Molecular Spectroscopy: Principles and Biophysical

Applications

BiCh132 Fall Quarter 2012

Jack BeauchampMany of the illustrations and tables used in these presentations were taken from the scientific literature and various WWW sites; the authors are collectively acknowledged.

This presentation is adapted in part from BiCh132 lectures of Professor Barton.

Recommended text: “Principles of Fluorescence Spectroscopy” by J. R. Lakowicz (3rd Edition; 2006)

Molecular Probes Handbook -11th Edition (Invitrogen)

Introduction to Fluorescence Spectroscopy

Useful probe of: environment structure dynamics chemical reactions

Timescales: visible absorption~ 10-15 sec vibrations ~ 10-14 sec emission~ 10-9 sec for allowed transitions 10-6-10-3 sec for forbidden transitions

On these timescales, emission is sensitive to competing processes

3210

3210

Absorption10-15 s Fluorescence

10-9 s

SolventCollisional vibrational dissipation

~ 10-12s

S1

T1

Phosphorescence10-6 – 10-3 s

S0

Intersystem crossing

Simplified Energy Level Diagram(Jablonski Diagram)

Franck–Condon principle energy diagram. Since electronic transitions are very fast compared with nuclear motions, vibrational levels are favored when they correspond to a minimal change in the nuclear coordinates. The potential wells are shown favoring transitions between v = 0 and v = 2.

Franck-Condon Principle for Electronic Transitions

Schematic representation of the absorption and fluorescence spectra corresponding to the energy diagram in previous slide. The symmetry is due to the equal shape of the ground and excited state potential wells. The narrow lines can usually only be observed in the spectra of dilute gases. The darker curves represent the inhomogeneous broadening of the same transitions as occurs in liquids and solids. Electronic transitions between the lowest vibrational levels of the electronic states (the 0-0 transition) have the same energy in both absorption and fluorescence.

Franck-Condon Principle for Electronic Transitions

Edward Condon

Franck-Condon Principle for Electronic

Transitions (1926)

Classically, the Franck–Condon principle is the approximation that an electronic transition is most likely to occur without changes in the positions of the nuclei in the molecular entity and its environment. The resulting state is called a Franck–Condon state, and the transition involved, a vertical transition. The quantum mechanical formulation of this principle is that the intensity of a vibronic transition is proportional to the square of the overlap integral between the vibrational wavefunctions of the two states that are involved in the transition.

Edward Condon

F = fluorescence quantum yield = fraction of singlets relaxing from excited state via fluorescence # photons emitted by fluorescence

unless some catalytic chemiluminescent process

Fluorescence Intensity x # excited state molecules x c I0

kF = rate of spontaneous emission P00 = transition probability

same path for excitation and emission

10 F

# photons absorbed=

FF

othersF

FF kk

k=

Rate constant for emission

kF + (rate constants for non-radiative pathways)

Fluorescence Quantum Yields

1. Internal conversion, kic

collision with solventdissipation of energy through internal vibrational modesbasically transfer into excited vibrational states of S0

Note - kic increases with Ttherefore FF decreases with T

What processes compete with fluorescence?

kF kic kq

kis

S0

S1

T1

kF kic kq

kis

S0

S1

T1

2. Intersystem crossing, kis

spin exchange converts S to Tget slow spin-forbidden phosphorescencefor metal complexes often a mixture of states

so “luminescence”

3. Collision with quencher, kq

e.q. S1+Q S0+Q*

molecules can quench excited state by: energy transfer spin exchange (paramagnetic + spin orbit coupling) electron transfer or proton transfer (+ energy)

Note - kF is not temperature dependent but all else is

][Qkkkk

k

qisicF

FF

So, what matters are the rates of these competing processes

Also,

Decay Kinetics of S1

Suppose initially have concentration in S1 of S1(0) then turn off light

Integrating,

where

If no other processes except fluorescence,

11 )( Skk

dt

dSothersF

tkk othersFeStS)(

11 )0()(

F

t

eS )0(1

othersFF

kk1

F fluorescence lifetime (measurable)

then RF

F k

1Radiative lifetime

R

FF

Can measure steady state or time resolved emission

For lifetimes: - then flash and turn off light and measure decay as a function of time- flash photolysis- single photon counting- streak cameras- time resolution depends on flash(also frequency domain measurements - phase modulation)

For quantum yields, need geometry constant and correct for emission detectors-use standards (actinometry)

Practical things:

