Linked Functions and Reciprocal Effects in Hemoglobin: A Second Look

We have used thermal and chemical denaturation to characterize the thermodynamics of unfolding for turkey ovomucoid third domain (OMTKY3). Thermal denaturation was monitored spectroscopically at a number of wavelengths and data were subjected to van't Hoff analysis; at pH 2.0, the midpoint of denaturation (T,) occurs at 58.6 -t 0.4 "C and the enthalpy of unfolding at this temperature (AH,) is 40.8 -t 0.3 kcal/mol. When T, was perturbed by varying pH and denaturant concentration, the resulting plots of AH, versus T, yield a mean value of 590 5 120 cal/(mol .K) for the change in heat capacity upon unfolding (AC,). A global fit of the same data to a n equation that includes the temperature dependence for the enthalpy of unfolding yielded a value of 640 k 110 cal/(mol .K). We also performed a variation of the linear extrapolation method described by Pace and Laurents, which is an independent method for determining ACp (Pace, C.N. & Laurents, D., 1989, Biochemistry 28, 2520-2525). First, OMTKY3 was thermally denatured in the presence of a variety of denaturant concentrations. Linear extrapolations were then made from isothermal slices through the transition region of the denaturation curves. When extrapolated free energies of unfolding (AG,) were plotted versus temperature, the resulting curve appeared linear; therefore, AC, could not be determined. However, the data for AG, versus denaturant concentration are linear over an extraordinarily wide range of concentrations. Moreover, extrapolated values of AG, in urea are identical to values measured directly.Keywords: heat capacity; linear extrapolation method; protein folding; van't Hoff analysis One facet of "the protein folding problem" is understanding how proteins maintain their unique native conformations. While both the energetics and conformation of the native structure are clearly dictated by the primary amino acid sequence, the contributions of individual residues are at present ill-defined. Comprehensive studies of such contributions can most easily be addressed in small proteins, such as ovomucoid third domain (56 amino acid residues, MW = 6.1 kDa). Our ultimate goal is to quantitatively describe the contribution of every amino acid residue to theglobal stability of ovomucoid third domain. Key information for these studies is knowledge of the free energy of unfolding (AG,) over a wide range of experimental conditions. AG, can be directly measured over only a narrow temperature range but, for a two-state process, can be predicted at any temperature from the modified Gibbs-Helmholtz equation:AG, = AH,-(l -T/T,)

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“…As described by Wyman (1964), the linkage relationship between changes in pH and protein stability is…”

Section: Discussionmentioning

confidence: 99%

Thermodynamics of unfolding for turkey ovomucoid third domain: Thermal and chemical denaturation

1993

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“…For the native protein at 48 "C, however, the same is true only up to 20% sorbitol, above which further addition of sorbitol reverses the effect and any infinitesimal increase in solvent composition renders the thermodynamic interaction favorable. Quantitatively, the effect on the unfolding reaction at any given solvent composition with reference to that solvent composition is expressed by the linkage equation (Wyman, 1964):…”

Section: Stabilization Is Caused By General Nonspecific Interactionsmentioning

confidence: 99%

Mechanism of the stabilization of ribonuclease a by sorbitol: Preferential hydration is greater for the denatured than for the native protein

1997

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The effect of interactions of sorbitol with ribonuclease A (RNase A) and the resulting stabilization of structure was examined in parallel thermal unfolding and preferential binding studies with the application of multicomponent thermodynamic theory. The protein was stabilized by sorbitol both at pH 2.0 and pH 5.5 as the transition temperature, Tm, was increased. The enthalpy of the thermal denaturation had a small dependence on sorbitol concentration, which was reflected in the values of the standard free energy change of denaturation, SAG" = AC"(sorbito1) -AG"(water). Measurements of preferential interactions at 48 "C at pH 5.5, where protein is native, and pH 2.0, where it is denatured, showed that sorbitol is preferentially excluded from the denatured protein up to 40%, but becomes preferentially bound to native protein above 20% sorbitol. The chemical potential change on transferring the denatured RNase A from water to sorbitol solution is larger than that for the native protein, A p f > A&, which is consistent with the effect of sorbitol on the free energy change of denaturation. The conformity of these results to the thermodynamic expression of the effect of a co-solvent on denaturation, AG; + A p f = AG; + A p f , indicates that the stabilization of the protein by sorbitol can be fully accounted for by weak thermodynamic interactions at the protein surface that involve water co-solvent exchange at thermodynamically non-neutral sites. The protein structure stabilizing action of sorbitol is driven by stronger exclusion from the unfolded protein than from the native structure.

