Biological Photochemistry: The fate of electronic excited states

in proteins, DNA, and the role of quenching

Robert J. StanleyRobert J. Stanley

DOE Workshop on Aqueous ScintillatorsJanuary 19, 2010

emple Chemistry DepartmentPhiladelphia, PAwww.chem.temple.edu

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 DNA…a polymer of nucleotides connected by phosphodiester linkagesphosphodiester linkages

5’

3’

Nucleic acid bases A, T, C, & G

Voet and Voet, Biochemistry, 2nd Ed. Wiley, New York, 1995

B-DNA is double-stranded (ds) DNA,B-DNA is double-stranded (ds) DNA,

forming the famous double helixforming the famous double helix

(1954 - Watson, Crick, Franklin)(1954 - Watson, Crick, Franklin)Watson-Crick base pairing

(complementarity)

DNA absorbs UV radiationDNA absorbs UV radiation

240 260 280 300 320 340 360 380 400

0.0

0.2

0.4

0.6

0.8 5'-CTCCPACTTGC-3' 5'-GCAAGTTGGAG-3' dsDNA

Abs

orba

nce

(cor

rect

ed)

Wavelength (nm)

P=6MAP

* transition

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– *G8-oxo-G– T-T T<>T pyrimidine dimerization– Cancer, apoptosis…BAD

UV light damages DNAUV light damages DNA

BadBad photochemistry photochemistry

h

N

NH

O

T-T

O

O O

HN

N

CH 3

N

HN

O

O N

O

O

T<>Tor CPD

CH3 CH3

NH

C3H

< 320 nm

2+2 photo-cycloaddition

If DNA damage is left unrepaired If DNA damage is left unrepaired then mutations, cell death, and cancer then mutations, cell death, and cancer

can developcan develop

http://toms.gsfc.nasa.gov/ery_uv/euv.html

Förster orDexter Transfer(singlets)

Triplet Energy Transfer

Fluorescence

D*A

hD

DA

3

3 1

*

or

ISCD A DA

D A D A

DA*

hA

BrightDarkBright or Dark

Pathways involving energy transfer

D = G*, A*, C*, T*

A = G, A, C, T

ConicalIntersection

Intramolecularvibrationalrelaxation

Fluorescence

D*ABrightDark

hD

DA

“Structural” quenching pathways

DhotA

PhotoinducedElectron Transfer(PET)

Exciplex (EX) formation(charge transfer) Fluorescence

D*A

hD

DA

or D A D A

Pathways involving electron transfer

or D A D A

BrightDarkBright or Dark

hEX?

•Repair of the thymidines is Repair of the thymidines is

direct: direct:

T<>TT<>T T-T without modifying T-T without modifying

the DNA backbonethe DNA backbone

•Wide spread: E. coli, Frogs, Wide spread: E. coli, Frogs, Rice, Kangaroos…Humans (no!)Rice, Kangaroos…Humans (no!)

Enzymatic Repair of CPDs by Enzymatic Repair of CPDs by DNA PhotolyaseDNA Photolyase uses uses blue light as an energy source (Good photochemistry)blue light as an energy source (Good photochemistry)

Sancar, A. Structure and function of DNA photolyase. Biochemistry 33, 2-9 (1994).

Possible Applications:•Photosomes® (AGI Dermatics)•transgenic crops (wheat?)

Mees, A., et al (2004) Science 306, 1789-1793.

• PL functions efficiently with PL functions efficiently with only only FADFAD (required for (required for repair repair andand binding binding

• PL binds the CPD with high PL binds the CPD with high affinity (no light required):affinity (no light required):

KKAA = 10 = 109 9 MM-1 -1 for dsDNA with CPDfor dsDNA with CPD

DNA Photolyase (PL) is aDNA Photolyase (PL) is a flavoprotein flavoprotein (Vitamin B(Vitamin B22) that binds and repairs CPDs) that binds and repairs CPDs

Park, H.-W., Kim, S.-T., Sancar, A., and Deisenhofer, J. (1995) Science 268, 1866-72.

Biochemistry 2Biochemistry 2ndnd Ed., Voet and Voet, J. Wiley & Sons Ed., Voet and Voet, J. Wiley & Sons

Flavin Structure and Flavin Structure and Oxidation StatesOxidation States

•Flavins can transfer 1 Flavins can transfer 1 oror 2 electrons (unlike 2 electrons (unlike nicotinamide) and are nicotinamide) and are used in a large number of used in a large number of redox reactions in the cellredox reactions in the cell

•Surprisingly, flavins Surprisingly, flavins are a major biological are a major biological chromophore (DNA chromophore (DNA repair, circadian rhythms, repair, circadian rhythms, phototropism, etc.)phototropism, etc.)

