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Time-resolved room temperature protein phosphorescence: nonexponential decay from single emitting tryptophans
B D Schlyer1, J A Schauerte, D G Steel
1Institute of Gerontology, University of Michigan, Ann Arbor 48109.
Biophysical Journal
|September 1, 1994
Summary
Horse liver alcohol dehydrogenase and alkaline phosphatase exhibit complex phosphorescence decays, indicating multiple molecular states. Temperature influences these dynamics, revealing insights into protein flexibility and conformational changes.
Area of Science:
- Biophysics
- Protein Dynamics
- Spectroscopy
Background:
- Tryptophan phosphorescence provides insights into the local environment of proteins.
- Nonexponential decays in phosphorescence can indicate complex molecular dynamics or multiple emitting species.
Purpose of the Study:
- To investigate the nonexponential room temperature phosphorescence (RTP) decays of horse liver alcohol dehydrogenase (LADH) and alkaline phosphatase (AP).
- To elucidate the underlying causes of heterogeneous triplet-state kinetics in these proteins.
Main Methods:
- Analysis of phosphorescence decays using the maximum entropy method (MEM).
- Temperature-dependent phosphorescence measurements.
- Wavelength-selective excitation experiments.
Main Results:
- LADH phosphorescence decay shows two amplitude packets, while AP shows a single Gaussian distribution, analyzed via MEM.
- Nonexponential decay is an intrinsic property of the proteins, not due to impurities.
- Phosphorescence decay kinetics are dependent on excitation wavelength for LADH, suggesting excitation of different molecular sub-ensembles.
- Increasing temperature broadens the decay rate distributions for both proteins.
Conclusions:
- Nonexponential phosphorescence decays in LADH and AP are attributed to conformational heterogeneity and varying local rigidity around the tryptophan residues.
- Interconversion between conformational states occurs on a timescale longer than phosphorescence decay.
- Temperature influences the population and/or dynamics of these conformational states, impacting protein dynamics.