Mpi Lecture

Elektron-Paramagnetic Resonance Spectroscopy Basics & Applications to Biological Systems § Basics (What is EPR ?) § Historical Introduction (NMR/EPR) § Application Fields § Basic Principle § Technical Requirements § Advanced Methods § EPR Parameters § Applications to Proteins (What can we learn from EPR?) § Organic radicals in proteins Semiquinone radicals § Metal centres in protein complexes Mn+II, Cu+II , Mo V § Spin labels Mobility, Access, Distances

Elektron-Paramagnetic Resonance Spectroscopy What is EPR ?

EPR is a spectroscopical technique that detects: • unpaired electrons (electron spins : ESR) • identity of the molecule • and information of the molecular structure (structure, dynamics, bounding) • the molecular environment (< 0.8 nm for nuclear spins and up to 50 nm for other electron spins)

EPR is nondestructive, needs 100 µl sample (or less!), concentrations of >100 µmolar paramagnetic species

Elektron-Paramagnetic Resonance Spectroscopy Application fields

Ø Physics: Susceptibility, Semiconductors, Quantum Dots, Defect Centres ... Ø Chemistry: ET-Reaction Kinetics, Organo-Metallic, Catalysis, Molecular Magnets... ØIonization Radiation: Alanin radiation dosimetry, Radiation damage, Irradiated food .. Ø Material research: Polymers, Glases, Superconductors, Corrosion, Fullerenes, Dating ... Ø Biology: Enzyme Reaction, ET-Reaction, Folding&Dynamics, Metal centres ...

Paramagnetic metal ions (Cu, Mn, Ni, Co, Mo, Fe) and complexes in enzymes Hemes and FeS clusters in electron transfer reactions in protein Amino acid radicals of the protein backbone (as tyrosine, triptophane and glycil) protein bound cofactor radicals (as semiquinones and flavines) Transient paramagnetic chormophores in light driven processes Nitroxide spin labels attached to cysteines or nucleic acids

Elektron-Paramagnetic Resonance Spectroscopy

Energie E

Basic physics

+1/2

Magnetic Resonance Condition

∆E = g ⋅ µ B ⋅ B0 = h ⋅ν MW Under resonance conditons microwave gets absorbed -> Detection signal

-1/2 magnetic field B

0

Elektron-Paramagnetic Resonance Spectroscopy Instrumentation, Basic Principle

EPR Experiment

Elektron-Paramagnetic Resonance Spectroscopy Historical introduction

1897:

Pieter Zeeman Line splitting in external magnetic field

1922:

Otto Stern, Walter Gerlach Quantisation in external magnetic field

1925:

Goldsmith, Uhlenbeck Spin of electron

1945:

Zavojski First EPR experiment

1946:

Block, Purcell, Pound First NMR experiment

1950:

Erwin Hahn First pulse NMR experiment

1958:

Bill Mims First pulse EPR experiment

1965:

Richard Ernst First FT-NMR experiment

1976:

Richard Ernst First 2D-NMR experiment

1986:

Jack Freed First FT- & 2D EPR experiment

1994:

Wrachtrup, Köhler, Groenen, Borzyskowski First single molecule EPR experiment

Elektron-Paramagnetic Resonance Spectroscopy Instrumentation

Frequency: Factor 1000 larger in EPR ! (GHz instead of MHz) Coupling strength: Factor 1 000 000 larger in EPR ! (MHz instead of Hz) Relaxation Times: Factor 1000 000 smaller in EPR ! (ns instead of ms) a much higher techniqual requirements !!

Sensitivity : Factor 1 000 000 better than in NMR !! (1nM instead of 1mM )

Elektron-Paramagnetic Resonance Spectroscopy EPR Parameters : G-Tensor Reflects symmetry of the electronic orbital of unpaired electron

Spherical symmetric orbital

Cylinder symmetrical orbitalre

Lower symmetry orbital

Elektron-Paramagnetic Resonance Spectroscopy EPR Parameters : A-Tensor (HF-Tensor)

Electron spin density at nucleus: isotropic a

e

Spin density at n

n

Dipolare coupling to distant nucleus: anisotropic A Distance to n

n e

Elektron-Paramagnetic Resonance Spectroscopy Advanced Methods 0.1

0.3

1

3.4

6.4

Multifrequency-EPR

Magnetfeld [T] B ν Mikro360 wellen[subfrequenz mm] [GHz]

12.8

0

M

3 [S]

9 [X]

35 [Q]

180 [G]

95 [W]

Mikrowellen Pulse

t

Pulse-EPR

Pulsabstände

t

1

W

2

14.9

6.0

3.7

2.2

2.0

1.1

t

ENDOR (Electron Nuclear Double Resonance

KernZeeman Frequenz (im X-Band)

