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)

Fluorescence: Instrumentation

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

Fluorescence: Temperature Effects

UV/Visible and CD are Absorption techniques: The electron absorbs energy from the photon, gets to an excited state.