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

Abstract

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,)

Denaturant m values and heat capacity changes: Relation to changes in accessible surface areas of protein unfolding

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Conformational stability of ribonuclease T1 determined by hydrogen‐deuterium exchange

“…Proteins generally exhibit increased stability in D 2 0 (Swint & Robertson, 1993). For this reason, urea denaturation studies were performed to determine the stability of RNase TI in D 2 0 under the conditions identical to those used for the HX experiments.…”

Thermodynamic Analysis of the Structural Stability of Phage 434 Cro Protein

“…Extrapolation of data from chemical denaturation (e.g., urea and guanidine hydrochloride) is based either on the linear free energy model (LEM) 1 found to be valid for a number of proteins (1, 2) or on the denaturant-protein binding model (DBM) (3, 4). Combined analyses of chemical and thermal denaturation data which assume a temperatureindependent ∆C p,U and the thermodynamic equivalence of thermally and chemically denatured states have been reported for various proteins (2,(5)(6)(7)(8)(9)(10)(11)(12).The conformational stability and the principal thermodynamic parameters (∆G U , ∆H U , ∆S U , T m , and ∆C p,U ) which describe the folding transitions of bacteriophage 434 Cro protein are examined here. In vivo, Cro and the repressor proteins regulate the switch between phage lysis and lysogeny by sequence-specific DNA binding (13).…”