Steady state tryptophan fluorescence emission intensity of G proteins increases 1.5-2.5 fold upon activation by GTP. We have used the two tryptophan G protein, transducin to analyze the origin of this effect. Absolute quantum yields for W127 and W207, were, determined by combining precise measurement of integral emission intensity calibrated against NATA (?=0.14) with the products of lifetime amplitude and exponential decay time for 295nm excitation and 345 nm emission. Four different ligand states of the protein with GDP, GTP?S, AlF4 and Mg2+ were examined in pH 7.0, 10 mM PO4 buffer at 220C. Two lifetimes dominated each state; one nearly constant at 3.210.1 ns and the other varying from 6.4ns for G?GDP to 7.6 for G?GTP?S?Mg. ??of the 3.2ns state proved to be constant at 0.27 in all states while ??of the longer lived component ranged from 0.13 (G?GDP) to 1.010.05 (G?GTP?S?Mg). We have identified W127 as the ?=0.27 ?=3.2 ns component. Failure of net steady state emis sion maximum to change despite the 8 fold increase in ?W207 with its apparent folding into a more apolar environment (crystal structure) implies a fast, non-radiative, powerful excited state energy conduit out of W207 in G?GDP.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR001348-20
Application #
6480849
Study Section
Project Start
2001-08-01
Project End
2002-07-31
Budget Start
1997-10-01
Budget End
1998-09-30
Support Year
20
Fiscal Year
2001
Total Cost
$155,764
Indirect Cost
Name
University of Pennsylvania
Department
Type
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
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