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
Q
FFK
Combined Dynamic and Static Quenching
MACMAC
1000
][)/(
)0(
3 ANQVcmmolecules
volumeunitperquenchersmean
where
eP
Therefore
)1000/]([0 ])[1( AVNQD eQK
F
F
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/ƒ
where 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