ν Radio-frequenz [MHz] R F

1 4

1 7

2

N O H

1 3

C

3 1

P

1

H

Elektron-Paramagnetic Resonance Spectroscopy Microwave frequency bands

m S= -1/2 0,11 T 3GHz (S-Band)

1 T/35GHz (Q-Band) 3,4 T/95GHz (W-Band)

6,4 T/180GHz (G-Band)

0,34 T 9,5GHz (X-Band) B0

m S= +1/2

High-field EPR Spectral resolution Resolution of G-anisotropy: Orientation selection mS = +½

O

gzz

CH3

gyy O

mS = -½

gxx

gxx

gyy gzz

X-Band

G-Band

Elektron-Paramagnetic Resonance Spectroscopy Field dependence of spectra

Distringuishes field dependent and field independent parameters

Nitroxid spectra as a function of magnetic field

Elektron-Paramagnetic Resonance Spectroscopy Instrumentation: 180 GHz Spectrometer

Pulsed EPR Hahn Echo sequence

Refocusing technique eliminates inhomogeneous linewidth

t

3

t

0

t microwave

1

t

π

π/2

2

ECHO

FID t

0

t

1

t

2

t

3

time

ESEEM Spectroscopy Small Hyperfine couplings

T

H O 3

4 5

2

Me O

1

O

C H3

6

H CH3

0

2

4

6

n

H

The semiquinone interacts with 14N nitrogen

8 T im e

1 0 1 2 (µ s )

1 4

1 6

1 8

FFT F F T A m p l i t u d e ( a. u. )

Me O

Echo Amplitude

Measure of the echo amplitude as a function of T

0

0

F

r

2 e

q

u

e

n

4

c

y

(

M

6 H

z )

8

E

Electron Nuclear Double Resonance (ENDOR) Anisotropic Hyperfine interactions

m

S

“NMR detected by EPR” Simultaneous irradiation of the sample with microwave and radio frequencies Enhanced spectral resolution Simplification of hyperfine spectra

m

I

Electron Nuclear Double Resonance (ENDOR) ENDOR spectra

6

x 10

-3

N H 17O P CH3

4 2 0 -2 -100

-80

-60

-40

-20

0

20

40

60

80

100

10

20

30

40

50

60

70

80

90

100

20

40

60

80

100

120

140

160

180

200

0.3 0.2 0.1 0 -0.1 0.3 0.2 0.1 0 -0.1

Pulsed Electron Double Resonance (PELDOR) Dipolare coupling between paramagnetic molecules

distances of r AB between 10Å - 50Å

rAB = 29.1Å O N

O

O

O

O

1.2

O

1.2

S-Band (3.6 GHz) X-Band (9.7 GHz)

1.1 Echoamplitude [a.u.]

N

1

1 0.8

0.9 0.6

0.8

ωDip = 2.1 MHz

0.4

0.7 0.2

0.6 0.5

0

0

1

2 3 Pump Pulse Position [µs]

4

5

r AB = 29.2 Å 0

5 10 Frequency [MHz]

15

Elektron-Paramagnetic Resonance Spectroscopy Instrumentation: Pulsed X-band EPR/ENDOR

The Theunpaired unpairedelectron electronas asaalocal localprobe probe

Puls-ESR,PELDOR

ENDOR, ESEEM

cw-ESR

Elektron-Paramagnetic Resonance Spectroscopy Detection methods

Microwave transmission detection:

sensitivity >1014 spins

Microwave bridge detection:

sensitivity >1011 spins (9 GHz) sensitivity >107 spins (100 GHz)

Electrical detection:

sensitivity >107 spins

Optical detection:

sensitivity >104 spins

Atomic force microscope

sensitivity >103 spins

Confocal microscope fluoreszenz:

sensitivity >1 spins

Elektron-Paramagnetic Resonance Spectroscopy Applications to Biological Systems

§

G-Protein complex

§ Photosynthesis § Cytochrome c oxidase

Photosynthetic bacterial reaction centre of rhodobacter spheroides (BChl)2* BPh QA QB 4 ps (BChl)2+ BPh- QA QB 200 ps (BChl)2+ BPh QA- QB 100 µs (BChl)2+ BPh QA QB-

(BChl)2 BPh QA QB

High-Field-EPR High-Field-EPRmeasurements measurementson on bRC bRC 9330 GHz GHz

QB 95 GHz

95 GHz

Structure Structureofofthe thechormophores chormophoresininPSI PSIby byEPR EPR

P700

Abstand und Orientierung der Chromophore zueinander und zur Membranebene

2.5 nm 27°

A1 1.48 nm

Fe/S

Semiquinone radical QA in bRC

2

T [µs]

echo intensity

Dynamics of protein bound molecules

1.6 1.2

echo detected spectrum

ϕ O

O

relaxation 190 K time

θ

ϕ

0.8

O

120 K

0.4

O

magnet field B

0

P21ras · MnII+ · GDP protein complex § Molecular switch for signal transduction "active state" Hydrolysis

GDP exchangek factor (GEF)

k

d is s

c a t

Effector / GAP

GTP

P

i

p21:GDP "inactive state"

Oncogenic mutation at glycin12 position ⇒ strongly reduced catalytic rate constants !

