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.
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
Simplified Energy Level Diagram
(Jablonski Diagram)
Solvent
Collisional vibrational dissipation
~ 10-12s
S1
3
2
1
0
Intersystem crossing
T1
Absorption
10-15 s
S0
3
2
1
0
Fluorescence
10-9 s
Phosphorescence
10-6 – 10-3 s
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.
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
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
Edward Condon
Fluorescence Quantum Yields
F = fluorescence quantum yield
= fraction of singlets relaxing from excited state via fluorescence
# photons emitted by fluorescence
=
# photons absorbed
0  F 1
unless some catalytic chemiluminescent process
F 
Rate constant for emission
kF
kF 

k others
=
kF +
 (rate constants for non-radiative pathways)
Fluorescence Intensity   F x # excited state molecules
  F x  c I0
kF = rate of spontaneous emission 
P00 = transition probability
same path for excitation and emission
What processes compete with fluorescence?
S1
kis
T1
kF
kic
kq
S0
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 F decreases with T
S1
kis
T1
kF
kic
kq
S0
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)
So, what matters are the rates of these competing processes
F 
kF
k F  k ic  k is  k q [Q ]
Note - kF is not temperature dependent but all else is
Decay Kinetics of S1
Suppose initially have concentration in S1 of S1(0) then turn off light

dS 1
dt
 (k F 
k
others
) S1
Integrating,
S1 (t )  S1 ( 0 ) e
 S1 (0 )e
1
where
F
 kF 
(kF 
 t
 k others ) t
F
k
others
 F  fluorescence lifetime (measurable)
If no other processes except fluorescence,
then
Also,
F 
1
kF
R
F 
Radiative lifetime
F
R
Practical things:
Sample
Excitation
Monochromator
Light source
Emitted light
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)
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.
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.
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.
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
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
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.
Practical things (for a few $ more):
http://www.olympusfluoview.com/applications/fretintro.html
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.
Some Exceptions to Mirror Image Rule
Dimer excited state
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)
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)
Absorption
Probe
Dansyl chloride
Ethidium
lmax
Fluorescence
max (x10-3)
lmax
F
0.1-0.3
340-350
4.3
510-560
274
1.4
303
0.05
+ DNA ~1
F (ns)
10-15
2
20
when intercalated, yield and lifetime increase
Fluorescence Quenching
If you have 2 fluorescent components (probes), even two bound
components, they will have different rates of quenching, kq
F1
Q
kq for F1 > kq for F2
F2
kq gives measure of accessibility of chromophore
Stern-Volmer Analysis of Quenching
In the absence of quencher,
0 
1
kF 
k
others
in the presence of quencher,
Q 
1
kF 
k
others
 k q [Q ]
where quenching is the result of bimolecular collisions.
Stern-Volmer quenching with concentration of Q, [Q]
0
Q

k k
k k
kF 
others
F
q
[Q ]
others
 1
k q [Q ]
kF 
k
others
 1  k q 0 [ Q ]
 1  K SV [ Q ]
Stern-Volmer Plot
0
Q
 1  K SV [ Q ]
where KSV=kq
0
Q
Slope=KSV
1
[Q]
Values of kq reflect
collisional frequency and bimolecular diffusion controlled rate constant, k0
k0 
4 N
1000
( R F  R q )( D F  D q )
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.
D 
expect kq’s of 1010 M-1s-1 or less
kT
6  R
Consider equilibrium formation of a ground state complex which is not fluorescent:
KS 
[ FQ ]
Q+F
FQ
[ F ][ Q ]
The total conc. of fluorophore = [ F ]0  [ F ]  [ FQ ]
 KS 
or
[ F ]0  [ F ]
K S [Q ] 
[ F ][ Q ]
[ F ]0
1  K S [Q ] 
If FQ is not fluorescent, then
[F]
[ F ]0

F
1
[F]
[ F ]0
[F]
 fraction of fluorescence
F0
 1  K S [Q ] 
F0
F
F0
so that
F
F0

