Title: Biological Photochemistry
1Biological 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
2Electronic excited states in Biology
- Chemiluminescence
- Bioluminescence charge transfer? radicals?
- Photoinduced electron transfer
- Photosynthesis
- DNA repair
- Photochemistry
- DNA damage
- photosensors
3DNAa polymer of nucleotides connected by
phosphodiester linkages
Nucleic acid bases A, T, C, G
Voet and Voet, Biochemistry, 2nd Ed. Wiley, New
York, 1995
4B-DNA is double-stranded (ds) DNA, forming the
famous double helix (1954 - Watson, Crick,
Franklin)
Watson-Crick base pairing (complementarity)
5DNA absorbs UV radiation
??? transition
6Quenching 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 ? TltgtT pyrimidine dimerization
- Cancer, apoptosisBAD
7UV light damages DNA Bad photochemistry
22 photo-cycloaddition
lt 320 nm
8If DNA damage is left unrepaired then mutations,
cell death, and cancer can develop
http//toms.gsfc.nasa.gov/ery_uv/euv.html
9Pathways involving energy transfer
DA
D G, A, C, T
A G, A, C, T
h?D
DA
h?A
Triplet Energy Transfer
Förster or Dexter Transfer (singlets)
DA
Fluorescence
10Structural quenching pathways
DA
Bright Dark
DhotA
h?D
Intramolecular vibrational relaxation
Conical Intersection
DA
Fluorescence
11Pathways involving electron transfer
DA
h?D
h?EX?
Photoinduced Electron Transfer (PET)
Exciplex (EX) formation (charge transfer)
DA
Fluorescence
12Enzymatic Repair of CPDs by DNA Photolyase uses
blue light as an energy source (Good
photochemistry)
- Repair of the thymidines is direct
- TltgtT? T-T without modifying the DNA backbone
- Wide spread E. coli, Frogs, Rice,
KangaroosHumans (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).
13DNA 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.
14Flavin 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 - Surprisingly, flavins are a major biological
chromophore (DNA repair, circadian rhythms,
phototropism, etc.)
Biochemistry 2nd Ed., Voet and Voet, J. Wiley
Sons
15Photolyase functions by Photoinduced Electron
Transfer from the FAD to the CPD
Theres a cavity in the protein
FAD
16What happens to substrate conformation upon
binding to Photolyase?
Minor disruption
AA
Photolyase
Moderate disruption
Base Flipping
TltgtT
Severe disruption
17Fluorescent reporter approach to probing double
helical structure
5-probe approach
?
Base Flipping
3-probe approach
?
Base Flipping
The fluorescence quantum yield of the reporter
decreases when base stackedbut why?
186MAP is an attractive new fluorescent adenosine
analogue
4-amino-6-methyl-8-(2-deoxy-?-D-ribofuranosyl)-7(8
H)-pteridone
Properties1 ?fl 0.2 ?ex 330 nm (? 8,500
M-1cm-1) ?em 430 nm (large Stokes
shift) 1Hawkins, et al, Synthesis and
Fluorescence Characterization of Pteridine
Adenosine Nucleoside Analogs for DNA
Incorporation. Anal. Biochem.298, 231-240 (2001).
K. Yang, S. Matsika, and R.J. Stanley,
Biochemistry 2007
19Base flipping of the CPD monitored by 6MAP
PL
-PL
5-GCAAGTTGGAG-3 3-CGTTCAFCCTC-5
PL
5-GCAAGTTGGAG-3 3-CGTTCFACCTC-5
-PL
Why is the intensity pattern sequence-dependent?
20These data are consistent with disruption of base
stacking due to base flipping of the CPD by
Photolyase
?
Mees et al, Science v. 306, 1789-1793 (2004)
21Is the fluorescence quantum yield modulation of
6MAP due to PET?
Stern-Volmer quenching of 6MAP by G,A,C, and
T what is the rate of quenching, kq?
What are the redox potentials? Cyclic voltammetry
of 6MAP in aprotic organic solvents
submitted to Biochemistry
22The quenching of 6MAP proceeds through
nucleobase oxidation 6MAPNMP?6MAP?NMP?