FADH—

—

Photolyase functions by Photoinduced Electron Photolyase functions by Photoinduced Electron Transfer from the FAD to the CPDTransfer from the FAD to the CPD

•A large separation between the A large separation between the FADHFADH-- and the CPD (~16 and the CPD (~16 ÅÅ) ) would give a slow electron would give a slow electron transfer rate (ktransfer rate (keTeT, from Marcus , from Marcus

theory)theory)

kTGreT eek 4/)(2 2

Orbital overlap x Driving force

•Slow electron transfer would Slow electron transfer would compete poorly with compete poorly with 11FADHFADH—— deactivation (about 5 ns)deactivation (about 5 ns)

but but repairrepair > 0.7! > 0.7!

There’s a cavity in the protein

FAD

What happens to substrate conformation What happens to substrate conformation upon binding to Photolyase?upon binding to Photolyase?

Base Flipping

Photolyase

Minor disruption

Moderate disruption

Severe disruption

AA

T<>T

Fluorescent reporter approach to probing Fluorescent reporter approach to probing double helical structuredouble helical structure

Base Flipping5’

5’

3’

3’

5’

5’

3’

3’

5’-probe approach:

Base Flipping5’

5’

3’

3’

5’

5’

3’

3’

3’-probe approach:

The fluorescence quantum yield of the reporter decreases when base

stacked…but why?

6MAP is an attractive new fluorescent 6MAP is an attractive new fluorescent adenosine analogueadenosine analogue

Properties:1

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).

4-amino-6-methyl-8-(2-deoxy--D-ribofuranosyl)-7(8H)-pteridone

310 320 330 340 350 360 370 380 390 400 410

0.00

0.01

0.02

0.03

ss-3'-6MAP ds-3'-6MAP-TT ds-3'-6MAP-CPD ss-5'-6MAP ds-5'-6MAP-TT ds-5'-6MAP-CPD

Abs

orba

nce

(cor

rect

ed)

Wavelength (nm)

K. Yang, S. Matsika, and R.J. Stanley, Biochemistry 2007

N N

H

O

O

ThymineR

N8

7

6

N 4

N

2

N

N

O

6MAP

H H

CH3

3

CH3C 1'

Base flipping of the Base flipping of the CPD monitored by CPD monitored by 6MAP6MAP

5’-GCAAGTTGGAG-3’3’-CGTTCAFCCTC-5’

5’-GCAAGTTGGAG-3’3’-CGTTCFACCTC-5’

Why is the intensity pattern sequence-dependent?

-PL +PL

-PL +PL

These data are consistent with disruption of base These data are consistent with disruption of base stacking due to base flipping of the CPD by stacking due to base flipping of the CPD by

PhotolyasePhotolyase

Photolyase

Mees et al, Science v. 306, 1789-1793 (2004)

?

Is the fluorescence quantum yield modulation of Is the fluorescence quantum yield modulation of 6MAP due to PET?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

The quenching of 6MAP* proceeds through The quenching of 6MAP* proceeds through nucleobase oxidation:nucleobase oxidation:

6MAP*:NMP6MAP*:NMP6MAP6MAP¯:NMP¯:NMP++

(Scandola-Balzani relation)

FBA NB GET(eV) Eact(eV)

6MAP

G -0.63 0.000

A -0.16 0.003

C 0.021 0.048

dT -0.009 0.032

submitted to Biochemistry

What’s the mechanism for base analog quenching?What’s the mechanism for base analog quenching?Pathways for energy transduction in a model FBA oligo

ConicalIntersection

PhotoinducedElectron Transfer

ExciplexFluorescence

5’-NF*N-3’

BrightDark

5’-NFN-3’

5’-NF +N--3’

hConicalIntersection

PhotoinducedElectron Transfer

ExciplexFluorescence

5’-NF*N-3’

BrightDark

5’-NFN-3’

5’-NF +N--3’

h

280 300 320 340 360 380

-0.5

0.0

0.5

1.0

(

M-1 c

m-1)

Wavelength (nm)

2AP 3'-CCC2APGC-5' 2AP+4C+G

T = 77K=55

Absorption Stark spectra of ssDNA with 2AP (), a hexamer with 2AP () , and a mix of the individual bases ().

