Picosecond tryptophan fluorescence of thioredoxin: evidence for discrete species in slow exchange.
The steady-state tryptophan fluorescence and time-resolved tryptophan fluorescence of Escherichia coli thioredoxin, calf thymus thioredoxin, and yeast thioredoxin have been studied. In all proteins, the tryptophan residues undergo strong static and dynamic quenching, probably due to charge-transfer interactions with the nearby sulfur atoms of the active cysteines. The use of a high-resolution photon counting instrument, with a time response of 60 ps full width at half-maximum, allowed the detection of fluorescence lifetimes ranging from a few tens of picoseconds to 10 ns. The data were analyzed both by classical nonlinear least squares and by a new method of entropy maximization (MEM) for the recovery of lifetime distributions. Simulations representative of the experimental data were used to test the MEM analysis. Strong support was obtained in this way for a small number of averaged discrete species in the fluorescence decays. Wavelength studies show that each of these components spreads over closely spaced excited states, while the temperature studies indicate that they do not exchange significantly on the nanosecond time scale. The oxidized form of thioredoxin is characterized by a high content of a very short lifetime below 70 ps, the amplitude of which is sharply decreased upon reduction. On the other hand, the fluorescence anisotropy decays indicate that reduction causes an increase of the very fast tryptophan rotations in an otherwise relatively rigid structure. While the calf thymus and E. coli proteins have mostly similar dynamical fluorescence properties, the yeast thioredoxin differs in many respects.
Mérola
F
F
Department of Medical Biophysics, Karolinska Institutet, Stockholm, Sweden.
Rigler
R
R
Holmgren
A
A
Brochon
J C
JC
D000818:Animals; D001426:Bacterial Proteins; D001665:Binding Sites; D002417:Cattle; D004926:Escherichia coli; D005454:Fluorescence Polarization; D007700:Kinetics; D015394:Molecular Structure; D012441:Saccharomyces cerevisiae; D013045:Species Specificity; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013879:Thioredoxins; D014364:Tryptophan
D001426:Bacterial Proteins; D013879:Thioredoxins; D014364:Tryptophan