1. Some structures Dansyl chloride 1,5-I-AEDANS Fluorescein isothiocyante ANS Ethidium bromide 5-[2-[(2-iodoacetyl)amino]ethylamino] naphthalene-1-sulfonic acid 5-(dimethylamino)naphthalene -1-sulfonyl chloride

2. Another family of probes The “fluorescent proteins” – Green fluorescent protein or GFP -S 65 -Y 66 -G 67 - O2O2 “fused” chromophore λ ex,max (nm) λ em,max (nm) 398508 T>30 o C 398 nm H+ 508 nm H+

3. Formation of the GFP fluorophore

4. 2008 Nobel Prize in Chemistry Osamu Shimomura, Marine Biology Laboratory, Wood’s Hole Martin Chalfie, Biological Sciences, Columbia University Roger Tsien, HHMI, University of California, San Diego

5. Let’s build a fluorimeter Light source sample detector filter slit

6. Light source sample detector monochromators Scanning excitation and emission spectra Excitation (absorbance) and emission Spectra λ max,ex – excitation maximum λ max,em – emission maximum

7. Scanning excitation and emission spectra I Wavelength (nm) 260 320 380 440

8. Fluorescence intensity and concentration I Concentration (α A) expected observed As the concentration increases the signal strength does not increase proportionately Correction factor= antilog A ex + A em 2 A ex + A em < 0.1 Beware of changes to A ex + A em Inner filter effect 0.1 0.2 A

9. Environment and fluorescence Emission λ max - exposure to more polar solvent will shift emission E.g., Tryptophan emission from protein, more polar environment longer λ emission Fluorescence Intensity 300 350 400 Protein A λ max = 320 nm Protein B λ max = 350 nm Applications: Protein folding Protein-protein, protein-ligand interactions Wavelength nm

10. Environment and fluorescence -2 Quantum yield decreases with exposure to more polar environment. e.g., ANS in a series of solvents Application: ANS will partition into hydrophobic binding site on protein-this can be monitored by enhanced fluorescence Fluorescence Intensity 400 480 560 Wavelength nm octanol propanol methanol ethylene glycol water

11. Fluorescence polarization Light source monochromators detector I II - I I II + I Polarization, P = I II and I-Intensity resolved parallel and perpendicular to excitation I II - I I II + 2 I Anisotropy, A = I II -parallel to the polarization of Incident radiation I -perpendicular to polraization of Incident light

12. I II - I I II + 2 I Anisotropy, A = Is measured as a fraction of the total fluorescence and is independent from the fluorophore concentration Anisotropy can be measured in steady-state and in time-resolved modes. Depolarization will occur as molecules rotate and this can be used to learn about molecular motion Depolarization and molecular motion Protein + ligand Protein-ligand Rotational relaxation of protein ~ 10-100 ns Fluorescent, small molecule ligand ~ relaxation < 10 ns Time-averaged anisotropy of ligand will increase as it binds to the protein.

13. Fluorescence quenching Dynamic and static quenching Dynamic quenching involves collisions with quencher molecules to depopulate the excited state. Static quenching involves complex formation between the quencher and fluorophore prior to excitation. Recall, Φ F = k F / (k F + ∑k i ) = τ / τ F Quantum yield in the presence of quencher Q (Φ F ) Q = = k F / (k F + ∑k i + k[Q]) Ratio of fluorescence intensities in the absence and presence of Q, Φ F / (Φ F ) Q = (k F + ∑k i + k[Q]) / (k F + ∑k i ) = 1 + (k[Q]/(k F + ∑k i )) = 1 k[Q]τ

14. Dynamic quenching – Stern-Volmer constant Dynamic quenching usually measured as intensity in the absence and presence of quencher, I o /I Q = 1 + K[Q]Stern-Volmer equation K – Stern-Volmer constant (I o /I Q ) [O 2 ] (M) 1.0 1.4 1.8 e.g., O 2 is a quencher of W Fluorescence We can compare W in different proteins for their sensitivity to quenching by O 2 0.04 0.08 0.12 0.16

15. Fluorescence resonance energy transfer (FRET) Energy transfer is a result of interaction between donor and acceptor molecules- does not involve emission of a photon. The extent of energy transfer depends on distance (and other factors) and has seen extensive use to assess donor/acceptor distance. Donor molecule absorbs a photon (i.e., excitation) but instead of fluorescing energy transfer occurs to a neighboring, acceptor molecule. The acceptor must have an acceptable enegertic match for it to undergo excitation (i.e., resonance)