300 350 400 450

-0.6-0.4-0.20.00.20.40.60.81.01.2

I F a

nd

I F (

norm

.)

and

(

norm

.)

Wavelength (nm)

6-MI

Stark absorption and emission spectra of 6-MI (), a guanine analog, compared with their absorption and emission spectra ().

Stark and MRCI calculations (Matsika)Stark and MRCI calculations (Matsika)

380 400 420 440 460 480 500 520 540

0

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

Wavelength (nm)

ss-6MAP/PLox

ss-6MAP/TT/PLox

ss-6MAP/T<>T/PLox

380 400 420 440 460 480 500 520 540

0

200000

400000

600000

800000

1000000

1200000

1400000

1600000

1800000

Wavelength (nm)

Another possibility: Another possibility: 6MAP emission overlaps the absorption of the 6MAP emission overlaps the absorption of the FAD: FRET from 6MAP*FAD: FRET from 6MAP*FAD?FAD?

Yang et al, JPC B (2007)

60

6 60

ETDA

R

R r

R0 the Förster distance where ET = 0.5

rDA the distance between a donor (fluorescent analogue) and an acceptor (FAD in photolyase)

Fluorescence Energy Transfer Efficiency Fluorescence Energy Transfer Efficiency

R0 (Å) = 6/142 )(211.0 Jn D

The Förster distanceThe 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.

dF

dFJ

D

AD

)(

)()( 4

FD(): the fluorescence intensity of the donor as a function of wavelength.

εA(): the molar extinction coefficient of the acceptor at that wavelength;

The Overlap IntegralThe Overlap Integral

350 400 450 500

FD

A

Wavelength (nm)

22 )coscos3(cos ADT

θT: mD, mA

θD: mD , rDA

θA: mA, rDA

The Orientation FactorThe Orientation Factor

R

N8

7

6

N

4 N

2

N

H2N

O

6MAP in 3'-6MAP

3

H3C

8

76

5a

9a9

N5

4a

10a

N10 4

NH

2N

1

H3C

H3C

O

O

R

FADox in Photolyase

mD mArDA

The transition dipole moment directionThe transition dipole moment direction6MAP was calculated from TD-DFT6MAP was calculated from TD-DFT

Yang et al, JPC B (2007)

Orientation factors and Orientation factors and ETET between between

Probes and FADProbes and FADoxox

From the crystal structure, lit. and TDDFT calcs

crystal structure

experiment

Yang et al, JPC B (2007)

FRET efficiency vs. orientationFRET efficiency vs. orientation

60 70 80 90 100 1100.0

0.2

0.4

0.6

0.8

1.0

3'-6MAP/FAD (m1)

5'-6MAP/FAD (m1)

FRET

(deg.)

)6( MAPxtal

Yang et al, JPC B (2007)

NO FRET!NO FRET!

400 450 500 550 600

-0.04

-0.02

0.00

0.02

0.04

I [I

(6M

AP

/T<

>T

/PL)

-I(6

MA

P/T

<>

T)]

Wavelength (nm)

• 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)

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?

PLred : T<>T

1

5

4

3

2

6

1PLred : T<>T

PLsq• : T<>T •

PLsq• : T|_|T •

PLsq• : T-T •

PLred + T-T

PLred : T-T

7

eT

krec

1

2

kbeT

kdiss

kic, krad

h

c

MacFarlane and Stanley (2003) Biochemistry 42, 8558-8568

Ti:sapphire

CW Nd:YAG

ISO

Ti:Sapphire amplifier

Mode and wavelengthmonitor

Lasercontrol

YLF laser

CCD

Mo

no

chro

ma

to

r

Delay stagecontroller

SynchronizationDelay

Generator

ChopperController

M3

M1

M2

M4

M5

M6M

M12

M13

M7

M10

M9

M8M11

M15

M14

L1

L5L2

L4 L3

L6

L7 L8

W1 W3

W2

P1

B1

F1

F2

Transient absorption measurement layout

BBO

CaF2

Sample

0 1000 2000 3000

-0.003

-0.002

-0.001

0.000

0.001

0.002

0.003

1:5 PLred

_:(T<>T)5

A26

5

Time (ps)

PLred

_

0 20 40 60 80 100

pseT 2032

PET to the CPD substrate quenches the PET to the CPD substrate quenches the FADHFADH excited state in ~ 30 ps excited state in ~ 30 ps

MacFarlane and Stanley (2003) Biochemistry 42, 8558-8568

1

1 1

(3 ns)~ 0.01

(3 ns) (0.032 ns)

radfl

rad ET

k

k k

What’s are the intermediates?What’s are the intermediates?

