THE ENERGETICS AND COOPERATIVITY OF PROTEIN FOLDING - A SIMPLE EXPERIMENTAL-ANALYSIS BASED UPON THE SOLVATION OF INTERNAL RESIDUES

The reversible unfolding of two dissimilar proteins, phosphoglycerate kinase from Bacillus stearothermophilus (PGK) and Staphylococcus aureu s nuclease (SAN), was induced with two denaturants, urea and guanidini um chloride (GuHCl). For each protein, structural transitions were mon itored by intrinsic fluorescence intensity changes arising from a uniq ue tryptophan residue. In the case of SAN the single, native tryptopha n residue was used, whereas for PGK two versions, one with a tryptopha n at position 315 and one at 379, were constructed genetically. The re sultant folding curves were analyzed by considering the change in the solvation free energy of internal amino acid residues as the denaturan t concentration was varied. We derive the following simple relationshi p: -RT ln K = DELTAG(w) + nDELTAG(s,m)[D]/(K(den.) + [D]) where K is t he equilibrium constant describing the distribution of folded and unfo lded forms at a given denaturant concentration [D], DELTAG(w) is the f ree energy change for the transition in the absence of denaturant, and n is the number of internal side chains becoming exposed. DELTAG(s,m) and K(den.) are constants derived empirically from the solvation ener gies of model compounds and represent the behavior of an average inter nal side chain between 0 and 6 M GuHCl and 0 and 8 M urea. For protein s of known structure these values can easily be derived, and for other s, average values in guanidinium chloride (DELTAG(s,m) = 0.775 kcal/mo l and K(den.) = 5.4 M) or urea (DELTAG(s,m) = 1.198 kcal/mol and K(den ) = 25.25 M) can be used in the analysis. Results show that the parame ters n and DELTAG(w) are independent of the denaturant used for all 12 transitions studied. This supports the hypothesis that the unfolding activity of urea and GuHCl can be accounted for by their effect on the solvation energy of amino acid side chains which are buried in the fo lded but exposed in the unfolded protein. This simple analytical treat ment allows the 'cooperativity'' of protein folding to be interpreted in terms of the number of side chains becoming exposed to the solvent in a given step and allows accurate estimation of the free energy irre spective of the denaturant concentration needed to induce the transiti on.