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https://hdl.handle.net/2142/25447
Description
Title
Fluorescence lifetime distributions in proteins
Author(s)
Alcala, Jose Ricardo
Issue Date
1987
Doctoral Committee Chair(s)
Gratton, E.
Department of Study
Physics
Discipline
Physics
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
fluorescence lifetime distributions
single tryptophan residue proteins
tryptophan
double dye laser excitation
Language
en
Abstract
"State of the art frequency domain instrumentation was developed to measure the
natural fluorescence decay of single tryptophan residue proteins. The instrumentation
uses mode-locked, synchronously pumped, cavity dumped and frequency doubled dye
laser excitation. The detectors are modulated phase coherently with the mode-locked
laser pulses to accurately determine the phase delay and modulation of the fluorescence
response. The instrument achieves 1 ps time resolution. The resolvabz""l£ty provided by
the experimental data obtained with such instrumentation is determined. It is shown
that the discrete exponential analysis of the fluorescence decay overestimates the resolvability
of the data. It is shown that large sets of exponentially decaying components
with lifetimes distributed continuously in lifetime space can be recovered, within the
limits of resolvability provided by the frequency domain data, using probability density
functions. It is also shown based on the dynamic nature of the macromolecules that
the fluorescence decay of proteins can arise from distributions of exponentially decaying
components. Lzfetime distribution functions are derived based on (i) conformational
interconverting models with distributions of activation energies and (ii) statistical mechanical
models of the protein. The fluorescence lifetime distribution analysis of data
from four single tryptophan residue proteins show that the shape of the distribution is determined
by the structure of the protein. Such distributions become narrower and shifted
to shorter lifetime values with increasing temperature. These results can be explained
based on the dynamic origin of the fluorescence lifetime distribution in macromolecules.
The inadequacy of single conformational model to account for the fluorescence lifetime
distribution suggests that proteins fluctuate in a multitude of conformational sub-states."
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