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Lecture 4: Fluorescence
UV/Visible and CD are Absorption techniques: The electron absorbs energy from the photon, gets to an excited state.But what happens after that?
The electron can be completely kicked out of it’s orbital (which we can detect as a current – think PMT)The electron can lose it’s energy by exciting the vibrational modes of nearby atoms (heat)The electron can lose it’s energy by emitting a photon of lower energy than the original transition.
This last option is called Fluorescence, and where it occurs, we can use it to our advantage
Fluorescence:Physical Basis
The first step in the fluorescence process is absoprtion. As we know, this occurs as a normal/boltzmann distribution of energies
h1
*
AbsorptionRelaxation: Measured as the Fluorescence Lifetime (~ 1 – 25 ns)
h2
h
Fluorescence: Always at a higher wavelenth, can be anisotropic
Fluorescence: Quantum Yield
An important difference between measuring absorption and fluorescence is that the photo-emission pathway must compete with other energy decay pathwaysThis results in an ‘efficiency of Fluorescence’ called the Quantum Yield ()
absorbed
emitted
h
h
All of the decay pathways that contribute to are sensitive to the environment of the fluorophore including:The
solventTemperature
Nearby resonant electrons
Fluorescence Lifetime
The Quantum Yield can also be expressed as a function of the rates of the competing decay processes
iie
e
kk
kki = the rate associated with any non-radiative process
ke = rate of radiative decay
The non-radiative pathways are:Collisional quenching (Temperature): Collision excites vibrational state that is resonant with e-*Nearby dipole (Solvent/Quencher): Excited e- interacts with a nearby ground state e- dipoleInternal conversion (Fluorophore): Excited e- loses energy internally, through direct interactions with other electrons or by adopting a more favorable spin state
Fluorescence Lifetime
The Fluorescence Lifetime is the mean period for which an electron stays in the excited state before emitting a photon
i
ie kk
t e0[*][*][*] = population of excited e-[*]0 = initial population of excited e-
Fluorescence lifetime is commonly used as an imaging technique because it minimizes the effects of photon scattering
The lifetime is the inverse of the rate constant:
i ie kk
L1
Lifetimes are typically in the .1 – 30 ns range
Fluorescence Lifetime Data
There are two ways of measuring the fluorescence lifetime:
The ‘direct’ method
i
ie kk
t e0[*][*]
Phase Modulation
FT Fluorescence Lifetime.mw
Fluorescence Anisotropy
Fluorescence anisotropy results from preferential absorption of light that is polarized in the same plane as the excitation vector
During the relaxation phase, the analyte will ‘tumble’ in solution. The plane polarization of the photon emitted will reflect the new (random) orientation, thus:
90
90
II
IIA
p
pFFA is related to the rotational
diffusion coefficient by:tDtF
ReA 6)(
For real fluorophores, we must also deal with fluorescence relaxation and limiting anisotropy:
cfoFAFA /1)/(
Intrinsic Fluorescence in Proteins
Most biological molecules are essentially non-fluorescent (DNA and RNA are very weakly fluorescent @ 330 nm)But there are three fluorophoric amino acids:
Phenylalanine (257282
nm) = .04
Tyrosine (274303
nm) = .14
Tryptophan (280~348
nm) = .20
Of these, Tryptophan is by far the most useful, but is very sensitive. Can range from 0 – 1 depending on environment.
Tryptophan Fluorescence
Absolute fluorescence intensity doesn’t tell us anything specific about the environment of the fluorophore
Under most circumstances, tryptophan fluorescence is reduced upon exposure to water, but not always…
But the emission does: Tryptophan fluoresces at ~345 nm in water, but ~330 nm in apolar solvent
Fluorescence Quenching in Proteins
Tryptophan fluorescence is commonly quenched by two factors:
Proximity to protonated acidic residues such as Asp or Glu
Proximity to resonant ligand (usually Heme):
Artificial Fluorescent Probes
Oftentimes, especially with other biological macromolecules, there is no suitable fluorophore, so:Covalently bind fluorescent
‘probe’:
Non-covalent probes:
Do the measured properties of the molecule remain the same??
Förster Resonance Energy Tranfer (FRET)Förster resonance energy transfer occurs when one fluorophore ‘donates’ it’s excitation energy to a second fluorophore.The efficiency of FRET pairs depends on the overlap integral between the emission spectrum of the donor and the absorption spectrum of the acceptor:
dI ADoverlap 4)()(
FRET overlap integral.mw
FRET is a ‘Molecular Ruler’
The reason that FRET is so useful is that, assuming fluorophores and solvent conditions are constant, the transfer efficiency is directly related to the proximity.
