degrees remains unaffected in the presence of the osmolyte. This observation on DeltaG(D) degrees in the presence of osmolytes has been considered in the physiological context.     

Thermodynamics of RNA duplexes with tandem mismatches containing a uracil-uracil pair flanked by C.G/G.C or G.C/A.U closing base pairs

The thermodynamics governing the denaturation of RNA duplexes containing 8 bp and a central tandem mismatch or 10 bp were evaluated using UV absorbance melting curves. Each of the eight tandem mismatches that were examined had one U-U pair adjacent to another noncanonical base pair. They were examined in two different RNA duplex environments, one with the tandem mismatch closed by G.C base pairs and the other with G.C and A.U closing base pairs. The free energy increments (Delta Gdegrees(loop)) of the 2 x 2 loops were positive, and showed relatively small differences between the two closing base pair environments. Assuming temperature-independent enthalpy changes for the transitions, (Delta Gdegrees(loop)) for the 2 x 2 loops varied from 0.9 to 1.9 kcal/mol in 1 M Na(+) at 37 degrees C. Most values were within 0.8 kcal/mol of previously estimated values; however, a few sequences differed by 1.2-2.0 kcal/mol. Single strands employed to form the RNA duplexes exhibited small noncooperative absorbance increases with temperature or transitions indicative of partial self-complementary duplexes. One strand formed a partial self-complementary duplex that was more stable than the tandem mismatch duplexes it formed. Transitions of the RNA duplexes were analyzed using equations that included the coupled equilibrium of self-complementary duplex and non-self-complementary duplex denaturation. The average heat capacity change (DeltaC(p)) associated with the transitions of two RNA duplexes was estimated by plotting DeltaH degrees and DeltaS degrees evaluated at different strand concentrations as a function of T(m) and ln T(m), respectively. The average DeltaC(p) was 70 +/- 5 cal K(-)(1) (mol of base pairs)(-)(1). Consideration of this heat capacity change reduced the free energy of formation at 37 degrees C of the 10 bp control RNA duplexes by 0.3-0.6 kcal/mol, which may increase Delta Gdegrees(loop) values by similar amounts.     

Heat capacity of proteins. II. Partial molar heat capacity of the unfolded polypeptide chain of proteins: protein unfolding effects

Using the heat capacity values for amino acid side-chains and the peptide unit determined in the accompanying paper, we calculated the partial heat capacities of the unfolded state for four proteins (apomyoglobin, apocytochrome c, ribonuclease A, lysozyme) in aqueous solution in the temperature range from 5 to 125 degrees C, with an assumption that the constituent amino acid residues contribute additively to the integral heat capacity of a polypeptide chain. These ideal heat capacity functions of the extended polypeptide chains were compared with the calorimetrically determined heat capacity functions of the heat and acid-denatured proteins. The average deviation of the experimental functions from the calculated ideal ones in the whole studied temperature range does not exceed the experimental error (5%). Therefore, the heat-denatured state of a protein, in solutions with acidic pH preventing aggregation, approximates well the completely unfolded state of this macromolecule. The heat capacity change caused by hydration of amino acid residues upon protein unfolding was also determined and it was shown that this is the major contributor to the observed heat capacity effect of unfolding. Its value is different for different proteins and correlates well with the surface area of non-polar groups exposed upon unfolding. The heat capacity effect due to the configurational freedom gain by the polypeptide chain was found to contribute only a small part of the overall heat capacity change on unfolding.     

Heat capacity-independent determination of differential free energy of stability between structurally homologous proteins

Under the assumption of equivalent heat capacity values, the differential free energy of stability for a pair of proteins midway between their thermal unfolding transition temperatures is shown to be independent of DeltaC(p) up to its cubic term in DeltaT(m). For model calculations reflecting the nearly 30 degrees C difference in T(m) for the adenylate kinases from the arctic bacterium Bacillus globisporus and the thermophilic bacterium Geobacillus stearothermophilus, the resultant error in estimating DeltaDeltaG by the formula 0.5 [DeltaS(T(m1))(1)+DeltaS(T(m2)) (2)] DeltaT(m) is less than 1%. Combined with the analogous thermal unfolding data for the adenylate kinase from Escherichia coli, these three homologous proteins exhibit T(m) and DeltaS(T(m)) values consistent with differential entropy and enthalpy contributions of equal magnitude. When entropy-enthalpy compensation holds for the differential free energy of stability, the incremental changes in T(m) values are shown to be proportionate to the changes in free energy.     

