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CSB Techniques Workshop CSB Techniques Workshop May 2008May 2008
Fluorescence MethodsFluorescence Methods
Jeremy MooreJeremy Moore
Why use Fluorescence Why use Fluorescence Spectroscopy?Spectroscopy?
Sensitive – each assay uses Sensitive – each assay uses μμg amounts of g amounts of proteinprotein
Versatile – useful for ligand binding, enzyme Versatile – useful for ligand binding, enzyme kinetics, protein-protein interactions, protein kinetics, protein-protein interactions, protein folding/unfolding, detergent associationfolding/unfolding, detergent association
Quick – assays can take less than 30 mins to Quick – assays can take less than 30 mins to 1 hour1 hour
Reproducible – if experiment designed right!Reproducible – if experiment designed right!
Basic PrincipleBasic Principle
EmissionExcitation
Excited state lifetime (1-10ns)
Ground
state
Excited electronic
singlet state Relaxed singlet excited
state
Quantum Yield – number of fluorescent photons emitted : number photons absorbed
Application to Proteins – Application to Proteins – Intrinsic fluorescenceIntrinsic fluorescence
Phe, Tyr and Trp all give rise to intrinsic Phe, Tyr and Trp all give rise to intrinsic fluorescence when excitedfluorescence when excited
Phe 280/310, Tyr 270/305, Trp 290/340Phe 280/310, Tyr 270/305, Trp 290/340 Phe emission is weak in comparison to Phe emission is weak in comparison to
Tyr and TrpTyr and Trp Fluorescent emission (quantum yield) is Fluorescent emission (quantum yield) is
sensitive to solvated state of sensitive to solvated state of fluorophore, ie solvent exposed = weak fluorophore, ie solvent exposed = weak emission, hidden = strongemission, hidden = strong
Ligand FluorescenceLigand Fluorescence
Fluorescence is dependent on Fluorescence is dependent on conjugationconjugation
Very useful for reduction/oxidation Very useful for reduction/oxidation reactions involving ligand and/or reactions involving ligand and/or cofactor cofactor
Perform excitation scan for likely Perform excitation scan for likely wavelengths to excite at (check wavelengths to excite at (check buffer spectrum first!)buffer spectrum first!)
Ligand/Protein Coupled Ligand/Protein Coupled FluorophoreFluorophore
Large number of commercially available fluorophores Large number of commercially available fluorophores from Molecular Probes (see Invitrogen website)from Molecular Probes (see Invitrogen website)
Can choose excitation/emission characteristicsCan choose excitation/emission characteristics Determine which coupling system is more suitable Determine which coupling system is more suitable
for your purpose, functional group, protein for your purpose, functional group, protein mutagenesis, buffer!mutagenesis, buffer!
Beware length of linker between protein and Beware length of linker between protein and fluorophore – alkyl chains like to associate with fluorophore – alkyl chains like to associate with detergents!detergents!
Highly hydrophobic fluorophores may associate with Highly hydrophobic fluorophores may associate with hydrophobic regions – non specific binding, also hydrophobic regions – non specific binding, also observed with detergents!!observed with detergents!!
Fluorescence Resonance Fluorescence Resonance Enhancement Transfer (FRET)Enhancement Transfer (FRET)
Occurs when emission wavelength of Occurs when emission wavelength of donor species overlaps with donor species overlaps with excitation wavelength of acceptorexcitation wavelength of acceptor
Only occurs if fluorophores are within Only occurs if fluorophores are within ~100 ~100 Å (Főrster radius: distance at Å (Főrster radius: distance at which energy transfer is 50% which energy transfer is 50% efficient)efficient)
FRETFRET
Excitation
Excited state lifetime
Ground
state
Excited electronic
singlet state Relaxed singlet excited
statedonor
donor
acceptor
Example – OpcA outer Example – OpcA outer membrane protein (membrane protein (N. N.
meningitidismeningitidis))
Excitation 280 nmExcitation 280 nm
Emission Max 340 Emission Max 340 nMnM
Trp emission from Trp emission from tyr excitation?tyr excitation?
