Uncovering the innate thermodynamic quantities in protein unfolding

The “cold denaturation” phenomenon is analyzed using an extension of the Planck-Benzinger thermal work function. For small molecules, reaction enthalpies are often obtained around room temperature, such that ΔH=ΔHo(To)+∫ ΔC po dT, and the heat of reaction is estimated in terms of the innate temperature-invariant enthalpy (inherent chemical bond energy), ΔHo(To). Cottrell (The Strength of Chemical Bonds; Academic Press; New York, London, 1958; Chapter 3; pp. 21–46; Chapter 4; pp. 47–70) pointed out 40 years ago that ΔH and ΔHo(T0) differ only by 1% in small molecules, but in 1971 Benzinger [Nature (London) 1971, 220, 100–103] made the crucial observation that this difference is large in biological macromolecules due to the large magnitude of the heat capacity integrals (thermal agitation energy). In the other words, for small molecules, [ΔH−ΔHo(T0)] is a correction of only a few percent, whereas for biological macromolecules, the heat capacity integrals can be large, from 10% up to 50% of the total heat of reaction. In the case of T4 phage lysozyme, the thermal unfolding of wild type and mutant [W138, W138Y, and 3W(128, 138, 158)3Y] forms have heat capacity integrals that are some 10 times greater than the innate temperature-invariant enthalpy. In cases of protein unfolding such as the phage T4 phage lysozyme mutants, no thermodynamic molecular switch, unique to biological systems, is observed. It is apparent that use of the Planck-Benzinger thermal work function to evaluate the innate temperature-invariant enthalpy can be tremendously helpful in differentiating between native wild-type and closely-related mutant forms of protein. Therefore, this thermodynamic application should be essential to any future studies involving the site-directed, mutagenic approach to an examination of structure-function problems in proteins.