0

0
gives same KS.V. as
Q
F
Slope=KS.V.
1
[Q]
[Q]
But could have
F0
0
F
Q
[Q]
or even
F0
0
F
Q
[Q]
F0
F
0
F0
Q
F
[Q]
0
Q
[Q]
Dynamic
Static
For dynamic quenching, quenching process is diffusion controlled
For static quenching
F0
F
 1  K SV [ Q ]
but no change in  – not a diffusion controlled process
Singlet-Singlet Energy Transfer
(Förster Transfer)
Singlet-Singlet Energy Transfer
(Förster Transfer)
Singlet-Singlet Energy Transfer
(Förster Transfer)
R
Donor
R
Very useful for “long range”
distance (20-80 Å)
Acceptor
Pick donor and acceptor to have appropriately matched energy levels:
D*
A*
A0
D0
kT= rate constant for transfer
D* +A0
kT
k-T
D0+A*
k-T is not likely given rapid
vibrational relaxation
Absorption
D
Emission
Absorption
A
D
Emission
A
l / nm
Energy transfer gives sensitized emission and donor deexcitation
Resonant interaction with acceptor excitation- weak coupling limit
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
V DA 
D A
R
3

3 (  D  R )( R   A )
R
5
Where R is distance between A + D and  D ,  A are dipole
moment operators

D A
R
3

lump 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.
 k T    Db  Aa V DA  Da  Ab 
2
for isoenergetic D(b
A(a
 k T    Db  Aa  D   A  Da  Ab 
2
   Db  D  Da    Aa  A  Ab 
emission
2

R
a) emission
b) excitation
3
2

2
R
6
absorption
2
  Db  D  Da   
2
0
3
D
  Aa  A  Ab    A 
1
quantum yield
lifetime of donor w/o acceptor
frequency of transition
extinction coefficient for A
 kT 

2
R
6
D
D
A
4
For general case, where transition involves a range of frequencies
kT 
1
D
(
R0
)
6
R
4
1
where R 0  9 . 7 x 10 ( J  n  D ) 6 cm
3
and
J
2
refractive index of medium
between donor and acceptor
4
  A (  )f D (  )  d
normalized fluorescence of donor
overlapping with acceptor
or
R 0  8 . 79 x 10
J
5
4
( J n  D )
2
  A (l )f D (l )l dl
4
1
6
A
Naively, looks like D is emitting and A is reabsorbing but that transfer is
trivial.
Also what would be effect on  D ?
D

A
D

k F   k others  k T
 1  kTD
k F   k others
R0
1
R
6
6
Usual to define efficiency
E 
kT

k T  k F   k others

R0
6
kT  1
R
1 R0
E 
kT
6
R0
6
R
6
R  R0
6
6
6

0
k T0
0k T  1
E 
R0
6
R  R0
6
6
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 donor
D alone
D+A
I
l
E= fraction of donors deexcited
therefore 1-E= fraction of donors remaining excited

F
DA
F
D
D
A

D
2.) Enhanced emission by acceptor
-should be sensitized emission: excite D, watch A emit
D absorb
I
Acceptor emission
l
watch here
In practice, want 3 replicas for study:
Dalone
donor quench
D+A
A sensitized
emission
Aalone
An example: Distance measurement in melittin
Depending upon solvent, can exist
as monomer or tetramer, -helix or
random coil
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
FDA
 1  E  0 . 55
FD
E=0.45
R=24.4Å
But there are issues1.)
2 is not known, nor directly measurable
for R  R 0  (  2 )1 6
so even rough estimate suffices
Dale Eisinger Method- exploit the jitter
macromolecule
acceptor
donor
κ is related to the
relative orientation of the
donor/acceptor pair
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%
2.) Imperfect Stoichiometry
kT1
A1
E 
D
k T1  k T 2
k T 1  k T 2  k F   k other
kT2
A2
(monomer/ tetramer equilibrium for example)
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
Classic papers: A Spectroscopic Ruler
Lubert Stryer and Richard Haugland
Proc. Natl. Acad. Sci USA, 58, 719-726 (1967)
A
D
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
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
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.
Single Molecule Fluorescence Experiments
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)