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“…At any given solvent composition, the effect of increasing infinitesimally the concentration of any ligand, in this case co-solvent, on protein stability is given by the linkage relation (Wyman, 1964): where d p 3 = RTd In a3. The stabilizing ability of a co-solvent is defined, therefore, by the difference between the chemical potential gradients for the protein in the native and denatured states.…”

Section: Thermodynamic Stabilizationmentioning

confidence: 99%

Temperature dependence of the preferential interactions of ribonuclease a in aqueous co‐solvent systems: Thermodynamic analysis

Xie

Timasheff

1997

Protein Science

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The temperature dependence of preferential solvent interactions with ribonuclease A in aqueous solutions of 30% sorbitol, 0.6 M MgCIB, and 0.6 M MgS04 at low pH (1.5 and 2.0) and high pH (5.5) has been investigated. This protein was stabilized by all three co-solvents, more so at low pH than high pH (except 0.6 M MgCI2 at pH 5.5). The preferential hydration of protein in all three co-solvents was high at temperatures below 30 "C and decreased with a further increase in temperature (for 0.6 M MgC12 at pH 5.5, this was not significant), indicating a greater thermodynamic instability at low temperature than at high temperature. The preferential hydration of denatured protein (low pH, high temperature) was always greater than that of native protein (high pH, high temperature). In 30% sorbitol, the interaction passed to preferential binding at 45% for native ribonuclease A and at 55 "C for the denatured protein. Availability of the temperature dependence of the variation with sorbitol concentration of the chemical potential of the protein, (dp2/drn3)~,p,m2, permitted calculation of the corresponding enthalpy and entropy parameters. Combination with available data on sorbitol concentration dependence of this interaction parameter gave (approximate) values of the transfer enthalpy, A&,r, and transfer entropy Transfer of ribonuclease A from water into 30% sorbitol is characterized by positive values of the transfer free energy, transfer enthalpy, transfer entropy, and transfer heat capacity. On denaturation, the transfer enthalpy becomes more positive. This increment, however, is small relative to both the enthalpy of unfolding in water and to the transfer enthalpy of the native protein from water to a 30% sorbitol solution.Keywords: preferential binding; protein-solvent interactions; protein stabilization by co-solvents; transfer enthalpy; transfer free energy In the companion paper (Xie & Timasheff, 1997), protein (RNase A) stabilization by sorbitol in aqueous solution was examined in concerted manner by thermal denaturation and measurement of preferential interactions at conditions in which the protein (RNase A) was either native or unfolded at identical temperatures. It was established that at the limited condition of 48 "C, RNase A was in the native state at pH 5.5, but was denatured at pH 2.0. At 48 "C sorbitol was preferentially excluded from the denatured protein, but preferentially bound to the native one. The higher preferential hydration of the denatured protein than that of the native one is the thermodynamic source of the stabilization.It is generally accepted that knowledge of the temperature dependence of the partial specific volume of proteins should provide important information on solvent-protein interactions (Kauzmann, 1959;Kupke, 1973;Xie & Timasheff, 1997). Gekko and Morikawa (1 98 1 a) measured the preferential interaction parameters of bovine serum albumin in 30% aqueous solutions of glycerol and sorbitol chemistry, Brandeis University, Waltham, Massachusetts 02254.Reprint requests to: Ser...

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Linked Functions and Reciprocal Effects in Hemoglobin: A Second Look

Cited by 1,654 publications

References 38 publications

Thermodynamics of unfolding for turkey ovomucoid third domain: Thermal and chemical denaturation

Thermodynamics of unfolding for turkey ovomucoid third domain: Thermal and chemical denaturation

Mechanism of the stabilization of ribonuclease a by sorbitol: Preferential hydration is greater for the denatured than for the native protein

Temperature dependence of the preferential interactions of ribonuclease a in aqueous co‐solvent systems: Thermodynamic analysis

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