Development of two-photon polarization microscopy into a protein - PowerPoint PPT Presentation

Development of two-photon polarization microscopy into a protein structure tool Mgr. Josef Melcr Project leader: Josef Lazar, Ph.D. Summer School in Molecular Biophysics and Systems Biology in Nové Hrady, Czech Republic, from July 8 th to July 28 th 2013 jointly organized by the Institute of Nanobiology and Structural Biology of GCRC, Academy of Sciences of the Czech Republic, the University of Szeged, Hungary, Jagiellonian University, Krakow, Poland, and Comenius University in Bratislava, Slovakia, and supported from International Visegrad Fund

Optical properties of molecules are anisotropic

Transition dipole moment TDM TDM

Optical properties of fluorescent proteins are anisotropic

Fluorescent proteins in living cells Horizontal polarization Vertical polarization

Fluorescent proteins in living cells Horizontal polarization Vertical polarization

Horizontal + vertical polarization

A single fluorescent dye in a model membrane 10 μm GUV: Giant Unilamellar Vesicle F2N12S

Experiment and simulations together

Agreement of the two approaches + simulations × microscopy

Polarization microscopy and simulations of fluorescent proteins Neg-cleGFP dleGFP cleGFP

Mammalian cells with overexpressed fluorescent constructs 2-photon polarization microscopy images cleGFP dleGFP dleGFP Neg30-cleGFP dichroic 0.97 0.17 1.07 ratio: r = F hor / F ver

Round mammalian cells with overexpressed fluorescent constructs 2-photon polarization microscopy images cleGFP dleGFP Neg30-cleGFP dichroic 1.06 0.69 1.27 ratio: r = F hor / F ver

Fluorescent proteins in giant unilamellar vesicles a single GUV + Titanium electrodes used for GUVs preparation fluorescent protein with a lipid tag

Fluorescent proteins in vesicles bright field 488nm

Molecular dynamics simulations of cleGFP and NegCleGFP + video

Tilt-angle analysis cleGFP – tilt angle in time

Tilt-angle analysis – histogram cleGFP Neg-cleGFP

Tilt-angle analysis – histogram cleGFP Neg-cleGFP Neg-cleGFP

Conclusions - all tested approaches appear feasible - cell membrane topology affects linear dichroism - surface charges on a protein molecule affect interactions with the cell membrane - more experiments needed to evaluate the capability of two-photon polarization microscopy to yield quantitative information on protein structure