Fluorescent proteins

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

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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

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Biological Questions on GPCR

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

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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

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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

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Restricted cAMP signaling in cardiac myocytes
cAMP indicator
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Promiscuous G protein Coupling by GPCR
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Go-CFP resistant to PTX, Cell pretreated with PTX
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CFP 43012.5nM 46520nM YFP 50010nM
53515nM FRET 43012.5NM, 53515nM
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Control
CFP 43012.5nM 46520nM YFP 50010nM
53515nM FRET 43012.5NM, 53515nM
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CFP 43012.5nM 46520nM YFP 50010nM
53515nM FRET 43012.5NM, 53515nM
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514nM laser line bleach YFP about
70-90 Concomitant CFP bleach about 7-10
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Figure 6, Quantitative Analyses of FRET
a2A-YFP Go-CFP
a2A-YFP Gs-CFP
Donor(Go)acceptor(receptor) at 11, 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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