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

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

Mar 24, 2024 39 likes •260 views

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 Nov Hrady, Czech Republic, from July 8 th to July

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Summer School in Molecular Biophysics and Systems Biology in Nové Hrady, Czech Republic, from July 8th to July 28th 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

Development of two-photon polarization microscopy into a protein structure tool

  • Mgr. Josef Melcr

Project leader: Josef Lazar, Ph.D.

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Optical properties of molecules are anisotropic

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Transition dipole moment

TDM TDM

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Optical properties of fluorescent proteins are anisotropic

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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

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A single fluorescent dye in a model membrane

F2N12S GUV: Giant Unilamellar Vesicle 10 μm

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Experiment and simulations together

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Agreement of the two approaches

× microscopy + simulations

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Polarization microscopy and simulations of fluorescent proteins

cleGFP dleGFP Neg-cleGFP

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r = Fhor / Fver

Mammalian cells with

  • verexpressed fluorescent

constructs

dleGFP cleGFP 0.97 Neg30-cleGFP 1.07

2-photon polarization microscopy images

dleGFP 0.17

dichroic ratio:

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Round mammalian cells with

  • verexpressed fluorescent

constructs

dleGFP 0.69 cleGFP 1.06 Neg30-cleGFP 1.27

2-photon polarization microscopy images dichroic ratio:

r = Fhor / Fver

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Fluorescent proteins in giant unilamellar vesicles

Titanium electrodes used for GUVs preparation a single GUV

+

fluorescent protein with a lipid tag

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Fluorescent proteins in vesicles

bright field 488nm

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Molecular dynamics simulations

  • f cleGFP and NegCleGFP

+ video

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Tilt-angle analysis

cleGFP – tilt angle in time

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Tilt-angle analysis – histogram

cleGFP Neg-cleGFP

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Tilt-angle analysis – histogram

Neg-cleGFP cleGFP Neg-cleGFP

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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

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