N-15 BACKBONE DYNAMICS OF THE S-PEPTIDE FROM RIBONUCLEASE-A IN ITS FREE AND S-PROTEIN BOUND FORMS - TOWARD A SITE-SPECIFIC ANALYSIS OF ENTROPY CHANGES UPON FOLDING

Backbone N-15 relaxation parameters (R1, R2, H-1-N-15 NOE) have been m easured for a 22-residue recombinant variant of the S-peptide in its f ree and S-protein bound forms. NMR relaxation data were analyzed using the ''model-free'' approach (Lipari & Szabo, 1982). Order parameters obtained from ''model-free'' simulations were used to calculate H-1-N- 15 bond vector entropies using a recently described method (Yang & Kay , 1996), in which the form of the probability density function for bon d vector fluctuations is derived from a diffusion-in-a-cone motional m odel. The average change in H-1-N-15 bond vector entropies for residue s T3-S15, which become ordered upon binding of the S-peptide to the S- protein, is -12.6 +/- 1.4 J/mol.residue.K. N-15 relaxation data sugges t a gradient of decreasing entropy values moving from the termini towa rd the center of the free peptide. The difference between the entropie s of the terminal and central residues is about -12 J/mol.residue.K, a value comparable to that of the average entropy change per residue up on complex formation. Similar entropy gradients are evident in NMR rel axation studies of other denatured proteins. Taken together, these obs ervations suggest denatured proteins may contain entropic contribution s from non-local interactions. Consequently, calculations that model t he entropy of a residue in a denatured protein as that of a residue in a di-or tri-peptide, might over-estimate the magnitude of entropy cha nges upon folding.