DETECTION OF INTERMEDIATE PROTEIN CONFORMATIONS BY ROOM-TEMPERATURE TRYPTOPHAN PHOSPHORESCENCE SPECTROSCOPY DURING DENATURATION OF ESCHERICHIA-COLI ALKALINE-PHOSPHATASE

The reversible denaturation of Escherichia coli alkaline phosphatase ( AP) was followed by monitoring changes in enzymatic activity as well a s by measurements of the time-resolved room temperature phosphorescenc e from Trp 109. It is well known that the denaturants, ethylene diamin e tetraacetic acid (EDTA), acid and guanidine hydrochloride (GdnHCl) i nactivate AP by different mechanisms as reflected by differences in th e time dependence of inactivation. However, further information about structural changes that result during inactivation is obtained by meas urement of the phosphorescence intensity and radiative decay rate. Tim e-resolved tryptophan phosphorescence is exquisitely sensitive to chan ges in the local environment of the emitting residue, unlike the stead y state phosphorescence intensity which is a composite of both the lif etime and concentration of the emitting protein species. The results s how that while inactivation in EDTA proceeds by loss of the zinc ion a s expected, denaturation in acid or GdnHCl produces a heterogeneous po pulation of AP molecules, detected by a distribution analysis of the p hosphorescence lifetime, which may reflect multiple pathways to the fi nal unfolded state. Time-resolved phosphorescence also demonstrates th e existence of an enzymatically active but structurally less rigid int ermediate state during unfolding. As the rigidity decreases, the susce ptibility to further denaturation decreases at lower pH but increases with GdnHCl concentration. The experiments provide new insight into th e mechanism of denaturation of AP and demonstrate the sensitivity of t ime-resolved room temperature phosphorescence to the structural detail s of intermediate states produced during unfolding of proteins.