Abstract
The relationship of structure to function in the recognition of ribonuclease S-peptide by S-protein was studied by several methods. Liquid phase peptide synthesis was employed to generate analogs of S-peptide in which from 1 to 8 residues were deleted from the NH2-terminal end of the S-peptide. Additional derivatives were made by substitutions in the NH2-terminal three amino acids or by modifying the S-peptide analogs by trifluoroacetylation. The analogs were generated in the following way. S-Peptide was cleaved with chymotrypsin. The fragment obtained, RNase(9-20), was purified and lengthened step by step using liquid phase peptide synthesis. A second set of analogs were prepared by cleavage of CF3CO-S-peptide with elastase and the resulting CF3CO-RNase(7-20), similarly lengthened. The various analogs of S-peptide were tested in their capacity to combine with S-protein and regenerate biological activity as measured by Vmax and Kb. This work shows a positive contribution of every one of the first 8 NH2-terminal residues of S-peptide to the molecular recognition of S-protein in the presence of RNA substrate. Substitution of the first 3 residues by alanine or blocking of the free amino groups decreases recognition, indicating that the original primary structure is the most favorable one.
Highlights
Since other workers have described similar situations in which a small peptide acts as an “activator” of a protein. These include: a staphylococcal nuclease system described by Taniuchi and Anfinsen (2) in which the enzyme was split by thermolysin, and a second RNase system in which the enzyme was digested by carboxypeptidase and activated by a tetradecapeptide (Lin et al (3)). These studies seem to indicate that a mutual “recognition” of various regions of the molecule is a consequence of the primary structure
Amino acid analyses of peptides were performed on a Beckman model 121b amino acid analyzer
(100 volts/cm, 30 min, pH 6.5) and the concentration of peptides based upon amino acid analysis
Summary
Ribonuclease A, five times crystallized type 1-A and cytidine cyclic phosphate were obtained from Sigma Chemical Co.; yeast RNA from Schwarz BioResearch Inc.; Nagarse from Teikoku Chemical Industries. Biochemical Corp.; phosphocellulose (Cellex P), 0.94 meq/g, from Bio-Rad Laboratories; cal Co. and dicyclohexylcarbodiimide. Porcine pancreatic elastase was the kind gift of Dr David M. Ethyl trifluorothioacetate was prepared as described by Haupschein et al (10) and distilled before use; Boc-Ala-ONSu’. As described by Anderson et al (11); Boc-Phe-ONSu as by Wuntsch and Drees (12); Nps-Ala,-ONSu, Boc-Thr-ONSu, and Boc-Ala,-ONSu as by Levit (13). Z-08-Bt’-Glu-ONSu physics Department, was the kind gift from Dr A. Was prepared as described by Potts et al (9). (Field = 110 volts/cm, pH 6.5, 1 hour, Whatman No 3) N = ninhydrin staining; P = Pauly staining; A = S-peptide; B = chymotryptic digest of S-peptide; C = peptide RNase( l-8) after purification on a phosphocellulose column (see Fig. 2); D = RNase(9-20)
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