Hydrogen exchange in RNase A. Heterogeneity of the tertiary structure inferred from the peptide H-atom exchange, dynamical properties, and evolutionary conservation

Distribution over the tertiary structure of RNase A in crystal and in solut ion was analyzed for peptide II-atoms with a varying degree of the hydrogen exchange (I-IE) retardation, Basing on the visual inspection of wire model s and computer determination of intramolecular contacts in crystal structur es, the V-shaped protein molecule is divided into two major structural doma ins with a globular character of organization of the tertiary structure of each of them. Domain I is formed by residues [(1-19) + (47-81) + (102-124)] , and domain II, consisting of residues [(20-46) + (82-101)], can in turn b e divided into a rigid subdomain and a flexible subdomain formed by jointly packed loop segments 36-40 and 90-95. Each domain has internal nonpolar st ructural core(s) (SC) with cluster-forming residues oriented radially towar d the center of the globular domain and the surrounding tangentially orient ed residues of the cluster envelope. The larger SC of domain I is much bigg er than the SC of domain II, and together with the smaller SC of domain I t hey have five times more peptide hydrogens with strongly retarded HE in the RNase A crystal as compared with the only SC of domain II. This determines the asymmetry in the distribution of nonexchangeable peptide hydrogens ove r the V-shaped RNase molecule. The organization of the large SC of domain I provides for elevated dynamical stability of its structure, which manifest s itself in the strong retardation of HE and admits highly conserved amino acid substitutions in pancreatic RNases of mammals and turtle, as compared with the dynamically less stable and almost completely invariant SC of doma in II. The role of interdomain contacts and SC in the formation and functio ning of the RNase A active center is discussed.