Thermodynamic and structural studies of cavity formation in proteins suggest that loss of packing interactions rather than the hydrophobic effect dominates the observed energetics

The hydrophobic effect is widely believed to be an important determinant of protein stability. However, it is difficult to obtain unambiguous experimental estimates of the contribution of the hydrophobic driving force to the overall free energy of folding. Thermodynamic and structural studies of large to small substitutions in proteins are the most direct method of measuring this contribution. We have substituted the buried residue Phe8 in RNase S with alanine, methionine, and norleucine. Binding thermodynamics and structures were characterized by titration calorimetry and crystallography, respectively. The crystal structures of the RNase S F8A, F8M, and F8Nle mutants indicate that the protein tolerates the changes without any main chain adjustments. The correlation of structural and thermodynamic parameters associated with large to small substitutions was analyzed for nine mutants of RNase S as well as 32 additional cavity-containing mutants of T4 lysozyme, human lysozyme, and barnase. Such substitutions were typically found to result in negligible changes in DeltaC(p)() and positive values of both DeltaDeltaH degrees and DeltaDeltaS of folding. Enthalpic effects were dominant, and the sign of DeltaDeltaS is the opposite of that expected from the hydrophobic effect. Values of DeltaDeltaG degrees and DeltaDeltaH degrees correlated better with changes in packing parameters such as residue depth or occluded surface than with the change in accessible surface area upon folding. These results suggest that the loss of packing interactions rather than the hydrophobic effect is a dominant contributor to the observed energetics for large to small substitutions. Hence, estimates of the magnitude of the hydrophobic driving force derived from earlier mutational studies are likely to be significantly in excess of the actual value.     

Ion concentration and temperature dependence of DNA binding: comparison of PurR and LacI repressor proteins.

Purine repressor (PurR) binding to specific DNA is enhanced by complexing with purines, whereas lactose repressor (LacI) binding is diminished by interaction with inducer sugars despite 30% identity in their protein sequences and highly homologous tertiary structures. Nonetheless, in switching from low- to high-affinity DNA binding, these proteins undergo a similar structural change in which the hinge region connecting the DNA and effector binding domains folds into an alpha-helix and contacts the DNA minor groove. The differences in response to effector for these proteins should be manifest in the polyelectrolyte effect which arises from cations displaced from DNA by interaction with positively charged side chains on a protein and is quantitated by measurement of DNA binding affinity as a function of ion concentration. Consistent with structural data for these proteins, high-affinity operator DNA binding by the PurR-purine complex involved approximately 15 ion pairs, a value significantly greater than that for the corresponding state of LacI (approximately 6 ion pairs). For both proteins, however, conversion to the low-affinity state results in a decrease of approximately 2-fold in the number of cations released per dimeric DNA binding site. Heat capacity changes (DeltaC(p)) that accompany DNA binding, derived from buried apolar surface area, coupled folding, and restriction of motional freedom of polar groups in the interface, also reflect the differences between these homologous repressor proteins. DNA binding of the PurR-guanine complex is accompanied by a DeltaC(p) (-2.8 kcal mol(-1) K(-1)) more negative than that observed previously for LacI (-0.9 to -1.5 kcal mol(-1) K(-1)), suggesting that more extensive protein folding and/or enhanced structural rigidity may occur upon DNA binding for PurR compared to DNA binding for LacI. The differences between these proteins illustrate plasticity of function despite high-level sequence and structural homology and undermine efforts to predict protein behavior on the basis of such similarities.     

Differential scanning calorimetric studies on binding of N-acetyl-D-glucosamine to lysozyme

Differential scanning calorimetric (DSC) measurements were performed on the thermal denaturation of lysozyme and lysozyme complexed with N-acetyl-D-glucosamine (GlcNAc) at pH 5.00 (acetate buffer), 4.25 and 2.25 (Gly-HCl buffer). DSC data have been analyzed to obtain denaturation temperature T(d), enthalpy of denaturation DeltaH(D), heat capacity of denaturation DeltaC(pd) and cooperativity index eta. From these thermodynamic parameters, the binding constant K(L) and enthalpy of binding DeltaH(L), for the weak binding of lysozyme with GlcNAc have been determined. The values of K(L) and DeltaH(L) at pH 5.00 and 298 K are 42 +/- 4 M(-1) and -24 +/- 4 kJ mol(-1), respectively, and agree very well with the experimentally determined values from equilibrium and other studies. The binding constant has also been estimated by simulating the DSC curve with varying values of K(L) (T(d)) until it matches the experimental curve.     

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.     

Electrostatic interactions contribute to reduced heat capacity change of unfolding in a thermophilic ribosomal protein l30e

The origin of reduced heat capacity change of unfolding (DeltaC(p)) commonly observed in thermophilic proteins is controversial. The established theory that DeltaC(p) is correlated with change of solvent-accessible surface area cannot account for the large differences in DeltaC(p) observed for thermophilic and mesophilic homologous proteins, which are very similar in structures. We have determined the protein stability curves, which describe the temperature dependency of the free energy change of unfolding, for a thermophilic ribosomal protein L30e from Thermococcus celer, and its mesophilic homologue from yeast. Values of DeltaC(p), obtained by fitting the free energy change of unfolding to the Gibbs-Helmholtz equation, were 5.3 kJ mol(-1) K(-1) and 10.5 kJ mol(-1) K(-1) for T.celer and yeast L30e, respectively. We have created six charge-to-neutral mutants of T.celer L30e. Removal of charges at Glu6, Lys9, and Arg92 decreased the melting temperatures of T.celer L30e by approximately 3-9 degrees C, and the differences in melting temperatures were smaller with increasing concentration of salt. These results suggest that these mutations destabilize T.celer L30e by disrupting favorable electrostatic interactions. To determine whether electrostatic interactions contribute to the reduced DeltaC(p) of the thermophilic protein, we have determined DeltaC(p) for wild-type and mutant T.celer L30e by Gibbs-Helmholtz and by van't Hoff analyses. A concomitant increase in DeltaC(p) was observed for those charge-to-neutral mutants that destabilize T.celer L30e by removing favorable electrostatic interactions. The crystal structures of K9A, E90A, and R92A, were determined, and no structural change was observed. Taken together, our results support the conclusion that electrostatic interactions contribute to the reduced DeltaC(p) of T.celer L30e.     

