Refinement of noncalorimetric determination of the change in heat capacity, DeltaC(p), of protein unfolding and validation across a wide temperature range.
The change in heat capacity, DeltaC(p), on protein unfolding has been usually determined by calorimetry. A noncalorimetric method which employs the Gibbs-Helmholtz relationship to determine DeltaC(p) has seen some use. Generally, in this method the free energy change on unfolding of the protein is determined at a variety of temperatures and the temperature at which DeltaG is zero, T(m), and change in enthalpy at T(m) are determined by thermal denaturation and DeltaC(p) is then calculated using the Gibbs-Helmholtz equation. We show here that an abbreviated method with stability determinations at just two temperatures gives values of DeltaC(p) consistent with values from free energy change on unfolding determination at a much wider range of temperatures. Further, even the free energy change on unfolding from a single solvent denaturation at the proper temperature, when coupled with the melting temperature, T(m), and the van't Hoff enthalpy, DeltaH(vH), from a thermal denaturation, gives a reasonable estimate of DeltaC(p), albeit with greater uncertainty than solvent denaturations at two temperatures. We also find that nonlinear regression of the Gibbs-Helmholtz equation as a function of stability and temperature while simultaneously fitting DeltaC(p), T(m), and DeltaH(vH) gives values for the last two parameters that are in excellent agreement with experimental values.
Talla-Singh
Deepika
D
Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas 72701-1201, USA.
Stites
Wesley E
WE
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004795:Enzyme Stability; D019791:Guanidine / Q000494:pharmacology; D006358:Hot Temperature; D008836:Micrococcal Nuclease / Q000378:metabolism*; D009154:Mutation; D011489:Protein Denaturation; D017510:Protein Folding; D012044:Regression Analysis; D015203:Reproducibility of Results; D013210:Staphylococcus / Q000201:enzymology; D013696:Temperature; D013816:Thermodynamics
D001426:Bacterial Proteins; D008836:Micrococcal Nuclease; D019791:Guanidine