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Optical and computational studies of membrane protein structure
Mikhail Proskurin, Štěpán Timr, Dzmitriy Turavets
2011/07/28
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Fluorescent proteins in living cells
Horizontal polarization Vertical polarization
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Horizontal polarization Vertical polarization
Fluorescent proteins in living cells
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Horizontal + vertical polarization
Dichroic ratio: Fh / Fv
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Two-photon transition moment
TM
TM orientation ?
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Measuring fluorescence of GFP crystals
Excitation light polarization rotated 0-180°
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F [a
.u.]
Polarization angle [deg] Polarization angle [deg]
F [a
.u.]
Two-photon excitation 800 nmOne-photon excitation 405 nm
Fluorescence of a GFP crystal
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F [a
.u.]
Polarization angle [deg] Polarization angle [deg]
F [a
.u.]
Two-photon excitation 800 nmOne-photon excitation 405 nm
Fluorescence of a GFP crystal
Mathematical processing → transition moment orientation
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GFP two-photon transition moment
φ
φ = 5.3˚±2.0˚ (mean±2SEM)
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Horizontal + vertical polarization
Dichroic ratio: Fh / Fv
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Transition moment orientation with respect to cell membrane
Searching for α0, σ
Transition moment of a fluorescent protein attached to cell membrane
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LD measurement
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Fluorescence anisotropy measurement
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Theoretical models
log2(Fh / Fv ) prediction for vertically oriented membrane
σ [rad]σ [rad]α0 [rad] α0 [rad]
Linear dichroism Fluorescence anisotropy
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The dlGFP construct
G. Miesenboeck & al., 2004
Side view Front view
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Preparing the dlGFP plasmid
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Transfecting mammalian cells with dlGFP
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Fluorescence of dlGFP-expressing cells
Horizontal polarizationof excitation light
Vertical polarizationof excitation light
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Horizontal polarizationof excitation light
colored red
Vertical polarizationof excitation light
colored green
Fluorescence of dlGFP-expressing cells
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Fluorescence of dlGFP-expressing cells
Vertical polarization of excitation light colored green,horizontal polarization of excitation light colored red
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Optical properties of dlGFP in living cells
Linear dichroism Fluorescence anisotropy
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Matching dlGFP optical properties with tilt angle 0, distribution width
Linear dichroism Fluorescence anisotropy
α0 [rad]α0 [rad]
σ [rad] S
Least square fitting – sum of squared residuals (S) of log2 (Fh / Fv )depending on α0 and σ
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Linear dichroism Fluorescence anisotropy
α0 [rad]α0 [rad]
σ [rad] S
Least square fitting – sum of squared residuals (S) of log2 (Fh / Fv )depending on α0 and σ
Matching dlGFP optical properties with tilt angle 0, distribution width
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Conclusions
In principle, the method works
The mathematical model needs to be improved
Applications include studies of membrane protein structure and function and design of probes of molecular processes in living cells
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Acknowledgments
Josef LazarKarolína Tošnerová
Alexey BondarUFB
UNSB