P21ras · MnII+ · GDP / GTP protein complex

C N

GDP

Inactive (GDP) state

loop L2 T35

loop L4

active (GDP) state

C N

GppNHp loop L2 T35

loop L4

2+ p21 --Mn 2+--GTP p21ras Mn GTPprotein proteinnucleotide nucleotidecomplex complex ras

Konformationsänderung bei

->

loop L2 loop L2

Thr 35

H O

Hydrolyse H O

NH

Thr 35

H 2O

Mn

NH O

H2O

H 2O

Ser 17

Ser 17

H2 O

Mn O

H 2O

O

H 2O

O

O

"Nukleophiler

O Angriff" H2O

Pγ O

O

Pβ

O

O Lys 16

NH

loop L4

GTP

Pα O

O

- Pi

Pβ O

Lys 16 NH

loop L4

O

Pα O

Mulitfrequency EPR of P21ras · MnII+ · GDP protein complex

180 GHz

δ

f

x 10

δ

1. Mn Hyperfine line

f

95 GHz x 10

9.5 GHz

Two states distinguishable by HF-EPR spectroscopy

2.7 GHz 5 mT

P21ras · MnII+ · GDP protein complex

No differences at active site for wt & oncogenic mutant by X-ray !

Thr35

Gly12

Lys16

Asp57

GDP

Mg

Ser17

[E. F. Pai et al. EMBO J. 9, 2351 (1990)]

HF- EPR of P21ras · MnII+ · GDP protein complex

4 H2O17 ligands

3 H2O17 ligands

-50

-25

0 25 relative magnetic field B [G]

50

HF- EPR of P21ras · MnII+ · GDP protein complex

G12V

wt x5

n=5

n=4

n=4

n=3

n=3

n=2

T35S

T35A

n=4 n=3

n=2

n=5 n=4 n=3

1 mT

Differences in ligand sphere determined by EPR spectroscopy !!

Cytochrom CytochromccOxidase Oxidaseof ofParaccocus Paraccocusdenitrificans denitrificans

Multifrequency-EPR Multifrequency-EPRon onCytochrom CytochromccOxidase Oxidase X -B a n d

3000

3500

Q -B a n d

11800

12000

12200

12400

W -B a n d

33200

33400

33600

M a g n e t fe ld [ G a u s s ]

33800

Application Application on on Cytochrome Cytochrome cc Oxidase: Oxidase: Distance and orientation between binuclear CuA centre and Mn binding site

Cys B216

CuA Ser B217

rAB Asp A404 Cys B220 Glu B218 H2O

Mn

His A403

H. Käß, F. MacMillan, B. Ludwig, T. Prisner Biochemistry 104, 5362-5371 (2000)

Structure determination by EPR spectroscopy 2D-ESEEM (HYSCORE) Experiment

15

F2 [MHz]

14

10

N

5

Experimental 1

H

Spectra

a Bestimmung der N und H Wechselwirkungen 1 4

0

1

-5 -10 2

4

6

8

10

F1 [MHz]

12

14

Parameters

MO-Calculations Molecular Structure

16

QM-Simulations

Electron ElectronParamagnetic ParamagneticResonance Resonance(EPR) (EPR) Multifrequency Multifrequencycw-EPR: cw-EPR: Identification Identificationand andCharacterisation CharacterisationofofRadicals Radicals

Structural StructuralInformation: Information:

PULSE-EPR PULSE-EPRand andENDOR: ENDOR: Identification IdentificationofofLigand LigandSphere Sphere(< (<0.8 0.8nm) nm) PELDOR: PELDOR: Distance Distancebetween betweenParamagnetic ParamagneticCentres Centres(< (<6nm) 6nm)

Time TimeResolvedResolved-and andFT-EPR: FT-EPR: Photoinduced PhotoinducedElectron-Transfer Electron-TransferKinetics Kinetics

Dynamic DynamicInformation: Information:

Pulsed-High-Field-EPR: Pulsed-High-Field-EPR: Librational LibrationalDynamics DynamicsofofProtein-Bound Protein-BoundQuinones Quinones PELDOR: PELDOR: Conformational ConformationalDynamics Dynamics

Literature

Methods: Carrington, McLauchlan

Introduction to Magnetic Resonance

Schweiger

Ang. Chemie (Int. Edit. Engl.) (1991) 30:265-92

Applications to Biology: Berliner

Biol. Magn. Reson.

Deligiannakis et al.

Coord. Chem. Rev. (2001) 204:1-112

Prisner et al.

Annu. Rev. Phys. Chem. (2001) 52:279-313

Prisner

in Essays in Contemporary Chemistry