FRET assays with genetically targeted labels • Protein-protein interactions – heterotrimeric G proteins, transcription factors… • GPCR conformational changes at ms time resoln. • GTP/GDP-bound status of small G proteins • cAMP, cGMP, NO, Zn 2+ • Proteases such as caspases • Ca 2+ : cytosol vs. ER • Protein kinase/phosphatase activities – EGFR, Src, Abl, PKA, PKB, PKC • PIP 2 /IP 3 , DAG, other lipid-related signals otein knockouts in seconds by CALI
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FRET assays with genetically targeted labels Protein-protein interactions –heterotrimeric G proteins, transcription factors… GPCR conformational changes.
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FRET assays with genetically targeted labels
• Protein-protein interactions– heterotrimeric G proteins, transcription factors…
• GPCR conformational changes at ms time resoln.• GTP/GDP-bound status of small G proteins• cAMP, cGMP, NO, Zn2+
• Proteases such as caspases• Ca2+: cytosol vs. ER• Protein kinase/phosphatase activities
– EGFR, Src, Abl, PKA, PKB, PKC
• PIP2/IP3, DAG, other lipid-related signals
Protein knockouts in seconds by CALI
Cameleons: Ca2+ indicators based on CaM + GFP mutants
Atsushi Miyawaki
A generic design for indicators of kinase/phosphatase activity
FHA2
CFP
ex
YFP
em
FHA2
CFP
YFP
ex em
-OH
CKAR FRET Ratio
60min, 450x Frame Rate Low FRET
High FRET
+PKC
substrate sequence designed with help from http:/scansite.mit.eduGGSGGRFRRFQTLKIKAKAGGSGG
cytosolic CKAR
PM-anchored CKAR
CKAR: C Kinase Activity Reporter
Jon Violin, Alexandra Newton (UCSD)
0 2 4 6 8 10 12 14 161.02
1.00
0.98
0.96
0.94
0.92
0.90
MyrPalmCKAR in HeLa
10M Histamine
Fur
a R
ed In
tens
ity
FR
ET
Rat
io
Time (Minutes)
88
84
80
76
72
68
0 20 40 60 80 100 120
0.96
0.95
0.94
0.93
0.92F
ura
Red
Inte
nsity
FR
ET
Rat
io
Time (Seconds)
78
76
74
72
70
MyrPalm-CKAR
averaged peaks
CKAR targeted to plasma membrane by acylation detects agonist-stimulated oscillations slightly lagging [Ca2+]c
2.5 mM ReAsH for 2 h in DMEM; 250 mM EDT wash for 30 minutes in DMEM
3 repeats of 10 second excitation
•600•400•200•0
Time (ms)•Sw 3/19
(pA
)
•-2400
•-2200
•-2000
•-1800
•-1600
•-1400
•-1200
•-1000
•-800
•-600
•-400
•-200
•0
•200
25 50 75 100 125 150 175
-2000
-1400
-800
-200
time (sec)
curr
ent
(pA
)
ReAsH-mediated photoinactivation of L-type Ca2+ channels
Oded Tour; channel cDNA and cell line from R.W. Tsien
Cl- channels in the membrane were simultaneously unaffected
Acute CALI reveals importance of synaptotagmin in endocytosis
559-563
Endocytosisassayed withFM4-64
Tetracysteine-biarsenical CALI• Compared to traditional CALI, eliminates need to
raise innocuous Abs, label with dye, microinject just the right amount
• Compared with noncovalent small molecule inhibitors, avoids need for custom drug development/med chem, allows isoform specificity
• Compared with gene knockout/RNAi: much higher temporal/spatial resolution, less chance for compensation or avalanche of effects
• But only eliminates exogenous tagged copies. Ultimately, one would knock out endogenous copies, replace by tagged copies, show function is normal until CALI suddenly initiated