Excitation

Monochromator

Emission

Monochromator

Detector

Light source Emitted light

Sample

Principle: When a fluorescent molecule is excited with plane polarized light, light is emitted in the same polarized plane, provided that the molecule remains stationary throughout the excited state (which has a duration of 4 nanoseconds for fluorescein). If the molecule rotates and tumbles out of this plane during the excited state, light is emitted in a different plane from the excitation light. If vertically polarized light is exciting the fluorophore, the intensity of the emitted light can be monitored in vertical and horizontal planes (degree of movement of emission intensity from vertical to horizontal plane is related to the mobility of the fluorescently labeled molecule). If a molecule is very large, little movement occurs during excitation and the emitted light remains highly polarized. If a molecule is small, rotation and tumbling is faster and the emitted light is depolarized relative to the excitationplane.

Fluorescence Polarization / Depolarization

Practical (sometimes annoying) things:

Schematic representation of FP detection. Monochromatic light passes through a vertical polarizing filter and excites fluorescent molecules in the sample tube. Only those molecules that are oriented properly in the vertically polarized plane absorb light, become excited, and subsequently emit light. The emitted light is measured in both the horizontal and vertical planes.

Fluorescence Polarization / Depolarization

Here Ill is the intensity of emitted light polarized parallel to the excitation light, and I⊥ is the intensity of emitted light polarized perpendicular to the excitation light. An important property of the polarization that emerges from this equation is that it is independent of the fluorophore concentration. Although thisequation assumes that the instrument has equal sensitivity for light in both the perpendicular and parallel orientations, in practice this is not the case.

Fluorescence Polarization / Depolarization

Sarah A. Weinreis, Jamie P. Ellis, and Silvia Cavagnero, Dynamic Fluorescence Depolarization: A Powerful Tool to Explore Protein Folding on the Ribosome, Methods. 2010 , 52(1): 57–73. doi:  10.1016/j.ymeth.2010.06.001

Schematic depiction of a protein folding reaction in the cytoplasm of an E. coli cell, showing vividly how different the environment is from dilute in vitro refolding experiments. The cytoplasmic components are present at their known concentrations. Features of particular importance to the folding of a protein of interest (in orange) are: the striking extent of volume exclusion due to macromolecular crowding, the presenceof molecular chaperones that interact with nascent and incompletely folded proteins (GroEL in green, DnaK in red, and trigger factor in yellow), and the possibility of co-translational folding upon emergence of the polypeptide chain from the ribosome (ribosomal proteins are purple; all RNA is salmon). The cytoplasm image is courtesy of A. Elcock.

Anne Gershenson and Lila M. Gierasch, Protein Folding in the Cell: Challenges and Progress, Curr Opin Struct Biol. 2011, 21(1):32–41. http://dx.doi.org/10.1016/j.sbi.2010.11.001

Practical things (for a few $ more):

http://www.olympusfluoview.com/applications/fretintro.html

Excitation Spectrum – the excitation wavelength is scanned while the emission wavelength is held constant

Emission Spectrum - the emission wavelength is scanned while the excitation wavelength is held constant

- often gives the mirror image of the absorption spectrum

Mirror generally holds because of similarity of the molecular structure and vibrational levels of S0 and S1

Given the Franck-Condon Principle, electronic transitions are vertical, that is they occur without change in nuclear positions. If a particular transition probability between 0 and 2 vibrational levels is highest in absorption, it will also be most probable in emission.

Stokes shift: fluorescent emission is red-shifted relative to absorption

Some Exceptions to Mirror Image Rule

1. Contaminants !!

2. Excitation to higher state(s) S2

3. Different geometry in excited state

4. Exciplexes (CT state)

5. Excimers

6. pK effects (excited state acid base properties)

Dimer excited state

Acid-base properties are modified in electronically excited states

Example- pKa for acridine in ground state= 5.5 pKa for acridine in excited state= 10.7

protonation can occur during excited state lifetime

Effects are quantified with use of the Förster Cycle

Think of some applications of this phenomenon

Förster Cycle: Quantifies changes in acid-base properties in electronically excited states

ArOH (aryl alcohol such as napthol) – The shift in absorption spectra of the acid and its conjugate base can be used to quantify the difference in pKa in the ground and excited electronic state

.

