Scanning excitation and emission spectra

I

Wavelength (nm)

260 320 380 440

1) Scan excitation with emissionset at 380 nm

-λex,max = 280 nm

2) Scan emission with excitationset at 280 nm

-λem,max = 335 nm

3 ) Scan excitation withemission set at 335 nm

Fluorescence polarization

Light source

monochromators

detector

III - IIII + IPolarization, P =

IIIand I -Intensity resolved paralleland perpendicular to excitation

III - IIII + 2 IAnisotropy, A =

III-parallel to thepolarization of Incident radiation

I -perpendicularto polraization ofIncident light

III - IIII + 2 IAnisotropy, A =

Is measured as a fraction of the total fluorescence and is independentfrom the fluorophore concentration

Anisotropy can be measured in steady-state and in time-resolved modes. Depolarization will occur as molecules rotate and this canbe 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 itbinds to the protein.

Fluorescence quenchingDynamic and static quenching

Dynamic quenching involves collisions with quencher molecules todepopulate the excited state.

Static quenching involves complex formation between the quencher and fluorophore prior to excitation.

Recall,ΦF = kF / (kF + ∑ki) = τ / τF

Quantum yield in the presence of quencher Q(ΦF)Q = kF / (kF + ∑ki+ k[Q])

Ratio of fluorescence intensities in the absence and presence of Q,

ΦF/ (ΦF)Q = (kF + ∑ki+ k[Q]) / (kF + ∑ki)

= 1 + (k[Q]/(kF + ∑ki))

= 1 + k[Q]τIn terms of intensity, I0/I = 1 + KQ , (K= k τ)

Dynamic quenching – Stern-Volmer constant

Dynamic quenching usually measured as intensity in the absence and presence of quencher,

Io/IQ = 1 + K[Q] Stern-Volmer equation K – Stern-Volmer constant

(Io/IQ)

[O2] (M)

1.0

1.4

1.8e.g., O2 is a quencher of Wfluorescence in a protein

We can compare W accessibilityin different proteins for theirsensitivity to quenching by O2

0.04 0.08 0.12 0.16

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 offluorescing energy transfer occurs to a neighbouring, acceptormolecule. The acceptor must have an acceptable energetic match for itto undergo excitation (i.e., resonance)

ExEm

donor

I

Wavelength (nm)

Spectral properties of donor acceptor pair

ExEm

acceptor

Efficiency of ET depends on distanceFörster equation relates transfer efficiency (ET) to distance,

ET =R0

6

R6 + R06

1-ET

ET

1/6

R = R0

Ro is defined as the distance at which ET is 50% efficient

ET

100

80

60

40

20

010 20 30 40

Distance Å

Determining R0

R0 = 9.78 x 103 (J n-4 κ2 ΦD )1/6 (in Å)

J – the overlap of donor emission and acceptor excitation

n – refractive index of the medium, assumed ~ 1.4 in aqueousmedia

κ2 – is the orientation between donor and acceptor dipolesusually not known with certainty, ~ 0.67

ΦD – is the quantum yield of the donor in the absence of the acceptor

Efficiency of ET depends on distanceFörster equation relates transfer efficiency (ET) to distance,

ET =R0

6

R6 + R06

1-ET

ET

1/6

R = R0

Ro is defined as the distance at which ET is 50% efficient

ET

100

80

60

40

20

010 20 30 40

Distance Å

R0 ~ 32 Å

D- A separationnear R0