Thermodynamic and structural consequences of changing a sulfur atom to a methylene group in the M13Nle mutation in ribonuclease-S

Abstract

Two fragments of pancreatic ribonuclease A, a truncated version of S-peptide (residues 1-15) and S-protein (residues 21-124), combine to give a catalytically active complex. We have substituted the wild-type residue at position 13, methionine (Met), with norleucine (Nle), where the only covalent change is the replacement of the sulphur atom with a methylene group. The thermodynamic parameters associated with the binding of this variant to S-protein, determined by titration calorimetry in the temperature range 10-40 °C , are reported and compared to values previously reported and compared to values previously reported [ Varadarajan, R., Connelly, P. R., Sturtevant, J. M., and Richards, F. M. (1992) Biochemistry 31, 1421-1426] for other position 13 analogs. The differences in the values for the Met/Ile and Met/Leu pairs. The structures of mutant complex was determined to 1.85 Å 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-factor than the corresponding Met side chain. In contrast, the B-factors of the atoms of the cage of residues surrounding position 13 are all somewhat lower in the Nle variant than in the Met wild-type. Thus, the large differences in the binding enthalpy appear to residue entirely in the difference in chemical properties or dynamic behaviour of the –S- and –CH₂- groups and not in differences in the geometry of the side chains or the internal cavity surface. In addition, a novel method of obtaining protein stability data by means of isothermal titration calorimetry is introduced.