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?