1. Scanning excitation and emission spectra I Wavelength (nm) 260 320 380 440 1)Scan excitation with emission set at 380 nm -λ ex,max = 280 nm 2) Scan emission with excitation set at 280 nm -λ em,max = 335 nm 3 ) Scan excitation with emission set at 335 nm

2. 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

3. 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 and interactions 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.

4. 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 In terms of intensity, I 0 /I = 1 + KQ, (K= k τ)

5. 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 in a protein We can compare W accessibility in different proteins for their sensitivity to quenching by O 2 0.04 0.08 0.12 0.16

6. 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 neighbouring, acceptor molecule. The acceptor must have an acceptable energetic match for it to undergo excitation (i.e., resonance)

7. Ex Em donor I Wavelength (nm) Spectral properties of donor acceptor pair Ex Em acceptor

8. Efficiency of ET depends on distance Förster equation relates transfer efficiency (E T ) to distance, E T = R06R06 R 6 + R 0 6 1-E T ETET 1/6 R = R 0 R o is defined as the distance at which E T is 50% efficient ETET 100 80 60 40 20 0 10 20 30 40 Distance Å

9. Determining R 0 R 0 = 9.78 x 10 3 (J n -4 κ 2 Φ D ) 1/6 (in Å) J – the overlap of donor emission and acceptor excitation n – refractive index of the medium, assumed ~ 1.4 in aqueous media κ 2 – is the orientation between donor and acceptor dipoles usually not known with certainty, ~ 0.67 Φ D – is the quantum yield of the donor in the absence of the acceptor

10. Efficiency of ET depends on distance Förster equation relates transfer efficiency (E T ) to distance, E T = R06R06 R 6 + R 0 6 1-E T ETET 1/6 R = R 0 R o is defined as the distance at which E T is 50% efficient ETET 100 80 60 40 20 0 10 20 30 40 Distance Å R 0 ~ 32 Å D- A separation near R 0