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Fluorescent proteins

Fluorescent proteins. Excitation and Emission Wavelengths of AFP proteins. Strategy of FRET between CFP and YFP. Biochemical Modulation of AFP fluorescence. The General Design of FRET-based Fluorescent Probes. Some early indicators for second messenger.

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Fluorescent proteins

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  1. Fluorescent proteins

  2. Excitation and Emission Wavelengths of AFP proteins

  3. Strategy of FRET between CFP and YFP

  4. Biochemical Modulation of AFP fluorescence

  5. The General Design of FRET-based Fluorescent Probes

  6. Some early indicators for second messenger

  7. Using fluorescence protein to imaging signaling transduction

  8. Biological Questions on GPCR • Dimer/oligomerization • Ligand induced Conformational change • G protein coupling • Second messenger signaling cAMP/PKA, PKC, DAG, IP3, Ca2+ Scaffold protein/signal complex

  9. Biological Questions on GPCR • Dimer/oligomerization • Homo/hetreo dimer • Constitutive vesus ligand induced dimer • Dimer in receptor maturation in ER • New agonism • Functional diversity and Signaling integration

  10. Biological Questions on GPCR • Ligand induced Conformational change • Structural functional study • Agonism/antagonism • Receptor activation

  11. Biological Questions on GPCR G protein coupling *Real time activation *Screen model for drug discovery *Signaling/function co-relation. *Single cell study, instead of biochemical assay of GTPgS binding

  12. Biological Questions on GPCR Second messenger signaling cAMP/PKA, PKC, DAG, IP3, Ca2+ • Spatial and temporal resolution • Localized signaling properties, specificity and efficiency. • Specific function

  13. Restricted cAMP signaling in cardiac myocytes cAMP indicator

  14. Promiscuous G protein Coupling by GPCR

  15. Go-CFP resistant to PTX, Cell pretreated with PTX

  16. CFP 430+12.5nM 465+20nM YFP 500+10nM 535+15nM FRET 430+12.5NM, 535+15nM

  17. Control CFP 430+12.5nM 465+20nM YFP 500+10nM 535+15nM FRET 430+12.5NM, 535+15nM

  18. CFP 430+12.5nM 465+20nM YFP 500+10nM 535+15nM FRET 430+12.5NM, 535+15nM

  19. 514nM laser line bleach YFP about 70%-90% Concomitant CFP bleach about 7-10%

  20. Figure 6, Quantitative Analyses of FRET a2A-YFP + Go-CFP a2A-YFP + Gs-CFP Donor(Go):acceptor(receptor) at 1:1, a proportion of receptor is not coupled to G protein Increaing the ration leads to higher degree of precoupling. In physiological conditions, G proteins are in significant excess compared to receptor, thus a large majority of receptor are precoupled.

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