A(A(,t) = ,t) = ccii(t)(t)ii(() = C(E - ) = C(E - 00))

where Ewhere Eii(() = True spectra of the intermediates) = True spectra of the intermediates

00(() = Ground state absorption spectrum) = Ground state absorption spectrum

• ConstructConstruct C(t) = CC(t) = C00eeKt Kt (from the K matrix)(from the K matrix)

• CalculateCalculate EEii ( () = C) = C-1-1A(A(,t),t)• MinimizeMinimize {{A(A(,t) – C(E- ,t) – C(E- 00)} using K )} using K

matrixmatrix

PLred : T<>T or T-T 1

4

3

2

1PLred : T<>T

PLsq• : T<>T •

PLsq• : T-T •

keT

krec

krepair

h krad

A unidirectional sequential model:A unidirectional sequential model:

rec

repairet

et

rec

k

kk

khv

khvhv

K

000

00

00

0

01000

20003000

400500

600700

0

0.005

0.01

0.015

0.02

0.025

0.03

Wavelength (nm)Time (ps)

de

lta

A

Pl-red+(TTT<>TT)The broadband The broadband

transient absorption transient absorption data:data:

01000

20003000

400

500

600

7000

0.005

0.01

0.015

0.02

0.025

0.03

0.035

Wavelength (nm)Time (ps)

de

lta A

Pl-red+(TTTTT)

400 450 500 550 600 650 700

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5x 10

4 Intermediate Spectra: PLred-CPD

Wavelength (nm)

Ext

inct

ion

(M

-1 c

m-1

)Spectrotemporal intermediates in the repair reaction:Spectrotemporal intermediates in the repair reaction:

E spectraE spectra

• Fitting the data does not rule out a sequential bond breaking Fitting the data does not rule out a sequential bond breaking mechanism...mechanism...• More complicated kinetics cannot be ruled out!More complicated kinetics cannot be ruled out!

PLSQ

PLred : T<>T or T-T 1

4

3

2

1PLred : T<>T

PLsq• : T<>T •

PLsq• : T-T •

53 ps

2753 ps

540 psh

620 ps

In conclusion…In conclusion…Quenching is a simple term for many possible mechanisms Quenching is a simple term for many possible mechanisms

to shunt electronic energy in excited moleculesto shunt electronic energy in excited molecules

PhotoinducedElectron Transfer(PET)

Fluorescence

D*A

h

DA

or D A D A

or D A D A

BrightDarkBright or Dark

A battery of approachesneed to be used to explore all possible

pathways

The Charge Separation Investigation Team

Goutham Kodali• Stark spectroscopy• Computational chemistry• “Vector dude”

Salim Siddiqui, M.D., Ph.D.•Stark spectroscopy•Computational chemistry

Dr. Zhanjia Hou•Ultrafast spectroscopy•Single molecule spectroscopy

Madhavan Narayanan•Ultrafast spectroscopy•Protein Chemistry

Dr. Alex MacFarlane IV•Ultrafast spectroscopy•Electric field effects

The GroupThe Group

CollaboratorsCollaboratorsProf. Aziz Sancar (UNC)Mary Hawkins (NIH)Prof. Spiridoula Matsika

FundingFundingNSF Molecular Biosciences, REUPetroleum Research Fund

Gone, but not forgotten..Gone, but not forgotten..

A closer look at the damage…A closer look at the damage…5’-GCTTAATTCG-3’5’-GCTTAATTCG-3’

3’-CGAA3’-CGAATTTTAAGC-5’AAGC-5’

Crystal structure: Park et al, PNAS 99, 15965-15970 (2002).

5’3’

AA

Base stacking is weakenedWatson-Crick base pairing is distorted

2.4Å1.9Å

DNA Photolyase (PL) binds its CPD DNA Photolyase (PL) binds its CPD substrate by base flippingsubstrate by base flipping

Mees, A., et al (2004) Science 306, 1789-1793.

Flavin Adenine Dinucleotide

CPD

350 400 450 500

0.0

0.2

0.4

0.6

0.8

1.0

Nor

mal

ized

Abs

orba

nce

FADox

(A)

3'-6map (D)

Nor

mal

ized

Flu

ores

ence

Wavelength (nm)

Spectral overlaps of probes and FADSpectral overlaps of probes and FAD

Does FRET explain the intensity pattern

difference?

S0S2 S0S1