1. CSB Techniques Workshop May 2008 Fluorescence Methods Jeremy Moore

2. Why use Fluorescence Spectroscopy? Sensitive – each assay uses μg amounts of protein Sensitive – each assay uses μg amounts of protein Versatile – useful for ligand binding, enzyme kinetics, protein-protein interactions, protein folding/unfolding, detergent association Versatile – useful for ligand binding, enzyme kinetics, protein-protein interactions, protein folding/unfolding, detergent association Quick – assays can take less than 30 mins to 1 hour Quick – assays can take less than 30 mins to 1 hour Reproducible – if experiment designed right! Reproducible – if experiment designed right!

3. Basic Principle Emission Excitation 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

4. Application to Proteins – Intrinsic fluorescence Phe, Tyr and Trp all give rise to intrinsic fluorescence when excited Phe, Tyr and Trp all give rise to intrinsic fluorescence when excited Phe 280/310, Tyr 270/305, Trp 290/340 Phe 280/310, Tyr 270/305, Trp 290/340 Phe emission is weak in comparison to Tyr and Trp Phe emission is weak in comparison to Tyr and Trp Fluorescent emission (quantum yield) is sensitive to solvated state of fluorophore, ie solvent exposed = weak emission, hidden = strong Fluorescent emission (quantum yield) is sensitive to solvated state of fluorophore, ie solvent exposed = weak emission, hidden = strong

5. Ligand Fluorescence Fluorescence is dependent on conjugation Fluorescence is dependent on conjugation Very useful for reduction/oxidation reactions involving ligand and/or cofactor Very useful for reduction/oxidation reactions involving ligand and/or cofactor Perform excitation scan for likely wavelengths to excite at (check buffer spectrum first!) Perform excitation scan for likely wavelengths to excite at (check buffer spectrum first!)

6. Ligand/Protein Coupled Fluorophore Large number of commercially available fluorophores from Molecular Probes (see Invitrogen website) Large number of commercially available fluorophores from Molecular Probes (see Invitrogen website) Can choose excitation/emission characteristics Can choose excitation/emission characteristics Determine which coupling system is more suitable for your purpose, functional group, protein mutagenesis, buffer! Determine which coupling system is more suitable for your purpose, functional group, protein mutagenesis, buffer! Beware length of linker between protein and fluorophore – alkyl chains like to associate with detergents! Beware length of linker between protein and fluorophore – alkyl chains like to associate with detergents! Highly hydrophobic fluorophores may associate with hydrophobic regions – non specific binding, also observed with detergents!! Highly hydrophobic fluorophores may associate with hydrophobic regions – non specific binding, also observed with detergents!!

7. Fluorescence Resonance Enhancement Transfer (FRET) Occurs when emission wavelength of donor species overlaps with excitation wavelength of acceptor Occurs when emission wavelength of donor species overlaps with excitation wavelength of acceptor Only occurs if fluorophores are within ~100 Å (Főrster radius: distance at which energy transfer is 50% efficient) Only occurs if fluorophores are within ~100 Å (Főrster radius: distance at which energy transfer is 50% efficient)

8. FRET Excitation Excited state lifetime Ground state Excited electronic singlet state Relaxed singlet excited state donor acceptor

9. Example – OpcA outer membrane protein (N. meningitidis) Excitation 280 nm Excitation 280 nm Emission Max 340 nM Emission Max 340 nM Trp emission from tyr excitation? Trp emission from tyr excitation?

10. FRET in action Ex 280 Em 340

11. Effect of detergent concentration on emission Β-octyl glucoside cmc 0.7% Β-octyl glucoside cmc 0.7% Emission increases until over cmc Emission increases until over cmc Trp association with hydrophobic region of detergent micelle / excludes solvent Trp association with hydrophobic region of detergent micelle / excludes solvent 1% 0.1% 0.5%

12. Find the reporter group! Y218F decreases by ~30% Y218F decreases by ~30% Y169F decreases by ~64% Y169F decreases by ~64% WT Y218F Y169F

13. Ligand-Fluorophores used Fluorescamine Dansyl 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

14. Ligand binding - monosaccharides Total volume 3 ml Total volume 3 ml 30 μg protein (10 µg/ml) 30 μg protein (10 µg/ml) 3 µl additions ligand 3 µl additions ligand 10 additions 10 additions 2-5 mins for mixing 2-5 mins for mixing ~30 mins total assay time ~30 mins total assay time

15. Analyse your data! Try to cover range of quench/enhancement Try to cover range of quench/enhancement Do appropriate controls – makes for an easier life later Do appropriate controls – makes for an easier life later Do repeats to ensure its not an artifact Do repeats to ensure its not an artifact

16. Binding site mutations 1 μMμM

17. Binding site mutations 2

18. Structure of binding site with sialic acid ? Recreate cystallisation conditions in cuvette – dissociation constants can change in different solutions! Recreate cystallisation conditions in cuvette – dissociation constants can change in different solutions!

19. Hints/Tips Make sure you have the correct cuvette: Make sure you have the correct cuvette: use quartz if using wavelengths below 340 nm use quartz if using wavelengths below 340 nm Clean cuvettes thoroughly – proteins can adhere to the optical windows Clean cuvettes thoroughly – proteins can adhere to the optical windows 1:1 mixture ethanol:glacial acetic acid works 1:1 mixture ethanol:glacial acetic acid works Do good controls, make sure experiments are reproducible – some experiments might only be observing ± 5-10 % difference in emission intensity Do good controls, make sure experiments are reproducible – some experiments might only be observing ± 5-10 % difference in emission intensity Keep your ligand additions as small as possible, make sure you are not observing a dilution effect! Keep your ligand additions as small as possible, make sure you are not observing a dilution effect! Allow for mixing/equilibriation – does cuvette holder have a magnetic stirrer? Is the stirrer moving? Allow for mixing/equilibriation – does cuvette holder have a magnetic stirrer? Is the stirrer moving?