emple Chemistry Department
Philadelphia, PA
www.chem.temple.edu
Biological Photochemistry:
The fate of electronic excited states
in proteins, DNA, and the role of quenching
Robert J. Stanley
DOE Workshop on Aqueous Scintillators
January 19, 2010
Electronic excited states in Biology
• Chemiluminescence
– Bioluminescence – charge transfer? radicals?
• Photoinduced electron transfer
– Photosynthesis
– DNA repair
• Photochemistry
– DNA damage
– photosensors
DNA…a polymer of nucleotides connected by
phosphodiester linkages
5’
Nucleic acid bases A, T, C, & G
3’
Voet and Voet, Biochemistry, 2nd Ed. Wiley, New York, 1995
B-DNA is double-stranded (ds) DNA,
forming the famous double helix
(1954 - Watson, Crick, Franklin)
Watson-Crick base pairing
(complementarity)
DNA absorbs UV radiation
* transition
Absorbance (corrected)
0.8
5'-CTCCPACTTGC-3'
5'-GCAAGTTGGAG-3'
dsDNA
0.6
0.4
0.2
P=6MAP
0.0
240
260
280
300
320
340
Wavelength (nm)
360
380
400
Quenching of excited states can be desirous or
devastating in living systems: DNA
• UV light absorbed by DNA is rapidly transformed
into heat
– Conical intersections in the potential surfaces of excited
and ground state nucleic acid bases leads to ultrafast
degradation of light into heat (10-12 sec.) …GOOD!
• Excited native DNA bases (Guanine, Adenine,
Thymine, Cytosine) can be either excited state donors
or acceptors
–
–
–
–
sequence dependent reaction
*G8-oxo-G
T-T  T<>T pyrimidine dimerization
Cancer, apoptosis…BAD
UV light damages DNA
Bad photochemistry
2+2 photo-cycloaddition
O
O
O
O
CH 3
CH 3 H 3 C
HN
O
NH
N
N
T-T
h
O
< 320 nm
CH 3
HN
O
NH
N
N
T<>T
or CPD
O
If DNA damage is left unrepaired
then mutations, cell death, and cancer
can develop
http://toms.gsfc.nasa.gov/ery_uv/euv.html
Pathways involving energy transfer
D = G*, A*, C*, T*
D*A
Bright
Dark
Bright or Dark
A = G, A, C, T
ISC
3
D * A 
DA
hD
DA*
 D 3 A or D 1 A
hA
Triplet Energy Transfer
Förster or
Dexter Transfer
(singlets)
DA
Fluorescence
“Structural” quenching pathways
D*A
Bright
Dark
DhotA
hD
Intramolecular
vibrational
relaxation
Conical
Intersection
DA
Fluorescence
Pathways involving electron transfer
D*A
Bright
Dark
Bright or Dark
D  A  or D  A 
hD
D A or D A
hEX?
Photoinduced
Electron Transfer
(PET)
Exciplex (EX) formation
(charge transfer)
DA
Fluorescence
Enzymatic Repair of CPDs by DNA Photolyase uses
blue light as an energy source (Good photochemistry)
•
Repair of the thymidines is direct:
T<>T T-T without modifying
the DNA backbone
• Wide spread: E. coli, Frogs, Rice,
Kangaroos…Humans (no!)
Possible Applications:
• Photosomes® (AGI Dermatics)
• transgenic crops (wheat?)
Mees, A., et al (2004) Science 306, 1789-1793.
Sancar, A. Structure and function of DNA photolyase. Biochemistry 33, 2-9 (1994).
DNA Photolyase (PL) is a flavoprotein
(Vitamin B2) that binds and repairs CPDs
• PL functions efficiently with
only FAD (required for
repair and binding
• PL binds the CPD with high
affinity (no light required):
KA = 109 M-1 for dsDNA with CPD
Park, H.-W., Kim, S.-T., Sancar, A., and Deisenhofer, J. (1995) Science 268, 1866-72.
Flavin Structure and
Oxidation States
• Flavins can transfer 1
or 2 electrons (unlike
nicotinamide) and are used
in a large number of redox
reactions in the cell
FADH—
—
Biochemistry 2nd Ed., Voet and Voet, J. Wiley & Sons
• Surprisingly, flavins are
a major biological
chromophore (DNA repair,
circadian rhythms,
phototropism, etc.)
Photolyase functions by Photoinduced Electron
Transfer from the FAD to the CPD
• A large separation between the
FADH- and the CPD (~16 Å)
would give a slow electron transfer
rate (keT, from Marcus theory)
There’s a cavity in
the protein
keT  e
 r 2
e
 ( G    ) 2 / 4kT
Orbital overlap x Driving force
FAD
• Slow electron transfer would
compete poorly with 1FADH—
deactivation (about 5 ns)
but repair > 0.7!
What happens to substrate conformation upon
binding to Photolyase?
Minor disruption
AA
Photolyase
Moderate disruption
Base Flipping
T<>T
Severe disruption
Fluorescent reporter approach to probing
double helical structure
5’-probe approach:
5’
3’
Base Flipping 5’
3’
5’
3’

3’
5’
3’-probe approach:
5’
3’
Base Flipping 5’
3’
5’
3’
The fluorescence quantum yield of
the reporter decreases when base
stacked…but why?

