Specific peptide-protein interactions in the ribonuclease S' system studied by 13C nuclear magnetic resonance spectroscopy with selectively 13C-enriched peptides.

Ribonuclease A: carbon-13 nuclear magnetic resonance assignments, binding sites, and conformational flexibility

“…We assign this peak to Met-13 Ce, by noting its closeness to Asp-14, because no other lie or Met methyl groups are close to a carboxylate group in the crystalline protein. The shift of 13.1 (pD 5.6) is fairly close to that observed for the same residue in the reconstituted protein by Niu et al (1979). Urea Binding at Low Concentration.…”

“…These partially serve to delineate the binding sites for Cu2+, Mn2+, phosphate, cytidine and its 2'-, 3'-, and S'-phosphates (Cyd and Cyd-2'-P, -3'-P, and -5'-P), and one or a few urea molecules at low concentration. Evidence is presented for a conformational change, and hence flexibility, in the active site region around the optimum pD for enzymic activity and another such change at around the optimum .Although there have been previous NMR1 studies of the aromatic and histidine !H resonances of ribonuclease A (Bradbury & Scheraga, 1966;Markley, 1975a;Shindo et al, 1976;Santoro et al, 1979) and of several unprotonated aromatic and carboxyl 13C resonances (Walters & Allerhand, 1980;Shindo et al, 1978;Niu et al, 1979), the aliphatic region of these spectra has never been explored at single-carbon resolution. Only four tentative assignments (Sadler et al, 1974) and one firm I3C assignment (Jaenck & Benz, 1979) have been made in the native protein.…”

Carbon-13 NMR studies of the molecular dynamics of selectively carbon-13-enriched ribonuclease complexes

“…In the present work, we have used the "model-free" approach (Lipari & Szabo, 1982a), which makes few assumptions about the nature of the molecular processes operating. We have utilized the selectively 13C-enriched RNase S' system (Niu et al, 1979), particularly focusing on the uniformly labeled Ala-5 complex. Analysis of the relaxation data for non-hydrogen-containing enriched carbon atoms (such as carbonyl or C' of His) was not attempted due to the complication of relaxation mechanisms other than dipole-dipole (Norton et al, 1977), although in general it was found that these values increased with observing field strength (Table I).…”

“…Materials. reported (Niu et al, 1979). Bovine pancreatic RNase S protein (type XII-S, grade XII-PR) and the sodium salt of cytidine cyclic 2',3'-phosphate were obtained from Sigma Chemical Co. RNase A (phosphate free) used to calibrate the assays was purchased from Worthington Biochemical Corp.…”

“…Methods. The three semisynthetic RNase S' complexes were prepared as described previously (Niu et al, 1979). A solution of the RNase (1-15) peptide was added to the S protein (ca.…”