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 as by measurements of the time-resolved room temperature phosphorescence from Trp 109. It is well known that the denaturants, ethylene diamine tetraacetic acid (EDTA), acid and guanidine hydrochloride (GdnHCl) inactive AP by different mechanisms as reflected by differences in the time dependence of inactivation. However, further information about structural changes that result during inactivation is obtained by measurement of the phosphorescence intensity and radiative decay rate. Time-resolved tryptophan phosphorescence is exquisitely sensitive to changes in the local environment of the emitting residue, unlike the steady state phosphorescence intensity which is a composite of both the lifetime and concentration of the emitting protein species. The results show that while inactivation in EDTA proceeds by loss of the zinc ion as expected, denaturation in acid or GdnHCl produces a heterogeneous population of AP molecules, detected by a distribution analysis of the phosphorescence lifetime, which may reflect multiple pathways to the final unfolded state. Time-resolved phosphorescence also demonstrates the existence of an enzymatically active but structurally less rigid intermediate state during unfolding. As the rigidity decreases, the susceptibility to further denaturation decreases at lower pH but increases with GdnHCl concentration. The experiments provide new insight into the mechanism of denaturation of AP and demonstrate the sensitivity of time-resolved room temperature phosphorescence to the structural details of intermediate states produced during unfolding of proteins.
Mersol
J V
JV
Department of Physics, Institute of Gerontology, University of Michigan, Ann Arbor 48109-2007.
Steel
D G
DG
Gafni
A
A
D000469:Alkaline Phosphatase / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D019791:Guanidine; D006146:Guanidines; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence / Q000379:methods; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry*
D001426:Bacterial Proteins; D006146:Guanidines; D014364:Tryptophan; D000469:Alkaline Phosphatase; D019791:Guanidine