3’
5’
6MAP is an attractive new fluorescent
adenosine analogue
4-amino-6-methyl-8-(2-deoxy--D-ribofuranosyl)-7(8H)-pteridone
CH3
O
H
H
N
H3C
N
6
N
H
3
C
1'
N
O
7
2
8
O
N
R
N
6MAP
Thymine
Properties:1
fl = 0.2
ex = 330 nm ( ~ 8,500 M-1cm-1)
em= 430 nm (large Stokes shift)
et al, “Synthesis and Fluorescence
Characterization of Pteridine Adenosine
Nucleoside Analogs for DNA Incorporation.”
Anal. Biochem.298, 231-240 (2001).
Absorbance (corrected)
N
4
0.03
ss-3'-6MAP
ds-3'-6MAP-TT
ds-3'-6MAP-CPD
ss-5'-6MAP
ds-5'-6MAP-TT
ds-5'-6MAP-CPD
0.02
0.01
0.00
310 320 330 340 350 360 370 380 390 400 410
Wavelength (nm)
1Hawkins,
K. Yang, S. Matsika, and R.J. Stanley, Biochemistry 2007
5’-GCAAGTTGGAG-3’
3’-CGTTCAFCCTC-5’
3.5x10
5
5'-6MAP
5'-6MAP/T<>T
5'-6MAP/T-T
a)
Fluorescence (corrected)
Base flipping of the
CPD monitored by
6MAP
3.0x10
5
2.5x10
5
2.0x10
5
1.5x10
5
1.0x10
5
5.0x10
4
5'-6MAP/PLox
b)
5'-6MAP/T<>T/PLox
5'-6MAP/T-T/PLox
+PL
-PL
Base Flipping
0.0
5’-GCAAGTTGGAG-3’
3’-CGTTCFACCTC-5’
Why is the intensity pattern
sequence-dependent?
Fluorescence (corrected)
350
6
2.0x10
1.5x10
400
450
a)
3'-6MAP
500 550 350 400
3'-6MAP/T<>T
Wavelength
b)(nm)
3'-6MAP/T-T
450
3'-6MAP/PL
500 550
ox
3'-6MAP/T<>T/PLox
3'-6MAP/T-T/PLox
6
+PL
-PL
1.0x10
6
5.0x10
5
Base Flipping
0.0
350
400
450
500
550 350
400
Wavelength (nm)
450
500
550
These data are consistent with disruption of base
stacking due to base flipping of the CPD by
Photolyase
Photolyase
?
Mees et al, Science v. 306, 1789-1793 (2004)
Is the fluorescence quantum yield modulation of
6MAP due to PET?
AMP
DMF
GMP
CMP
TMP
2.0
ACN
6MAP
-15
-25
6MAP
0
Current ( A)
I0/I
Stern-Volmer quenching of 0
1.5
6MAP by G,A,C, and T: 15
-15 DMAP
what is the rate of quenching,
1.0
0
kq?
15
-15
submitted to Biochemistry
-15
6MI
6MAP
0
0
-5
10
-425
10
-3
10
-2
10
10
-1
0
25
DMF
-25
0
25
15
-15
0
-25
ACN
15
nt ( A)
What are the redox
potentials?
Cyclic voltammetry
of 6MAP in aprotic
organic solvents
-25
Quencher Concentration (M)
3MI
0
25
2.0 1.5 1.0 0.5 0.0 -0.5 -1.0 -1.5 -2.0 -2.5 -25 2.0 1.5 1.0 0.5 0.0 -0.5 -1.0 -1.5 -2.0 -2.5
DMAP
0
Volts (NHE)
25
The quenching of 6MAP* proceeds through
nucleobase oxidation:
6MAP*:NMP6MAP¯:NMP+
9
7x10
9
6x10
FBA
9
5x10
kq (M s )
-1 -1
9
4x10
AMP
GMP
TMP
NB
GET(eV)
Eact(eV)
G
-0.63
0.000
A
-0.16
0.003
C
0.021
0.048
dT
-0.009
0.032
9
3x10
6MAP
9
2x10
CMP
(Scandola-Balzani relation)
9
10
-0.5
-0.4
-0.3
-0.2
-0.1
0.0
G°NBO (eV)
submitted to Biochemistry
0.1
0.2
What’s the mechanism for base analog quenching?
Pathways for energy transduction in a model FBA oligo
5’-NF*N-3’
h
Conical
Intersection
-1
0.5
0.0
 (M cm )
Bright
Dark
-1
1.0
2AP
3'-CCC2APGC-5'
2AP+4C+G
T = 77K
=55
-0.5
280
5’-NF +N--3’
300
320
340
360
380
Wavelength (nm)
Absorption Stark spectra of ssDNA with 2AP
(), a hexamer with 2AP () , and a mix of
the individual bases ().
Fluorescence
Exciplex
Stark and MRCI calculations (Matsika)
 and  (norm.)
5’-NFN-3’
1.2
1.0
0.8
0.6
0.4
0.2
0.0
-0.2
-0.4
-0.6
6-MI
300
IF and IF (norm.)
Photoinduced
Electron Transfer
350
400
450
Wavelength
(nm)
Stark absorption and emission spectra of 6-MI
(), a guanine analog, compared with their
absorption and emission spectra ().
Another possibility:
6MAP emission overlaps the absorption of the
FAD: FRET from 6MAP*FAD?
1800000
ss-6MAP/PLox
1600000
ss-6MAP/TT/PLox
1400000
ss-6MAP/T<>T/PLox
1200000
1000000
800000
600000
400000
200000
0
380
400
420
440
460
480
500
520
540
Wavelength (nm)
Yang et al, JPC B (2007)
Fluorescence Energy Transfer Efficiency
ET
6
R0
 6
6
R0  rDA
R0  the Förster distance where ET = 0.5
rDA  the distance between a donor (fluorescent
analogue) and an acceptor (FAD in photolyase)
The Förster distance
R0 (Å) = 0.211  ( n
D J )
2 4
1/ 6
2 : the orientation factor;
n : the refractive index of the medium;
D : the fluorescence quantum yield of the donor;
J : the overlap integral.
The Overlap Integral
F