Fluorescent Probes

Absorption and emission spectra of biomolecules. Top: Tryptophan emission from proteins. Middle: Spectra of extrinsic membrane probes. Bottom: Spectra of the naturally occurring fluorescence base, Yt base. DNA itself(---) displays very weak emission

Normally use extrinsic probes or modified bases/ unnatural amino acids (check out the Molecular Probes Catalogue)

when intercalated, yield and lifetime increase

Probe

Dansyl chloride

Ethidium

lmax max (x10-3) lmax F F (ns)

Absorption Fluorescence

340-350

274

4.3

1.4

510-560

303

0.1-0.3

0.05

10-15

2+ DNA ~1 20

If you have 2 fluorescent components (probes), even two bound components, they will have different rates of quenching, kq

kq gives measure of accessibility of chromophore

F1

F2

Q

kq for F1 > kq for F2

Fluorescence Quenching

In the absence of quencher,

in the presence of quencher,

where quenching is the result of bimolecular collisions.

Stern-Volmer Analysis of Quenching

othersF kk

10

][

1

Qkkk qothersFQ

othersF

q

othersF

qothersF

Q kk

Qk

kk

Qkkk ][1

][0

][1 0 Qkq

][1 QKSV

Stern-Volmer quenching with concentration of Q, [Q]

Stern-Volmer Plot

[Q]

Q 0

1

Slope=KSV

][10 QKSVQ

where KSV=kq

expect kq’s of 1010 M-1s-1 or less

Values of kq reflect

collisional frequency and bimolecular diffusion controlled rate constant, k0

))((1000

40 qFqF DDRR

Nk

Smoluchowski eqn.

R= collisional radiiD= diffusion coefficients

kq= fQk0 fQ = quenching efficiencyif fQ = 0.5, 50% of collisions lead to quenching

Can estimate D from Stokes-Einstein eqn.

R

kTD

6

]Q][F[

]FQ[KS Q + F FQ

Consider equilibrium formation of a ground state complex which is not fluorescent:

The total conc. of fluorophore = ]FQ[]F[]F[ 0

]Q][F[

]F[]F[K 0

S

1]F[

]F[]Q[K 0

S

]F[

]F[]Q[K1 0

S

or

If FQ is not fluorescent, then 00 F

F

]F[

]F[ fraction of fluorescence

F

F]Q[K1 0

S

so that

gives same KS.V. as

But could have

or even

00

F

F

[Q]

F

F0

[Q]

Q 0

1

Slope=KS.V.

[Q] [Q]

F

F0

F

F0

Q 0

Q 0

]Q[K1F

FSV

0

For dynamic quenching, quenching process is diffusion controlled

For static quenching

but no change in – not a diffusion controlled process

[Q] [Q]

F

F0

F

F0

Q 0

Q 0

Dynamic Static

Singlet-Singlet Energy Transfer(Förster Transfer)

Singlet-Singlet Energy Transfer(Förster Transfer)

Singlet-Singlet Energy Transfer(Förster Transfer)

Very useful for “long range” distance (20-80 Å)

R

R

Donor Acceptor

D*

D0

A*

A0

Pick donor and acceptor to have appropriately matched energy levels:

D D A A

Absorption AbsorptionEmission Emission

kT= rate constant for transfer

D* +A0 D0+A*kT

k-T

k-T is not likely given rapid vibrational relaxation

Energy transfer gives sensitized emission and donor deexcitation

Resonant interaction with acceptor excitation- weak coupling limit

nm/

Real world example: Cyan fluorescent protein/Red fluorescent protein

Absorption and emission spectra of cyan fluorescent protein (CFP, the donor) and red fluorescent protein (RFP or DsRed, the acceptor). Whenever the spectral overlap of the molecules is too great, the donor emission will be detected in the acceptor emission channel. The result is a high background signal that must be extracted from the weak acceptor fluorescence emission.

What’s the basis for the interaction?

-As in exciton coupling, dipole-dipole: just weak coupling limit

Can describe the potential operator

5AD

3AD

DA R

)R)(R(3

RV

Where R is distance between A + D and are dipole moment operators

AD ,

3

AD

Rlump all geometric and orientational parameters in here- really hard to know , lots of variability

= 0-4

According to Fermi’s Golden Rule:

-rate of transition is proportional to the square of the expectation value for the interaction causing the excitation.

