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Fluorescent proteins - PowerPoint PPT Presentation


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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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Presentation Transcript
biological questions on gpcr
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

biological questions on gpcr1
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
biological questions on gpcr2
Biological Questions on GPCR
  • Ligand induced Conformational change
    • Structural functional study
    • Agonism/antagonism
    • Receptor activation
biological questions on gpcr3
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

biological questions on gpcr4
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
slide27

Go-CFP resistant to PTX,

Cell pretreated with PTX

slide28

CFP 430+12.5nM 465+20nM

YFP 500+10nM 535+15nM

FRET 430+12.5NM, 535+15nM

slide29

Control

CFP 430+12.5nM 465+20nM

YFP 500+10nM 535+15nM

FRET 430+12.5NM, 535+15nM

slide30

CFP 430+12.5nM 465+20nM

YFP 500+10nM 535+15nM

FRET 430+12.5NM, 535+15nM

slide32

514nM laser line bleach YFP about 70%-90%

Concomitant CFP bleach about 7-10%

slide34

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.