INCORPORATION OF TRYPTOPHAN ANALOGS INTO STAPHYLOCOCCAL NUCLEASE - STABILITY TOWARD THERMAL AND GUANIDINE-HCL INDUCED UNFOLDING

The tryptophan analogues, 5-hydroxytryptophan, 7-azatryptophan, 4-fluo rotryptophan, 5-fluorotryptophan, and 6-fluorotryptophan, have been bi osynthetically incorporated into Staphylococcal nuclease, its V66W mut ant, and the Delta 137-149 fragment of the latter mutant. The guanidin e-HCl induced unfolding and thermal unfolding of these proteins were s tudied to characterize the effect of incorporation of these tryptophan analogues on the thermodynamic stability of the proteins. The three p roteins have tryptophan residues at positions 140 tin wild type) and 6 6 (in the Delta 137-149 fragment of V66W) and at both positions tin V6 6W). The unfolding data show that 5-hydroxytryptophan does not perturb the stability of wildtype nuclease, but it destabilizes the fragment and causes the V66W mutant to unfold in a more cooperative manner. 7-A zatryptophan is found to destabilize all three proteins. 4-Fluorotrypt ophan is slightly stabilizing of the three proteins, but the other two fluorotryptophans do not alter the stability of the proteins.