2

AbDaDAAaDbT Vk

2

3AbDaADAaDbT Rk

6

22

AbAAa

2

DaDDb R

D

032

DaDDb

1A

2

AbAAa

emission absorption

for isoenergetic D(b a) emission A(a b) excitation

quantum yieldlifetime of donor w/o acceptor

frequency of transition

extinction coefficient for A

4A

D

D6

2

T Rk

60

DT )

R

R(

1k

d)(f)(J 4DA

d)(f)(J 4DA

For general case, where transition involves a range of frequencies

cm)nJ(10x7.9R 61

D423

0 where

A)nJ(10x79.8R 61

D425

0

and

or

normalized fluorescence of donor overlapping with acceptor

refractive index of medium between donor and acceptor

DTothersF

TothersFAD

D k1kk

kkk

6

60

R

R1

1k

k1k

k

kkk

kE

T0

0T

0T

T

othersFT

T

660

660

RR1

RR

60

6

60

RR

RE

Naively, looks like D is emitting and A is reabsorbing but that transfer is trivial.

Also what would be effect on ?D

Usual to define efficiency

60

6

60

RR

RE

get 1/R6 dependence for E

can measure 10-100 Å distance separations depending on FRET pair

Want to measure donor-acceptor partners near R0 depending on experiment

This yields largest change in E for small changes in R that occur in the given experiment.

Very unique distance regime- FRET provides a spectroscopic ruler

Selected Applications of FRET

• Structure and conformation of proteins • Spatial distribution and assembly of protein complexes • Receptor/ligand interactions • Immunoassays • Probing interactions of single molecules • Structure and conformation of nucleic acids • Real-time PCR assays and SNP detection • Detection of nucleic acid hybridization • Primer-extension assays for detecting mutations

• Automated DNA sequencing • Distribution and transport of lipids • Membrane fusion assays • Membrane potential sensing • Fluorogenic protease substrates • Indicators for cyclic AMP and zinc

- Molecular Probes website

Different ways to carry out experiment:

monitor quenching of donor and/or enhanced emission by acceptor

1.) Quenching of donorD aloneD+A

E= fraction of donors deexcited

therefore 1-E= fraction of donors remaining excited

2.) Enhanced emission by acceptor-should be sensitized emission: excite D, watch A emit

D absorb

Acceptor emission

watch here

D

AD

D

DA

F

F

l

I

l

I

Dalone D+A Aalone

donor quench A sensitized emission

An example: Distance measurement in melittin

Depending upon solvent, can exist as monomer or tetramer, -helix or random coil

In practice, want 3 replicas for study:

Overlap integral (shaded area) for energy transfer from a tryptophan donor to a dansyl acceptor on melittin. R0=23.6 Å

Determine overlap integral for trp/dansyl pair:

R0= 23.6 Å

55.0E1F

F

D

DA

E=0.45 R=24.4Å

But there are issues-

1.) 2 is not known, nor directly measurable for so even rough estimate suffices

6120 )(RR

Dale Eisinger Method- exploit the jitter

Likely there is fast geometric averaging before transfer, blurring 2

often set 2=2/3 for dynamic avg. of all geometriesmeans uncertainty in R is < 15%

macromolecule

acceptor

donor

κ is related to the relative orientation of the donor/acceptor pair

2.) Imperfect Stoichiometry

3.) Does the probe perturb the structure?

if possible it is good to rely on intrinsic probes: in a protein tyr/trp energy transfer is possible

D

A1

A2

kT1

kT2

otherF2T1T

2T1T

kkkk

kkE

(monomer/ tetramer equilibrium for example)

Classic papers: A Spectroscopic RulerLubert Stryer and Richard HauglandProc. Natl. Acad. Sci USA, 58, 719-726 (1967)

DA

A = magnitude of excitation = a + E x d

Without the donor, excitation is that of acceptor; in the presence of donor, see sensitized emission and therefore excitation includes that of donor.

Mapping the Cytochrome c Folding LandscapeJulia G. Lyubovitsky, Harry B. Gray,* and Jay R. Winkler*

JACS, 2002, 124, 5481

Measurements of FRET between heme and C-terminal dansyl

There is a rapid equilibration among extendedconformations, enabling escape from frustratedcompact structures

Some population of extended conformations,with long distances remain even at long times.

Single Molecule Fluorescence Experiments

Example: Nucleic Aid Conformation and dynamics

Single molecule FRET study of Holliday junction by total internal reflectance microscopy. The nucleic acid is tethered to the surface via biotin-neutravidin conjugation. The conformational dynamics is shown in the fluorescence time trace.

McKinney, Declais, Lilley, Ha, Nature Structural Biol. 10 93-97 (2003)

TIRF

Area detector/camera