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лекция 2 методы анализа


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лекция 2 методы анализа

  1. 1. Методы анализа белокбелковых взаимодействий с использованием флуоресцентных белков
  2. 2. Interactoms Human protein-protein interaction network Ferrell Journal of Biology 2009 8:2 doi:10.1186/jbiol107
  3. 3. Interactoms Yeast SH3 domain protein-protein interaction network
  4. 4. Many protein interactions are dynamic and regulatable
  5. 5. Main methods to investigate protein–protein interactions Classical methods Co-immunoprecipitation Yeast two-hybrid screen Phage display Chemical cross-linking Fluorescence-based methods Fluorescence resonance energy transfer (FRET) Bimolecular fluorescence complementation (BiFC) Fluorescence cross-correlation spectroscopy (FCCS)
  6. 6. Förster (Fluorescence) Resonance Energy Transfer (FRET)
  7. 7. FRET is a non-radiative transfer of energy from excited donor chromophore molecule to an acceptor chromophore molecule
  8. 8. FRET is proportional to: • Donor quantum yield • Acceptor extinction coefficient • Overlap between donor emission and acceptor absorption spectra FRET is inversely proportional to: • Sixth power of the distance between donor and acceptor (1/R6). Thus, R < 10 nm.
  9. 9. How to measure FRET? FRET results in: Decrease of donor fluorescence intensity Increase of acceptor fluorescence intensity (exc at donor wavelength) Decrease of donor fluorescence lifetime Common approaches of FRET imaging are: 1. Filter-FRET or spectral imaging FRET 2. Donor dequenching by acceptor photobleaching 3. Fluorescence Lifetime Imaging Microscopy
  10. 10. Sensitized emission (three-filter) FRET imaging 1. Ex(Donor) -> Em(Donor) 1. Control: Donor only 2. Ex(Acceptor) -> Em(Acceptor) 2. Control: Acceptor only 3. Ex(Donor) -> Em(Acceptor) 3. Experiment: Donor + Acceptor Sorkin et al, Curr.Biol.2000
  11. 11. Donor dequenching by acceptor photobleaching SYFP2-mStrawberry fusion. J. Goedhart, U. Amsterdam CFP-Munc18-1 and cYFP-syntaxin1A interaction. Liu et al., JBC 2004
  12. 12. Fluorescence lifetime imaging microscopy (FLIM) - Absolute value - Quantitative - Concentration-independent - Special expensive equipment J. Goedhart, U. Amsterdam
  13. 13. Bimolecular fluorescence complementation (BiFC)
  14. 14. Bimolecular fluorescence complementation (split FPs) Fluorescent protein is separated onto two fragments, which develop fluorescence only after their association. This association is facilitated by the interaction between the proteins of interest that are fused to FP fragments. Kerppola TM. Nat. Rev. Mol. Cell. Biol. 2006, 7:449
  15. 15. Kerppola TM. Nat. Rev. Mol. Cell. Biol. 2006, 7:449
  16. 16. Bimolecular fluorescence complementation (BiFC): Split FPs Co-expression of 2 promoters Zhang, Ma & Chalfie, Cell 2004 Protein-protein interactions Shyu et al, Biotechniques 2006 Valencia-Burton et al. Nat Meth. 2007 Visualization of RNA molecules LacZ mRNA 5S rRNA
  17. 17. Fluorescence Correlation Spectroscopy (FCS) Fluorescence Cross-Correlation Spectroscopy (FCCS)
  18. 18. Principle of the method Detection of fluorescence fluctuations in a small volume at low concentrations of fluorophore(s). Further mathematical analysis of autocorrelations and cross-correlations.
  19. 19. Autocorrelation and cross-correlation analysis of GFP and mCherry-tagged proteins in live yeast cells Slaughter B D et al. PNAS 2007;104:20320-20325 ©2007 by National Academy of Sciences
  20. 20. FRET vs BiFC vs FCCS FRET BiFC FCCS Detection Real time Reversible Accumulative Irreversible Real time Reversible Image Yes Yes No Sensitivity Low High High FP concentration High High Low Important Not important Relative orientation of FPs Important Key properties of FPs Monomer Assembling Spectral overlap Maturation rate Brightness Photostability
  21. 21. Fluorescent Timers - proteins that changes color with time Mid-age organelles 5 Old organelles 4 Young organelles 3 2 1 500 600 Wavelength, nm 700 DsRed-E5 – tetrameric green-to-red timer (Terskikh et al., Science 2000) Early expression Late expression
  22. 22. DsRed-E5 Fluorescent Timer in C. elegans DIC FITC 2h 5h 10h 50h Terskikh et al., Science 2000 rhodamine overlay
  23. 23. Novel blue-to-red fluorescent timers Colorless Blue Red Mutagenesis FTs mCherry Subach et al. Nat Chem Biol. 2009
  24. 24. Intracellular trafficking of lysosomal membrane protein LAMP-2A with Fluorescent Timer Golgi -> plasma membrane -> early and recycling endosomes -> late endosomes and lysosomes. Subach et al. Nat Chem Biol. 2009.
  25. 25. ‘‘Fucci’’- fluorescent ubiquitination-based cell cycle indicator Miyawaki lab, Cell 2008
  26. 26. Fucci in the developing mouse head Miyawaki lab, Cell 2008
  27. 27. “Brainbow” mice Livet et al. Nature 2007 ~100 colors!
  28. 28. Application of fluorescent proteins for drug discovery Cell transfection with fluorescent protein genes linked to genes of interest Transfer of visible targets to mice Stably transfected tumor cells Discovery and evaluation of candidate drugs Target visualization Drug treatment control control drug1 drug2 treated
  29. 29. Whole body imaging of carcinogenesis and metastasis Glioma U87-RFP and GFP Pancreas cancer XPA1-RFP Prostate cancer PC-3-RFP Breast cancer MDA-MB-435-GFP Real-time non-invasive monitoring of tumor progression, evaluation of drug treatment efficiency From Anticancer Inc.