Molecular Spectroscopy: Principles and Biophysical Applications
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Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

Molecular Spectroscopy: Principles and Biophysical Applications

BiCh132 Fall Quarter 2012

Jack Beauchamp

Many 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.

Molecular Probes Handbook -11th Edition (Invitrogen)

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


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

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


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

Solvent

Collisional vibrational dissipation

~ 10-12s

S1

Intersystem crossing

T1

Absorption

10-15 s

Fluorescence

10-9 s

Phosphorescence

10-6 – 10-3 s

S0

Simplified Energy Level Diagram

(Jablonski Diagram)

3

2

1

0

3

2

1

0


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

Franck-Condon Principle for Electronic Transitions

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.


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

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.


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

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


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

Edward Condon


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

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

=

# photons absorbed

Rate constant for emission

=

kF + (rate constants for non-radiative pathways)

Fluorescence Quantum Yields


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

S1

kis

T1

kF

kic

kq

S0

What processes compete with fluorescence?

1. Internal conversion, kic

collision with solvent

dissipation of energy through internal vibrational modes

basically transfer into excited vibrational states of S0

Note - kic increases with T

therefore FF decreases with T


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

2. Intersystem crossing, kis

spin exchange converts S to T

get slow spin-forbidden phosphorescence

for 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)

S1

kis

T1

kF

kic

kq

S0


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

So, what matters are the rates of these competing processes

Note - kF is not temperature dependent but all else is


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

Decay Kinetics of S1

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

Integrating,

where

fluorescence lifetime (measurable)

If no other processes except fluorescence,

then

Radiative lifetime

Also,


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

Practical things:

Sample

Excitation

Monochromator

Emitted light

Light source

Emission

Monochromator

Detector

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)


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

Practical (sometimes annoying) things:

Fluorescence Polarization / Depolarization

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 excitation

plane.


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

Fluorescence Polarization / Depolarization

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.


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

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 this

equation assumes that the instrument has equal sensitivity for light in both the perpendicular and parallel orientations, in practice this is not the case.


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

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


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

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

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 presence

of 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.


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

Practical things (for a few $ more):

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


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

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

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.


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

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


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

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


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

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


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

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

.


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

Absorption

Fluorescence

Probe

lmax

max (x10-3)

lmax

F

F (ns)

Dansyl chloride

340-350

4.3

510-560

0.1-0.3

10-15

Ethidium

274

1.4

303

0.05

2

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

+ DNA ~1 20

when intercalated, yield and lifetime increase


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

F1

Q

F2

Fluorescence Quenching

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

kqfor F1 > kqfor F2

kq gives measure of accessibility of chromophore


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

Stern-Volmer Analysis of Quenching

In the absence of quencher,

in the presence of quencher,

where quenching is the result of bimolecular collisions.


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

Stern-Volmer Plot

Slope=KSV

1

[Q]

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

where KSV=kq


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

Values of kq reflect

collisional frequency and bimolecular diffusion controlled rate constant, k0

Smoluchowski eqn.

R= collisional radii

D= diffusion coefficients

kq= fQk0

fQ = quenching efficiency

if fQ= 0.5, 50% of collisions lead to quenching

Can estimate D from Stokes-Einstein eqn.

expect kq’s of 1010 M-1s-1 or less


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

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

Q + F FQ

The total conc. of fluorophore =

or

If FQ is not fluorescent, then

fraction of fluorescence


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

so that

gives same KS.V. as

Slope=KS.V.

1

[Q]

[Q]

But could have

or even

[Q]

[Q]


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

[Q]

[Q]

Dynamic

Static

For dynamicquenching, quenching process is diffusion controlled

For staticquenching

but no change in  – not a diffusion controlled process


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

Singlet-Singlet Energy Transfer

(Förster Transfer)


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

Singlet-Singlet Energy Transfer

(Förster Transfer)


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

Singlet-Singlet Energy Transfer

(Förster Transfer)

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

R

R

Donor Acceptor


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

Pick donor and acceptor to have appropriately matched energy levels:

D*

A*

kT= rate constant for transfer

A0

kT

D0

D* +A0 D0+A*

k-T

k-T is not likely given rapid vibrational relaxation

Emission

Absorption

Absorption

Emission

A

A

D

D

Energy transfer gives sensitized emission and donor deexcitation

Resonant interaction with acceptor excitation- weak coupling limit


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

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.


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

What’s the basis for the interaction?

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

Can describe the potential operator

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

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

= 0-4


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

According to Fermi’s Golden Rule:

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

for isoenergetic D(b a) emission

A(a b) excitation

emission absorption

quantum yield

lifetime of donor w/o acceptor

frequency of transition

extinction coefficient for A


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

For general case, where transition involves a range of frequencies

where

refractive index of medium between donor and acceptor

and

normalized fluorescence of donor overlapping with acceptor

or


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

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

Also what would be effect on ?

Usual to define efficiency


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

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.


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

Very unique distance regime

- FRET provides a spectroscopic ruler


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

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


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

Different ways to carry out experiment:

monitor quenching of donor and/or enhanced emission by acceptor

D alone

D+A

1.) Quenching of donor

I

l

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

I

Acceptor emission

l

watch here


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

In practice, want 3 replicas for study:

Dalone D+A Aalone

A sensitized

emission

donor quench

An example: Distance measurement in melittin

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


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

Determine overlap integral for trp/dansyl pair:

R0= 23.6 Å

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


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

E=0.45 R=24.4Å


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

But there are issues-

1.) 2 is not known, nor directly measurable

for

so even rough estimate suffices

Dale Eisinger Method- exploit the jitter

macromolecule

acceptor

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

donor

Likely there is fast geometric averaging before transfer, blurring 2

often set 2=2/3 for dynamic avg. of all geometries

means uncertainty in R is < 15%


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

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

A1

kT1

D

kT2

A2

(monomer/ tetramer equilibrium for example)


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

Classic papers: A Spectroscopic Ruler

LubertStryer and Richard Haugland

Proc. Natl. Acad. Sci USA, 58, 719-726 (1967)


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

A

D


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

Without the donor, excitation is that of acceptor;

in the presence of donor, see sensitized emission

and therefore excitation includes that of donor.

A = magnitude of excitation = a + E x d


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

Mapping the Cytochrome c Folding Landscape

Julia G. Lyubovitsky, Harry B. Gray,* and Jay R. Winkler*

JACS, 2002, 124, 5481

Measurements of FRET between heme and C-terminal dansyl


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

There is a rapid equilibration among extended

conformations, enabling escape from frustrated

compact structures

Some population of extended conformations,

with long distances remain even at long times.


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

Single Molecule Fluorescence Experiments


Molecular spectroscopy principles and biophysical applications bich132 fall quarter 2012 jack beauchamp

Example: Nucleic Aid Conformation and dynamics

TIRF

Area detector/

camera

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)


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