Measurement of Fluorescent Lifetimes: Time-Domain

Time-Correlated Single Photon Counting (TCSPC) vs.

Stroboscopic (Boxcar) Techniques.

Deconvolution

F(t) = ie-t/I

 

 

S(t) = L(t)F(t)dt

Time-Resolved Emission Spectra

Measurement of Fluorescent Lifetimes: Frequency-Domain

Measurement of Fluorescent Lifetimes: Frequency-Domain

Exciting Light: L(t) = a + bsin(t) ( = 2 x freq.) 

Emitted Light: F(t) = A + Bsin(t - ) Lifetimes: tan = x p

 m = (B/A)/(b/a) = [1 + ²m²]-½

Measurement of Fluorescent Lifetimes: Frequency-Domain

HN

Fluorescent Molecules

HN

Intrinsic Fluorescent Probes (i.e. tryptophan):•Sensitive to local environment•Relatively small•Readily available in proteins•Generated by site-directed mutagenesis

Covalent Extrinsic Probes (i.e. TAMRA):•Broad range of spectral properties•Bright, relatively photostable•Well characterized conjugation chemistry

Non-covalent Probes (i.e. mant-ATP):•Similar properties to covalent probes•No need to permanently modify protein•Target active site or ligand binding sites

Fluorescent Molecules

Intrinsic Fluorophores

Intrinsic Fluorophores

Extrinsic Fluorophores

Protein Structural Dynamics:effects on fluorescence emission spectra

Polarization experiments are sensitive to changes in orientation of a fluorescent probe.

Spectral Shifts depend on the environment around a fluorescent probe. A more polar environment tends to red shift the emission spectrum and a less polar environment tends to blue shift the emission spectrum.

Dynamic Quenching experiments are a quantitative way to measure the accessibility of a fluorescent probe to quenching molecules in the solvent.

FRET (Fluorescence Resonance Energy Transfer) experiments can measure the distance between a donor probe and an acceptor probe on the protein.

Dynamic Quenching: measure accessibility to solvent and rates of diffusion. kq - bimolecular quenching constant, proportional to rate of diffusion of quencher or fluorophore. 

= kF/(kF + kNR) 

Q = kF/(kF + kNR + kq[Q]) 

kq[Q] = pseudo-first order rate constant (M-1s-1) since [Q] >> [F]. 

/Q = (kF + kNR + kq[Q])/ (kF + kNR) = 1 + kq[Q]/ (kF + kNR) 

and since = 1/(kF + kNR)

/Q = 1 + kq[Q]Stern-Volmer Equation: 

F0/F = 1 + KD[Q] 

KD = kq = Stern-Volmer quenching constant.  Plot of (F0/F - 1) vs. [Q] is linear with slope = KD.

hv kF kNR kq[Q]

S1

S0

F0/F = = 1 + kq[Q] = 1 + KD[Q]

Stern-Volmer Plots

Static Quenching 

F0/F = 1 + KS[Q] Ks = equilibrium constant for quencher binding to fluorophore ([F-Q]/[F][Q]). Static quenching can be differentiated from dynamic quenching by: 1.) lifetime measurements - static quenching alters intensity, not lifetime.

dynamic quenching alters both. 2.) temperature effects – 

Combined Dynamic and Static Quenching:

Stern-Volmer plot is concave upward. 

F0/F = (1 + KD[Q]) x (1 + KS[Q])

= 1 + (KD + KS)[Q] + KDKS[Q]2 = 1 + Kapp[Q]

therefore 

)(][][

1)/( 0SDSDapp KKQKK

QFF

K

Combined Dynamic and Static Quenching

MACMAC

1000][

)/(

)0(

3 ANQVcmmolecules

volumeunitperquenchersmeanwhere

eP

Therefore

)1000/]([0 ])[1( AVNQD eQK

FF

Quenching Sphere of Action

Two Populations of Fluorophores: one accessible to solvent, one not.

F = Fa + Fb = (F0a/(1 + Ka[Q])) + F0b

(F0/(F0 – F)) = 1/(ƒK[Q]) +1/ƒwhere ƒ = F0a/( F0a + F0a), a = accessible and b = buried.

 

Electrostatic Effects on Dynamic Quenching

Dynamic Quenching – Two Populations of Fluorophores

1. Apoazurin Pf12. Ribnuclease T1

3. Staphylococcus nuclease4. Glucagon

Collisional Quenching in Proteins

BuriedResidue

ExposedResidue