60 )/(1
1
RrEFRET
r = the distance between donor and acceptor and R0 is the Förster radius (distance at
which FRET efficiency is 50%)overlapDInR 42286
0 10*8.8
2 = dipole orientation factor (0-4), n = refractive index of medium (1.333 in H2O @ 20°C), D quantum yield of the donor and
Ioverlap is the overlap integral
Artificial FRET Probes
There are lots of companies selling FRET probe pairs. The goal is to get the right excitation and emission while maximizing the overlap integral.
CellTrace™ calcein violetAlexaFluor™ 514
Good FRET Pair!
CellTrace™ calcein violetAlexaFluor™ 680
Bad FRET Pair!
http://www.probes.com/servlets/spectraviewer?fileid1=453h2o
FRET Probes: GFP Family FRET
The family of fluorescent proteinsGFP with fluorophore.pse
The GFP chromophore
S65T is much more efficient
Different chromphores give different ex and em
Fluorescence: Instrumentation
A basic fluorimeter should look a lot like a UV/visible Spectrophotometer. We need:
A light source (Xenon - we don’t care if it’s a bit noisy) A monochromator (sometimes not Czerny-Turner: We don’t require <1 nm resolution of excitation)A sample compartment
A photomultiplier (usually of the ‘red sensitive’ variety to help with measurements at longer wavelengths)
Applications: Equilibrium Protein FoldingFluorescence is the preferred spectroscopic technique for equilibrium protein folding experimentsIt is most often used as a ‘tryptophan environment’ probeWhich depends more on tertiary structure than secondary structureSo Fluorescence is often used in conjunction with CD222nm as a means of detecting equilibrium folding intermediates
Biophys. J., 93 (5): 1707-1718, 2007
Applications: Time Resolved FluorescenceThe extreme sensitivity and high duty cycle of Fluorescence measurements make it an excellent tool for ‘time resolved’ kinetic studies
Biophys. J. 93 (1): 208-217 2007
Fluorescence increasese upon zinc binding
FIA
Biophys. J. 93 (1): 218-224 2007
Hydrophobic collapse?3D
Fluorimetry uses CCD detector
Fluorescence Applications: FRET FoldingFRET probes are very useful in protein folding experiments as they permit direct measurements of inter-residue distances during foldingNucleosome Core
Particle
With FRET probes (W donor, modified C acceptors)
Applications: Single Molecule
Fluorescence is so sensitive, detection of single molecules is possible. Common applications are in biophysics:
Applications: Single Molecule FRET
Single moleucle FRET is a power tool for protein folding studies
Proc. Nat. Sci. USA (2007) 104 (1): 123-127
Looking to prove ‘downhill’ folding
A ‘bimodal’ distribution indicates two states
Time-Resolved, Single Molecule FRETSingle molecules can be monitored as a function of time by trapping them in tethered large lipid vesicles Labeled GCN4, TMR
Donor, Texas Red acceptorBiotinilated Lipid
VesicleStreptavidin monolayerDenaturant concentration near Tm
Single Molecule Instrumentation
Instruments for single molecule studies are complex, often due to the need to ‘trap’ the sample using (red) lasers
Fluorescence: Anisotropy
Fluorescence Anisotropy can be used to measure binding stoichiometry
Protein HU titrated into saturating DNA
Biochemistry (2003) 42 (10): 3096-3104
Anisotropy goes up due to rotational restriction of fluorescent probes on binding
Kinetics of Anisotropy Decay
Fluorescence anisotropy decays at a rate that depends on the rotational diffusion of the analyte
Biophys J. (1990), 57(4): 759–764.
Fluoresence Parallel Fluorescen
ce 90°
Anisotropy
tDtF
ReA 6)(
Fluorescence: Cellular Methods
Fluorescence microscopy is a very popular way of getting ‘sub-cellular localization’ of a protein. The analyte is usually made a GFP fusion.
Virology (2008), 370 (1)
Co-localized proteins (HEV replicase and ER proteins) are Yellow!
Fluorescence: Super Cool TechnologyDr. Robert Birge has invented a number of computer component devices using the fluorescence of Bacteriorhodopsin