mpi lecture

8/14/2019 Mpi Lecture

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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 complexesMn+II, Cu+II , MoV

Spin labels

Mobility, Access, Distances


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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 l sample (or less!),concentrations of >100 molar paramagnetic species


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Application fields

Physics: Susceptibility, Semiconductors, Quantum Dots, Defect Centres ...

Chemistry: ET-Reaction Kinetics, Organo-Metallic, Catalysis, Molecular Magnets...

Ionization Radiation: Alanin radiation dosimetry, Radiation damage, Irradiated food ..

Material research: Polymers, Glases, Superconductors, Corrosion, Fullerenes, Dating ...

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


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Instrumentation, Basic Principle

EPR Experiment


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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, Khler, Groenen, Borzyskowski

First single molecule EPR experiment


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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)

amuch higher techniqual requirements !!

Sensitivity : Factor 1 000 000 better than in NMR !!

(1nM instead of 1mM )


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EPR Parameters : G-Tensor

Sphericalsymmetric orbital

Cylindersymmetricalorbitalre

Lower symmetryorbital

Reflects symmetry of the electronic orbital of unpaired electron


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EPR Parameters : A-Tensor (HF-Tensor)

Electron spin density at nucleus: isotropic a

n

n

e

e

Dipolare coupling to distant nucleus: anisotropic A

Spin densityat n

Distance to n


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Advanced Methods

Magnetfeld[T]

Mikro-wellen-frequenz[GHz]

Radio--frequenz[MHz]

3 9 35 95 180 360

[S] [sub-mm]

[G][W][Q][X]

0.1 12.86.43.410.3

t

t1

t2

14.9

1.1

2.0

2.2

3.7

6.0

1 4

N1 7

O1 3

C3 1

P1

H2

H

Kern-ZeemanFrequenz(im X-Band)

Mikro-wellenPulse

B0

Puls-abstnde

M W

R F

Multifrequency-EPR

Pulse-EPR

ENDOR(Electron Nuclear

Double Resonance


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Microwave frequency bands

0,34 T9,5GHz

(X-Band)

0,11 T3GHz

(S-Band)3,4 T/95GHz

(W-Band)

6,4 T/180GHz

(G-Band)

1 T/35GHz(Q-Band)

B 0 m = +1/2S

m = -1/2S


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O

O

CH3

gxx

gyy

gzz

X-Band G-Band

gxx

gzz

gyy

mS = +

mS = -

High-field EPRSpectral resolution

Resolution of G-anisotropy: Orientation selection


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Field dependence of spectra

Distringuishes field dependent and field independent parameters

Nitroxid spectra as afunction of magnetic field


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Instrumentation: 180 GHz Spectrometer


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t0

t1

t1

t2

t2

t3

t3

time

microwave /2

ECHOFID

t0

Pulsed EPRHahn Echo sequence

Refocusing technique eliminatesinhomogeneous linewidth


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ESEEM SpectroscopySmall Hyperfine couplings

T

Measure of the echo amplitude as a function of T

C H 3

O

O

C H 3

H

M e O

M e O

H

H

1

3

2

45

6

n

0 2 4 6 8 1 0 1 2 1 4 1 6 1 8E

cho

A

mp

litude

T i m e ( s )

0 2 4 6 8

0

FFT

Amp

litude(a.u.

)

F r e q u e n c y ( M H z )

The semiquinone interactswith 14N nitrogen

FFT


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mS

mI

E

NMR detected by EPR

Simultaneous irradiation of the samplewith microwave and radio frequencies

Enhanced spectral resolution

Simplification of hyperfine spectra

Electron Nuclear Double Resonance (ENDOR)Anisotropic Hyperfine interactions


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Electron Nuclear Double Resonance (ENDOR)ENDOR spectra

-100 -80 -60 -40 -20 0 20 40 60 80 100-2

0

2

4

6 x 10-3

N H 17O P CH3

10 20 30 40 50 60 70 80 90 100-0.1

0

0.1

0.2

0.3

20 40 60 80 100 120 140 160 180 200-0.1

0

0.10.2

0.3


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0 5 1 0 1 50

0 . 2

0 . 4

0 . 6

0 . 8

1

1 . 2

F r e q u e n c y [ M H z ]

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

distances of rAB between 10 - 50

0 1 2 3 4 50.5

0 .6

0 .7

0 .8

0 .9

1

1.1

1 .2

P u m p P u l s e P o s i t i o n [ s ]

Echoamplitude[a.u.]

