Fluorescent proteins. Excitation and Emission Wavelengths of AFP 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

Using fluorescence protein to imaging signaling transduction

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 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 GPCR

• Ligand induced Conformational change– Structural functional study– Agonism/antagonism– Receptor activation

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 GTPS binding

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

Restricted cAMP signaling in cardiac myocytes

cAMP indicator

Promiscuous G protein Coupling by GPCR

Go-CFP resistant to PTX,

Cell pretreated with PTX

CFP 430+12.5nM 465+20nMYFP 500+10nM 535+15nMFRET 430+12.5NM, 535+15nM

Control

CFP 430+12.5nM 465+20nMYFP 500+10nM 535+15nMFRET 430+12.5NM, 535+15nM

CFP 430+12.5nM 465+20nMYFP 500+10nM 535+15nMFRET 430+12.5NM, 535+15nM

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

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 proteinIncreaing 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.