(Scandola-Balzani relation)
submitted to Biochemistry
23Whats the mechanism for base analog
quenching?Pathways for energy transduction in a
model FBA oligo
Absorption Stark spectra of ssDNA with 2AP (?), a
hexamer with 2AP (?) , and a mix of the
individual bases (?).
Stark and MRCI calculations (Matsika)
Stark absorption and emission spectra of 6-MI
(?), a guanine analog, compared with their
absorption and emission spectra (?).
24Another possibility 6MAP emission overlaps the
absorption of the FAD FRET from 6MAP?FAD?
Yang et al, JPC B (2007)
25Fluorescence Energy Transfer Efficiency
R0 ? the Förster distance where ?ET 0.5
rDA ? the distance between a donor (fluorescent
analogue) and an acceptor (FAD in photolyase)
26The Förster distance
?2 the orientation factor n the
refractive index of the medium ?D the
fluorescence quantum yield of the donor J
the overlap integral.
27The Overlap Integral
FD(?) the fluorescence intensity of the donor as
a function of wavelength. eA(?) the molar
extinction coefficient of the acceptor at that
wavelength
28The Orientation Factor
mD
mA
rDA
?T ? mD, mA ?D ? mD , rDA ?A ? mA, rDA
29The transition dipole moment direction 6MAP was
calculated from TD-DFT
Yang et al, JPC B (2007)
30Orientation factors and ?ET between Probes and
FADox
From the crystal structure, lit. and TDDFT calcs
experiment
crystal structure
Yang et al, JPC B (2007)
31FRET efficiency vs. orientation
Yang et al, JPC B (2007)
32NO FRET!
- The FAD is quenched 100x in the protein
(acceptor is dark) - A work-around time-resolved FRET?
- Quenching mechanism is different for the two
probes - photoinduced electron transfer vs. ultrafast
internal conversion? - Does FAD undergo PET to tryptophan???
Yang et al, JPC B (2007)
33Can we identify the kinetics and mechanism of
repair? Two color pump probe femtosecond
spectroscopy
- What is the electron transfer lifetime (?eT)?
- Does repair proceed by a concerted or sequential
mechanism?
?c
MacFarlane and Stanley (2003) Biochemistry 42,
8558-8568
34 Transient absorption measurement layout
BBO
CaF2
Sample
35PET to the CPD substrate quenches the FADH?
excited state in 30 ps
MacFarlane and Stanley (2003) Biochemistry 42,
8558-8568
36Whats are the intermediates?
A unidirectional sequential model
- ?A(?,t) ?ci(t)??i(?) C(E - ?0)
- where Ei(?) True spectra of the intermediates
- ?0(?) Ground state absorption spectrum
- Construct C(t) C0eKt (from the K matrix)
- Calculate Ei (?) C-1?A(?,t)
- Minimize ?A(?,t) C(E- ?0) using K matrix
37Pl-red(TTTltgtTT)
The broadband transient absorption data
Pl-red(TTTTT)
38Spectrotemporal intermediates in the repair
reaction E spectra
1PLred? TltgtT
53 ps
2
PLsq TltgtT ?
3
540 ps
620 ps
PLsq T-T ?
4
2753 ps
PLred ? TltgtT or T-T
1
- Fitting the data does not rule out a sequential
bond breaking mechanism... - More complicated kinetics cannot be ruled out!
39In conclusionQuenching is a simple term for
many possible mechanisms to shunt electronic
energy in excited molecules
A battery of approaches need to be used to
explore all possible pathways
40The 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
41The Group
Gone, but not forgotten..
Funding NSF Molecular Biosciences, REU Petroleum
Research Fund
Collaborators Prof. Aziz Sancar (UNC) Mary
Hawkins (NIH) Prof. Spiridoula Matsika
42A closer look at the damage 5-GCTTAATTCG-3 3-C
GAATTAAGC-5
5 3
A A
Crystal structure Park et al, PNAS 99,
15965-15970 (2002).
43DNA Photolyase (PL) binds its CPD substrate by
base flipping
CPD
Flavin Adenine Dinucleotide
Mees, A., et al (2004) Science 306, 1789-1793.
44Spectral overlaps of probes and FAD
S0?S2
S0?S1
Does FRET explain the intensity pattern
difference?