S - B a n d ( 3 . 6 GHz )X - B a n d ( 9 . 7 GHz )

rAB = 29.1

rAB = 29.2

N

OO

O

NOO

O

Dip = 2.1 MHz


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Instrumentation: Pulsed X-band EPR/ENDOR


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The unpaired electron as a local probeThe unpaired electron as a local probe

cw-ESR

ENDOR, ESEEM

Puls-ESR,PELDOR


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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 spinsConfocal microscope fluoreszenz: sensitivity >1 spins


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Applications to Biological Systems

G-Protein complex

Photosynthesis

Cytochrome coxidase


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(BChl)2 BPh QA QB

(BChl)2* BPh QA QB

(BChl)2+ BPh

-QA QB

(BChl)2+

BPh QA-

QB

(BChl)2+ BPh QA QB

-

4 ps

200 ps

100 s

Photosynthetic bacterial reaction centre of rhodobacter spheroides


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High-Field-EPR measurements on bRCHigh-Field-EPR measurements on bRC

QB

9 GHz330 GHz

95 GHz95 GHz


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Structure of the chormophores in PSI by EPRStructure of the chormophores in PSI by EPR

2.5 nm

27

1.48 nm

P700

A1

Fe/S

Abstand und Orientierung

der Chromophore zueinander

und zur Membranebene


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Semiquinone radical QA in bRC

Dynamics of protein bound molecules

0.4

0.8

1.2

1.6

magnet field B0

echo detectedspectrum

relaxationtime

]

2

e

cho

intensity

190 K

120 K

O

O

O

O


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p21:GDP

GDP

"active state"

"inac tive state"

GTP Pi

exchange-

fac tor (GEF)Effector

/ GAPk

d i s sk

c a t

Hydrolysis

P21rasMnII+ GDP protein complex

Molecular switch for signal transduction

Oncogenic mutation at glycin12 position

strongly reduced catalytic rate constants !


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C

loop L4

GppNHpN

loop L2

T35

T35

C

loop L2

loop L4

GDPN Inactive (GDP) state

active (GDP) state

P21rasMnII+ GDP / GTP protein complex


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p21ras - Mn2+ - GTP protein nucleotide complexp21ras - Mn2+ - GTP protein nucleotide complex

PP PP

- PiP OO

OOO

OH O

H

Ser 17Ser 17Thr 35

Thr 35

NH

NH

NH

NH

OOO

O

Lys 16Lys 16

MnMn

OO

H O2H O2H O2

H O2

H O2H O2

"Nukleophiler

Angriff" H O2

OO

OO

GTP

loop L2loop L2

loop L4loop L4

Konformationsnderung bei -> Hydrolyse


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180 GHz

95 GHz

9.5 GHz

2.7 GHz

5 m T

x 10

x 10

f

f

Mulitfrequency EPR of P21rasMnII+ GDP protein complex

1. Mn

Hyperfine

line

Two states distinguishableby HF-EPR spectroscopy


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GDP

Thr35 Gly12

Asp57 Ser17

Lys16 Mg

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

P21rasMnII+ GDP protein complex

No differences atactive site for wt &oncogenic mutantby X-ray !


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-50 -25 0 25 50relative magnetic field B [G]

HF- EPR of P21rasMnII+ GDP protein complex

4 H2O17 ligands

3 H2O17 ligands


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1 mT

wt G12V

T35S T35A

n=4

n=2

n=2

n=4

n=3

n=3 n=4

n=4

n=5

n=5

n=3

n=3

x 5

HF- EPR of P21rasMnII+ GDP protein complex

Differences in ligand sphere determined by EPR spectroscopy !!


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Cytochrom cOxidase of Paraccocus denitrificansCytochrom cOxidase of Paraccocus denitrificans


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Multifrequency-EPR on Cytochrom cOxidaseMultifrequency-EPR on Cytochrom cOxidase

3 3 8 0 03 3 6 0 03 3 4 0 03 3 2 0 0

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

1 2 4 0 01 2 2 0 01 2 0 0 01 1 8 0 0

3 5 0 03 0 0 0

X - B a n d

Q - B a n d

W - B a n d


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Application on Cytochrome c Oxidase:Application on Cytochrome c Oxidase:

rAB

CuA

Mn His A403H2O

Glu B218

Asp A404

Cys B216

Ser B217

Cys B220

Distance and orientation between binuclearCuA centre and Mn binding site

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

S d i i b EPR


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2 4 6 8 10 12 14 16

-10

-5

0

5

10

15

F [MHz]

F

[MHz] HN

14 1

1

2

2D-ESEEM(HYSCORE)

Experiment

a Bestimmung der

N und HWechselwirkungen

1 4 1

Parameters

Experimental

Spectra

Molecular

Structure

QM-Simulations

MO-Calculations

Structure determination by EPR spectroscopy


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Electron Paramagnetic Resonance (EPR)Electron Paramagnetic Resonance (EPR)

Structural Information:Structural Information:

Dynamic Information:Dynamic Information:

Multifrequency cw-EPR:Identification and Characterisation of Radicals

Multifrequency cw-EPR:

Identification and Characterisation of Radicals

PULSE-EPR and ENDOR:

Identification of Ligand Sphere (< 0.8 nm)

PULSE-EPR and ENDOR:

Identification of Ligand Sphere (< 0.8 nm)

PELDOR:

Distance between Paramagnetic Centres (< 6nm)

PELDOR:

Distance between Paramagnetic Centres (< 6nm)

Time Resolved- and FT-EPR:

Photoinduced Electron-Transfer Kinetics

Time Resolved- and FT-EPR:

Photoinduced Electron-Transfer Kinetics

Pulsed-High-Field-EPR:Librational Dynamics of Protein-Bound Quinones

Pulsed-High-Field-EPR:

Librational Dynamics of Protein-Bound Quinones

PELDOR:

Conformational Dynamics

PELDOR:

Conformational Dynamics


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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

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