0
100
200
300
400
500
600
700
800
900
1000
200 250 300 350 400 450 500
Wavelength
Inte
nsity
(A
.U)
FRET in actionFRET in action
Ex Ex 280280
Em Em 340340
Effect of detergent Effect of detergent concentration on emissionconcentration on emission
ΒΒ-octyl glucoside -octyl glucoside cmc 0.7%cmc 0.7%
Emission increases Emission increases until over cmcuntil over cmc
Trp association Trp association with hydrophobic with hydrophobic region of detergent region of detergent micelle / excludes micelle / excludes solventsolvent
0
200
400
600
200 300 400 500
Wavelength (nm)
Inte
nsity
(a.
u.)
1%
0.1%
0.5%
Find the reporter group!Find the reporter group!
0
200
400
600
800
1000
200 300 400 500
Wavelength (nm)
Inte
nsity
(a.
u.)
Y218F decreases Y218F decreases by ~30%by ~30%
Y169F decreases Y169F decreases by ~64%by ~64%
WT
Y218F
Y169F
Ligand-Fluorophores usedLigand-Fluorophores used
FluorescamineDansyl
Coupled with heparin (~3K MW)
Dansyl group inserted in detergent micelle; titrated into inverse CPCL micelle to confirm
Coupled with heparin derived disaccharide
Sulphate groups on disaccharide formed ionic interactions with surface of protein – could not be titrated off
Ligand binding - Ligand binding - monosaccharidesmonosaccharides
Total volume 3 mlTotal volume 3 ml 30 30 μμg protein (10 g protein (10
µg/ml)µg/ml) 3 3 µl additions ligandµl additions ligand 10 additions10 additions 2-5 mins for mixing2-5 mins for mixing ~30 mins total assay ~30 mins total assay
timetime 0
100
200
300
400
500
600
700
800
900
1000
200 300 400 500
Wavelength (nm)
Inte
nsity
(a.
u.)
Analyse your data!Analyse your data!
Try to cover range of Try to cover range of quench/enhancemenquench/enhancement t
Do appropriate Do appropriate controls – makes for controls – makes for an easier life lateran easier life later
Do repeats to ensure Do repeats to ensure its not an artifactits not an artifact
Binding site mutations 1Binding site mutations 1
K61AK61AK31AK31A
1.171.17±±0.460.46
K29AK29A
0.830.83±±0.100.10
K27AK27A
3.813.81±±0.130.13K239AK239A
V85AV85A
0.470.47±±0.080.08
Y169FY169F
Y218FY218F
WTWT--OpcAOpcA
0.670.67±±0.100.10
0.910.91±±0.420.424.954.95±±0.850.85
5.055.05±±0.460.46
1.701.70±±0.070.07
μM
Binding site mutations 2Binding site mutations 2
Y169Y169
Y218Y218
P238AP238AT83AT83A
L81AL81AF224AF224A
0.820.82±±0.160.16
R134AR134A
1.801.80±±0.710.71
I237A 1I237A 10.400.40±±0.680.68
P82AP82A
0.070.07±±0.040.04
1.001.00±±0.090.09
3.813.81±±0.310.31
Structure of binding site with Structure of binding site with sialic acidsialic acid
?? Recreate cystallisation conditions in Recreate cystallisation conditions in
cuvette – dissociation constants can cuvette – dissociation constants can change in different solutions!change in different solutions!
Hints/TipsHints/Tips Make sure you have the correct cuvette:Make sure you have the correct cuvette:
use quartz if using wavelengths below 340 nmuse quartz if using wavelengths below 340 nm Clean cuvettes thoroughly – proteins can adhere Clean cuvettes thoroughly – proteins can adhere
to the optical windowsto the optical windows 1:1 mixture ethanol:glacial acetic acid works1:1 mixture ethanol:glacial acetic acid works
Do good controls, make sure experiments are Do good controls, make sure experiments are reproducible – some experiments might only be reproducible – some experiments might only be observing observing ± ± 5-10 % difference in emission 5-10 % difference in emission intensityintensity
Keep your ligand additions as small as possible, Keep your ligand additions as small as possible, make sure you are not observing a dilution effect!make sure you are not observing a dilution effect!
Allow for mixing/equilibriation – does cuvette Allow for mixing/equilibriation – does cuvette holder have a magnetic stirrer? Is the stirrer holder have a magnetic stirrer? Is the stirrer moving?moving?