Synthetic Studies of Structure-Function Relationships in Staphylococcal Nuclease

Abstract The solid phase technique of peptide synthesis has been used to study structural features related to the activity of Fragment P2 (Residues 6 through 48) of nuclease-T. Binding of synthetic analogues to the complimentary native Fragment P3 (Residues 49 through 149) may be detected by affinity chromatography upon an agarose column containing covalently bound P3. Binding in free solution was detected by changes in the fluorescence spectrum of the single tryptophan residue in P3. Previous studies have shown that the synthetic 6-47 peptide, after purification by specific binding to an agarose P3 column, can generate approximately 30% of the maximum enzymic activity when added in an equivalent molar quantity to Fragment P3 Deletion of the NH2-terminal tripeptide from synthetic P2 (yielding synthetic 9-47) leaves both binding and activating abilities intact. Two shorter analogues, synthetic 18-47 and synthetic 33-47, have no activity, although the 18-47 analogue appears to retain some binding affinity for native P3. The methionine residues at positions 26 and 32 may be replaced by norleucine to give an analogue capable of both binding to and activating P3. Replacement of glutamic acid by glutamine at position 43 leads to an inactive derivative which retains its ability to bind specifically to Fragment P3.