The Specific Binding of Three Fragments of Staphylococcal Nuclease

Abstract

Abstract Previous observations on the productive complementation by several sets of two inactive and disordered fragments of Staphylococcal nuclease (149 residues without cysteines and disulfide bonds) indicated that peptide bonds either between residues 48 and 50 or at some point between residues 113 and 124 can be cleaved without causing destruction of the information to determine the functional structure. For example, nuclease-T-(6–48) (residues 6 to 48) binds to nuclease-T-(49–149) and to nuclease-T-(50–149) and forms nuclease-T', the enzymically active and ordered complexes ((6–48) + (49–149)) and ((6–48) + (50–149)), respectively. Nuclease-(1–126) and nuclease-(99–149) yield another functional complex ((1–126) + (99–149)). In the present study, nuclease-(49, 50–126) (the mixture of residues 49 to 126 and 50 to 126) was prepared by limited trypsin digestion of trifluoroacetylated nuclease-T-(49, 50–149) (the mixture of nuclease-T-(49–149) and nuclease-T-(50–149)). It is shown that nuclease-T-(6–48), nuclease-(49, 50–126), and nuclease-(99–149) bind in equimolar ratio below 20° to form a population of three-fragment complexes, presumably the mixture of ((6–48) + (49–126) + (99–149)) and ((6–48) + (50–126) + (99–149)). The three-fragment complex appears to have intrinsic enzymic activity, although at very low level, and affinity for the nuclease ligand, deoxythymidine 3',5'-diphosphate in the presence of Ca2+. The heat stability of the three-fragment complex is less than that of the enzymically active derivatives with only one discontinuity of the polypeptide chain, although the latter themselves show lower heat stability than that of intact nuclease. These observations support the view that specific non-covalent binding of the three fragments to form an enzymically active structure can still take place if the simultaneous cleavages of the peptide bonds are at the two permissible sites.