Origins of protein denatured state compactness and hydrophobic clustering in aqueous urea: inferences from nonpolar potentials of mean force

Free energies of pairwise hydrophobic association are simulated in aqueous solutions of urea at concentrations ranging from 0-8 M. Consistent with the expectation that hydrophobic interactions are weakened by urea, the association of relatively large nonpolar solutes is destabilized by urea. However, the association of two small methane-sized nonpolar solutes in water has the opposite tendency of being slightly strengthened by the addition of urea. Such size effects and the dependence of urea-induced stability changes on the configuration of nonpolar solutes are not predicted by solvent accessible surface area approaches based on energetic parameters derived from bulk-phase solubilities of model compounds. Thus, to understand hydrophobic interactions in proteins, it is not sufficient to rely solely on transfer experiment data that effectively characterize a single nonpolar solute in an aqueous environment but not the solvent-mediated interactions among two or more nonpolar solutes. We find that the m-values for the rate of change of two-methane association free energy with respect to urea concentration is a dramatically nonmonotonic function of the spatial separation between the two methanes, with a distance-dependent profile similar to the corresponding two-methane heat capacity of association in pure water. Our results rationalize the persistence of residual hydrophobic contacts in some proteins at high urea concentrations and explain why the heat capacity signature (DeltaC(P)) of a compact denatured state can be similar to DeltaC(P) values calculated by assuming an open random-coil-like unfolded state.     

Thermodynamic stability of DNA tandem mismatches

The thermodynamics of nine hairpin DNAs were evaluated using UV-monitored melting curves and differential scanning calorimetry (DSC). Each DNA has the same five-base loop and a stem with 8-10 base pairs. Five of the DNAs have a tandem mismatch in the stem, while four have all base pairs. The tandem mismatches examined (ga/ga, aa/gc, ca/gc, ta/ac, and tc/tc) spanned the range of stability observed for this motif in a previous study of 28 tandem mismatches. UV-monitored melting curves were obtained in 1.0 M Na(+), 0.1 M Na(+), and 0.1 M Na(+) with 5 mM Mg(2+). DSC studies were conducted in 0.1 M Na(+). Transition T(m) values were unchanged over a 50-fold range of strand concentration. Model-independent enthalpy changes (DeltaH degrees ) evaluated by DSC were in good agreement (+/-8%) with enthalpy values determined by van't Hoff analyses of the melting curves in 0.1 M Na(+). The average heat capacity change (DeltaC(p)) associated with the hairpin to single strands transitions was estimated from plots of DeltaH degrees and DeltaS degrees with T(m) and ln T(m), respectively, and from profiles of DSC curves. The average DeltaC(p) values (113 +/- 9 and 42 +/- 27 cal x K(-1) x mol(-1) of bp), were in the range of values reported in previous studies. Consideration of DeltaC(p) produced large changes in DeltaH degrees and DeltaS degrees extrapolated from the transition region to 37 degrees C and smaller but significant changes to free energies. The loop free energy of the five tandem mismatches at 37 degrees C varied over a range of approximately 4 kcal x mol(-1) for each solvent.     

Direct kinetic evidence for folding via a highly compact, misfolded state.

The 2 S seed storage protein, sunflower albumin 8 (SFA-8), contains an unusually high proportion of hydrophobic residues including 16 methionines (some of which may form a surface hydrophobic patch) in a disulfide cross-linked, alpha-helical structure. Circular dichroism and fluorescence spectroscopy show that SFA-8 is highly stable to denaturation by heating or chaotropic agents, the latter resulting in a reversible two-state unfolding transition. The small m(U) (-4.7 M(-1) at 10 degrees C) and DeltaC(p) (-0.95 kcal mol(-1) K(-1)) values indicate that relatively little nonpolar surface of the protein is exposed during unfolding. Commensurate with the unusual distribution of hydrophobic residues, stopped-flow fluorescence data show that the folding pathway of SFA-8 is highly atypical, in that the initial product of the rapid collapse phase of folding is a compact nonnative state (or collection of nonnative states) that must unfold before acquiring the native conformation. The inhibited folding reaction of SFA-8, in which the misfolded state (m(M) = -0.95 M(-1) at 10 degrees C) is more compact than the transition state for folding (m(T) = -2.5 M(-1) at 10 degrees C), provides direct kinetic evidence for the transient misfolding of a protein.