Download bchill_313_march9

Some structures
Dansyl chloride
1,5-I-AEDANS
5-(dimethylamino)naphthalene
-1-sulfonyl chloride
5-[2-[(2-iodoacetyl)amino]ethylamino]
naphthalene-1-sulfonic acid
ANS
Ethidium bromide
Fluorescein
isothiocyante
Another family of probes
The “fluorescent proteins” – Green fluorescent protein or GFP
-S65-Y66-G67O2
T>30oC
“fused” chromophore
λex,max(nm) λem,max(nm)
398
508
H+
398 nm
508 nm
H+
Formation of the GFP fluorophore
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
Let’s build a fluorimeter
sample
filter
Light source
slit
detector
Scanning excitation and emission spectra
sample
Light source
monochromators
Excitation (absorbance) and emission
Spectra
λmax,ex – excitation maximum
λmax,em – emission maximum
detector
Scanning excitation and emission spectra
I
260
320
Wavelength (nm)
380
440
Fluorescence intensity and concentration
As the concentration increases
the signal strength does not
increase proportionately
I
expected
Inner filter effect
observed
Aex + Aem
Correction factor= antilog
2
Aex + Aem < 0.1
0.1
0.2
Concentration
A
Beware of changes to Aex + Aem
(α A)
Environment and fluorescence
Emission λmax - exposure to more polar solvent will shift emission
E.g., Tryptophan emission from protein, more polar environment
longer λ emission
Fluorescence Intensity
Protein A λmax = 320 nm
Protein B λmax = 350 nm
Applications:
Protein folding
300
350
Wavelength nm
400
Protein-protein,
protein-ligand interactions
Environment and fluorescence -2
Quantum yield decreases with exposure to more polar environment.
e.g., ANS in a series of solvents
Fluorescence Intensity
octanol
propanol
methanol
Application: ANS will partition
into hydrophobic binding site
on protein-this can be monitored
by enhanced fluorescence
ethylene glycol
water
400
480
Wavelength nm
560
Fluorescence polarization
III - I
Polarization, P =
III + I
IIIand I -Intensity resolved parallel
and perpendicular to excitation
I -perpendicular
to polraization of
Incident light
Light source
monochromators
Anisotropy, A =
III - I
III + 2 I
III-parallel to the
polarization of
Incident radiation
Depolarization and molecular motion
III - I
Anisotropy, A =
III + 2 I
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
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.
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
Recall, prior to excitation.
and fluorophore
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]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
e.g., O2 is a quencher of W
Fluorescence
(Io/IQ) 1.8
We can compare W in different
proteins for their sensitivity to
quenching by O2
1.4
1.0
0.04
0.08 0.12
[O2] (M)
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 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)