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Biological Photochemistry

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Title: Biological Photochemistry


1
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
2
Electronic excited states in Biology
  • Chemiluminescence
  • Bioluminescence charge transfer? radicals?
  • Photoinduced electron transfer
  • Photosynthesis
  • DNA repair
  • Photochemistry
  • DNA damage
  • photosensors

3
DNAa polymer of nucleotides connected by
phosphodiester linkages
Nucleic acid bases A, T, C, G
Voet and Voet, Biochemistry, 2nd Ed. Wiley, New
York, 1995
4
B-DNA is double-stranded (ds) DNA, forming the
famous double helix (1954 - Watson, Crick,
Franklin)
Watson-Crick base pairing (complementarity)
5
DNA absorbs UV radiation
??? transition
6
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 ? TltgtT pyrimidine dimerization
  • Cancer, apoptosisBAD

7
UV light damages DNA Bad photochemistry
22 photo-cycloaddition
lt 320 nm
8
If DNA damage is left unrepaired then mutations,
cell death, and cancer can develop
http//toms.gsfc.nasa.gov/ery_uv/euv.html
9
Pathways 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
10
Structural quenching pathways
DA
Bright Dark
DhotA
h?D
Intramolecular vibrational relaxation
Conical Intersection
DA
Fluorescence
11
Pathways involving electron transfer
DA
h?D
h?EX?
Photoinduced Electron Transfer (PET)
Exciplex (EX) formation (charge transfer)
DA
Fluorescence
12
Enzymatic 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).
13
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.
14
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
  • Surprisingly, flavins are a major biological
    chromophore (DNA repair, circadian rhythms,
    phototropism, etc.)

Biochemistry 2nd Ed., Voet and Voet, J. Wiley
Sons
15
Photolyase functions by Photoinduced Electron
Transfer from the FAD to the CPD
Theres a cavity in the protein
FAD
16
What happens to substrate conformation upon
binding to Photolyase?
Minor disruption
AA
Photolyase
Moderate disruption
Base Flipping
TltgtT
Severe disruption
17
Fluorescent 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?
18
6MAP 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
19
Base 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?
20
These 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)
21
Is 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
22
The quenching of 6MAP proceeds through
nucleobase oxidation 6MAPNMP?6MAP?NMP?
(Scandola-Balzani relation)
submitted to Biochemistry
23
Whats 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 (?).
24
Another possibility 6MAP emission overlaps the
absorption of the FAD FRET from 6MAP?FAD?
Yang et al, JPC B (2007)
25
Fluorescence 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)
26
The 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.
27
The 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
28
The Orientation Factor
mD
mA
rDA

?T ? mD, mA ?D ? mD , rDA ?A ? mA, rDA
29
The transition dipole moment direction 6MAP was
calculated from TD-DFT
Yang et al, JPC B (2007)
30
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)
31
FRET efficiency vs. orientation
Yang et al, JPC B (2007)
32
NO 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)
33
Can 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
35
PET to the CPD substrate quenches the FADH?
excited state in 30 ps
MacFarlane and Stanley (2003) Biochemistry 42,
8558-8568
36
Whats 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

37
Pl-red(TTTltgtTT)
The broadband transient absorption data
Pl-red(TTTTT)
38
Spectrotemporal 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!

39
In 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
40
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

41
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
42
A 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).
43
DNA Photolyase (PL) binds its CPD substrate by
base flipping
CPD
Flavin Adenine Dinucleotide
Mees, A., et al (2004) Science 306, 1789-1793.
44
Spectral overlaps of probes and FAD
S0?S2
S0?S1
Does FRET explain the intensity pattern
difference?
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