Single molecule techniques:

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• Single molecule techniques
• Dynamics of fluorescence fluctuations in green
  fluorescent protein observed by fluorescence
  correlation spectroscopy
• U. Haupts et al.
• Proc. Natl. Acad. Sci., Nov 1998, 95(23),
  13573-13578

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• Green fluorescent protein shows strong
  fluorescence at physiological pH values. The
  analyzed mutant (EGFP) is excited at 488 nm and
  emits at 509 nm. At low pH a 398 nm peak appears
  in the absorption spectrum, but not in the
  excitation spectrum. The 398 nm species (A form)
  of EGFP does not fluoresce. Formation (by
  protonation) of this species can be measured in
  bulk or on single molecules.

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The simplest model for the fluorescence decrease
at low pH is protonation of a fluorescent species
A- at higher pH to a non-fluorescent species AH
at lower pH. This can be described with a
Henderson-Hasselbalch equation.
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From other mutants and from structural analysis
it is presumed that even at high pH there is an
equilibrium between two species. Is the second
species at high pH the AH (even though the pH is
several units above the pKa) or is it a third
species? How can this be distinquished?
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Model for a three species system
In this model there is a pH independent
equilibrium between A- and a non-fluorescent AH.
If the fraction of AH is 0.1, the averaged
fluorescence would only be 90 of A-. If there
was no AH and A-would fluoresce at 90
intensity, wouldnt it look the same?
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This could be checked, if we could measure single
molecules. In one case they would always
fluoresce at 90, in the other case they would
change between 100 and 0. Can we measure the
fluorescence of single molecules? Yes with FCS
An introduction by Petra Schwille and Elke
Haustein is given in http//www.biophysics.org/ed
ucation/schwille.pdf A brief overview follows
here.
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blinking
diffusion
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Which part of the autocorrelation function has to
do with diffusion? When the focal spot is
enlarged, the molecules need more time to diffuse
through it. Moving in and out turning
fluorescence on and off for molecule i takes
longer. At shorter times the autocorrelation
function is independent of the focal spot size.
Here, the molecules turn on and off while they
are in the focal spot.
diffusion
blinking
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The time rate tc and the fraction F of dark state
(AH and AH species) for the blinking can be
fitted. Time rates for diffusion tD are fitted,
but not used, because they are not only molecule
(MW) but also geometry of the light path)
dependent. The equation for the autocorrelation
function was taken from the literature without
modification
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The time rate and the fraction of dark state (AH
and AH species) for the blinking can be fitted.
At high pH (9-11) the fraction of the dark state
stays at 12. This can only be explained with a
pH independent equilibrium A- lt-gt AH.
pH 5.5 pH 9.0 Chemical relaxation tC 0.1
ms 0.35 ms Chemical relaxation tC 3.3 ms 4.2
ms Fraction in dark state F 0.65 0.13