BIOSYNTHETIC INCORPORATION OF 7-AZATRYPTOPHAN INTO THE PHAGE-LAMBDA LYSOZYME - ESTIMATION OF TRYPTOPHAN ACCESSIBILITY, EFFECT ON ENZYMATIC-ACTIVITY AND PROTEIN STABILITY

The phage lambda lysozyme (lambda L) contains four tryptophans. These have been efficiently replaced by 7-azatryptophan (7aW) through biosyn thetic incorporation into the overexpressed protein. Comparative analy sis of the effect of temperature or pH on the fluorescence of the wild -type lambda L and 7aWs-containing protein (a lambda L) shows that the stability of the protein is only mildly reduced by 7aW incorporation above pH 5 but that it is strongly decreased below pH 4 on protonation of inaccessible 7aWs. The a lambda L fluorescence depends on pH as a consequence of its effect on the denaturation equilibrium, on the stat e of protonation of accessible 7aWs in the native state and of all 7aW s in the denatured state. The pH dependence of the fluorescence is use d to estimate the number of accessible tryptophans in the protein. The result agrees with that derived from tryptophan NH exchange measureme nts by H-1-NMR. The acid limb of the activity-pH profile is characteri zed by a sharp drop that might arise from a cooperative acid-induced d enaturation. The difference in acid stability of a lambda L versus lam bda L is used to rule out this acid denaturation hypothesis as tryptop han replacement does not affect the lytic activity on chloroform-sensi tized Escherichia coli cells or its pH profile.