J
D
( ) A ( ) d
4
F
D
( )d
A
FD
350
400
450
500
Wavelength (nm)
FD(): the fluorescence intensity of the donor as a function of wavelength.
εA(): the molar extinction coefficient of the acceptor at that wavelength;
The Orientation Factor
  (cos T  3 cos  D cos  A )
2
2
O
H2N
mD
N
4
3
N
2
6
H3C
1
N
R
10a
rDA
O
N
10
9
4
9a
4a
8
N
H3C
5a
R
6MAP in 3'-6MAP
NH
N
N
8
7
2
O
5
7
6
H3C
FADox in Photolyase
θT:  mD, mA
θD:  mD , rDA
θA:  mA, rDA
mA
The transition dipole moment direction
6MAP was calculated from TD-DFT
Yang et al, JPC B (2007)
Orientation factors and ET between
Probes and FADox
From the crystal
structure, lit.
and TDDFT calcs
experiment
crystal structure
Yang et al, JPC B (2007)
FRET efficiency vs. orientation
1.0
 xtal ( 6 MAP )
0.8
0.6
FRET
0.4
3'-6MAP/FAD (m1)
0.2
5'-6MAP/FAD (m1)
0.0
60
70
80
90
 (deg.)
Yang et al, JPC B (2007)
100
110
I [I(6MAP/T<>T/PL)-I(6MAP/T<>T)]
NO FRET!
• The FAD is quenched 100x in
the protein (acceptor is dark)
0.04
• A work-around : timeresolved FRET?
0.02
0.00
• Quenching mechanism is
different for the two probes
-0.02
-0.04
400
450
500
550
Wavelength (nm)
600
• photoinduced electron
transfer vs. ultrafast internal
conversion?
• Does FAD* undergo PET
to tryptophan???
Yang et al, JPC B (2007)
Can we identify the kinetics and mechanism of repair?
Two color pump probe femtosecond spectroscopy:
2
1PL 
red
• What is the electron transfer
lifetime (eT)?
: T<>T
• Does repair proceed by a
concerted or sequential
mechanism?
eT
PLsq• : T<>T •
kic, krad

c
1
3
PLsq• : T|_|T • 
h
2
4
krec
kbeT
kdiss

PLred : T-T
PLred + T-T
1
PLred  : T<>T
7
PLsq• : T-T • 
5
6
MacFarlane and Stanley (2003)
Biochemistry 42, 8558-8568
Transient absorption measurement layout
F1
BBO
M9
Mode and wavelength
monitor
Laser
control
ISO
Ti:sapphire
M6
M10
M11
M13
P1
W1
M2
M3
M
W2
M8
W3
B1
F2 L4
CaF2
L3
Delay stage
M12 controller
M7
L1
M4
YLF laser
CW Nd:YAG
L5
Ti:Sapphire
amplifier
L6
M15
M5
M14
Sample
Chopper
Controller
Synchronization
Delay
Generator
L7 L8
Monochromator
L2
M1
CCD
PET to the CPD substrate quenches the
FADH excited state in ~ 30 ps
0.003
0.002
 fl 
_
1:5 PLred :(T<>T)5
A265
0.001
krad
krad  k ET
(3 ns) 1

