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Single pair Forster resonance energy transfer (Protein undergo conform…
Single pair Forster resonance energy transfer
Protein undergo conform changes
why do we know?
NMR
cryoelectron spectroscopy
molecular dynamics simulations
FRET
ensemble
single molecule measurements
determine intermediate states
measurement options
molecules diffusing in solution
only nmolecules with low disociation constant Kd can be investigated
they are still complexes in nanomolar concentration range
molecules immobilized on surface
subpopulations
reactive pathways
FRET efficiency is inversly proportional to R^6
R - Forster radius
R=f(quatum yield, spectral overlap (donor, acceptor), adsorption spectra of acceptor)
FRET eff determination
photon counts
need to have correction factors
laser excitation
crosstalk of donor in the acceptor channel
difference in the donor/acceptor detection efficiency
quantum yield
fluorophores
for low FRET stated lifetime of donors is higher
donor lifetime with/without acceptor
if deviate from this value
high FRET eff, low donot lifetime
photophysical effects
TIRF
objective-type
prism-type
waveguide
confocal
better time resolution
uses APD detectors
less molecules detected (sequential measurements)
detectors
point detectors
can detect individual photons
photon converts to electrone-hole pair
good time resolution, bad position resolution
photomultiplier tubes (PMT)
TR 50 ps
QY 20%
Avanance photodiodes (APD)
QU 60% (vis) 90% (IR)
TR 50-350 ps
Hybrid photodetector
photomultipliers + APD
QY 50% vis
TR 23 ps
cameras
collect charges in cell arrays
good position resolution, bad time resolution
can increase SNR sacrificing with localization accuracy
CCDs
cooled CCD or EMCCD
QY 90-95%
TR 20-33 ms
CMOS cameras
beter TR
QY 30-60%, new are 90%
what can be detected?
FRET eff
if in dilluted solutions
1-5 fluor bursts per second - only one molecule is in volume
several hours of measuremenst
stoichiometry
by comb several lasers
PIE - pulsed interleaved excitation
syncro several pulsed lasers
lifetime
anisotropy
by polarizing waves
excitation sources
lasers
pulsed
fluorescence lifetime information
donor-acceptor stoich information
continious
FCS FRET
can see timescale of conformations
detects the same molecule (by measuring fluor bursts shortly after initial burst)
as volume and timerange are small the probability of detecting the same molecule is high
uses nanomolar concentration
functions depend on them
transtiton time
determided via max likelyhood mechanism
few mictoseconds
Sample preparation
labeling
Tag-labeling
specific site but large size cause structural changes and position limitations
non-natural AA
no position limitations (small size), still site-specific
natural cysteins
don't cause structural changes but if molecule has cysteins they have to be removed
cysteine rarely occurs in proteins
stochastic labeling (mistakes)
doesn't influences measurements
if influences - this can be corrected
important: maleimide bound
buffer
oxygen scavenger (increase photostability)
Dye properties
photostable, bright, monoexponential lifetime, low micro-milli second blinking
Vitamin E - Trolox used to quench dark/dim dye states
geminate recombinations
emit = 100 photons
cover vis-near IR spectrum
QY 10%
fluor lifetime - few ns
size 1-2 nm
chemicall y inert