Optical and computational studies of membrane protein structure

Mikhail Proskurin, Štěpán Timr, Dzmitriy Turavets

2011/07/28

Fluorescent proteins in living cells

Horizontal polarization Vertical polarization

Horizontal polarization Vertical polarization

Fluorescent proteins in living cells

Horizontal + vertical polarization

Dichroic ratio: Fh / Fv

Two-photon transition moment

TM

TM orientation ?

Measuring fluorescence of GFP crystals

Excitation light polarization rotated 0-180°

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

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

GFP two-photon transition moment

φ

φ = 5.3˚±2.0˚ (mean±2SEM)

Horizontal + vertical polarization

Dichroic ratio: Fh / Fv

Transition moment orientation with respect to cell membrane

Searching for α0, σ

Transition moment of a fluorescent protein attached to cell membrane

LD measurement

Fluorescence anisotropy measurement

Theoretical models

log2(Fh / Fv ) prediction for vertically oriented membrane

σ [rad]σ [rad]α0 [rad] α0 [rad]

Linear dichroism Fluorescence anisotropy

The dlGFP construct

G. Miesenboeck & al., 2004

Side view Front view

Preparing the dlGFP plasmid

Transfecting mammalian cells with dlGFP

Fluorescence of dlGFP-expressing cells

Horizontal polarizationof excitation light

Vertical polarizationof excitation light

Horizontal polarizationof excitation light

colored red

Vertical polarizationof excitation light

colored green

Fluorescence of dlGFP-expressing cells

Fluorescence of dlGFP-expressing cells

Vertical polarization of excitation light colored green,horizontal polarization of excitation light colored red

Optical properties of dlGFP in living cells

Linear dichroism Fluorescence anisotropy

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

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

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

Acknowledgments

Josef LazarKarolína Tošnerová

Alexey BondarUFB

UNSB