THERMODYNAMIC AND STRUCTURAL CONSEQUENCES OF CHANGING A SULFUR ATOM TO A METHYLENE GROUP IN THE M13N1E MUTATION IN RIBONUCLEASE-S

Two fragments of pancreatic ribonuclease A, a truncated version of S-p eptide (residues 1-15) and S-protein (residues 21-124), combine to giv e a catalytically active complex. We have substituted the wild-type re sidue at position 13, methionine (Met), with norleucine (Nle), where t he only covalent change is the replacement of the sulfur atom with a m ethylene group. The thermodynamic parameters associated with the bindi ng of this variant to S-protein, determined by titration calorimetry i n the temperature range 10-40 degrees C, are reported and compared to values previously reported [Varadarajan, R., Connelly, P. R., Sturteva nt, J. M., & Richards, F. M. (1992) Biochemistry 31, 1421-1426] for ot her position 13 analogs. The differences in the free energy and enthal py of binding between the Met and Nle peptides are 0.6 and 7.9 kcal/mo l at 25 degrees C, respectively. These differences are slightly larger than, but comparable to, the differences in the values for the Met/Il e and Met/Leu pairs. The structure of the mutant complex was determine d to 1.85 Angstrom resolution and refined to an R-factor of 17.4% The structures of mutant and wild-type complexes are practically identical although the Nle side chain has a significantly higher average B-fact or than the corresponding Met side chain. In contrast, the B-factors o f the atoms of the cage of residues surrounding position 13 are all so mewhat lower in the Nle variant than in the Met wild-type. Thus, the l arge differences in the binding enthalpy appear to reside entirely in the difference in chemical properties or dynamic behavior of the -S- a nd -CH2- groups and not in differences in the geometry of the side cha ins or the internal cavity surface. In addition, a novel method of obt aining protein stability data by means of isothermal titration calorim etry is introduced.