~ 0.01
1
1
(3 ns)  (0.032 ns)
0.000
-0.001
-0.002
_
PLred
 eT  32  20 ps
-0.003
0
0
1000
20
40
2000
60
80
100
3000
Time (ps)
MacFarlane and Stanley (2003)
Biochemistry 42, 8558-8568
What’s are the intermediates?
A unidirectional sequential model:
1PL
A(,t) = ci(t)i() = C(E - 0)

red
: T<>T
2
keT
PLsq• : T<>T •
where Ei() = True spectra of the intermediates
0() = Ground state absorption spectrum
•
Construct
C(t) = C0eKt
(from the K matrix)

3
krepair
h
krad
PLsq• : T-T • 
4
 hv hv
 hv  k
et
K 
 0
k et

0
 0
•
•
Calculate
Minimize
0
0
 k repair
0
k rec 
0 
0 

 k rec 
krec
1
PLred  : T<>T or T-T
Ei () = C-1A(,t)
{A(,t) – C(E- 0)} using K matrix
Pl-red+(TTT<>TT)
0.03
0.025
delta A
0.02
0.015
0.01
0.005
0
0
1000
700
600
2000
500
3000
400
Time (ps)
Wavelength (nm)
Pl-red+(TTTTT)
0.035
0.03
0.025
delta A
The broadband
transient absorption
data:
0.02
0.015
0.01
0.005
0
700
0
1000
600
2000
3000
Time (ps)
500
400
Wavelength (nm)
Spectrotemporal intermediates in the repair reaction:
E spectra
4.5
x 10
4
1PL
Intermediate Spectra: PLred-CPD

red
: T<>T
53 ps
2
4
PLsq• : T<>T •
Extinction (M-1 cm-1)
3.5

PLSQ
3
3
2.5
h
2
620
ps
540 ps
PLsq• : T-T • 
4
1.5
1
2753 ps
0.5
0
400
450
500
550
600
Wavelength (nm)
650
700
1 PLred  : T<>T or T-T
• Fitting the data does not rule out a sequential bond breaking
mechanism...
• More complicated kinetics cannot be ruled out!
In conclusion…
Quenching is a simple term for many possible mechanisms
to shunt electronic energy in excited molecules
D*A
D  A  or D  A 
h
DA
Bright
Dark
Bright
or Dark
 
D A or D A
Photoinduced
Electron Transfer
(PET)
Fluorescence
A battery of approaches
need to be used to explore all possible
pathways
The Charge Separation Investigation Team
Madhavan Narayanan
•Ultrafast spectroscopy
•Protein Chemistry
Dr. Zhanjia Hou
•Ultrafast spectroscopy
•Single molecule spectroscopy
Goutham Kodali
• Stark spectroscopy
• Computational chemistry
• “Vector dude”
Dr. Alex MacFarlane IV
•Ultrafast spectroscopy
•Electric field effects
Salim Siddiqui, M.D., Ph.D.
•Stark spectroscopy
•Computational chemistry
The Group
Gone, but not forgotten..
Funding
NSF Molecular Biosciences, REU
Petroleum Research Fund
Collaborators
Prof. Aziz Sancar (UNC)
Mary Hawkins (NIH)
Prof. Spiridoula Matsika
A closer look at the damage…
5’-GCTTAATTCG-3’
3’-CGAATTAAGC-5’
A
A
5’
3’
2.4Å
1.9Å
Watson-Crick base pairing is distorted
Base stacking is weakened
Crystal structure: Park et al, PNAS 99, 15965-15970 (2002).
DNA Photolyase (PL) binds its CPD
substrate by base flipping
CPD
Flavin Adenine Dinucleotide
Mees, A., et al (2004) Science 306, 1789-1793.
Spectral overlaps of probes and FAD
S0S2
S0S1
0.8
0.6
Does FRET explain
the intensityFADpattern
(A)
3'-6map (D)
difference?
Normalized Absorbance
Normalized Fluoresence
1.0
ox
0.4
0.2
0.0
350
400
Wavelength (nm)
450
500