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
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
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
Basic physics
Energie E
Magnetic Resonance Condition

+1/2
∆E = g ⋅ µ B ⋅ B0 = h ⋅ν MW
Under resonance conditons

microwave gets absorbed
-> Detection signal
-1/2
magnetic field B 0
Instrumentation, Basic Principle
EPR Experiment
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
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 )
EPR Parameters : G-Tensor
Reflects symmetry of the electronic orbital of unpaired electron
Spherical
symmetric orbital
Cylinder
symmetrical
orbitalre
Lower symmetry
orbital
EPR Parameters : A-Tensor (HF-Tensor)
Electron spin density at nucleus: isotropic a
Spin density
n at n
e
Dipolare coupling to distant nucleus: anisotropic A

Distance to n
n
e
Advanced Methods
0.1 0.3 1 3.4 6.4 12.8 Magnetfeld

Multifrequency-EPR [T]
B 0
ν M W
3 9 35 95 180 360 Mikro-

[S] [X] [Q] [G] [sub- wellen-
[W]
mm] frequenz
[GHz]
Mikro-
wellen
Pulse
Puls-
Pulse-EPR
t 1
t 2 abstände
Kern-
Zeeman
14.9
3.7
6.0
2.2
1.1
2.0
Frequenz
(im X-Band)
ENDOR
(Electron Nuclear νR F
Double Resonance 1 4 1 7 2 1 3 3 1 1
Radio--
N O H C P H frequenz
[MHz]
Microwave frequency bands
m S= -1/2
1 T/35GHz
0,11 T (Q-Band)
3GHz
(S-Band) 6,4 T/180GHz
3,4 T/95GHz
(W-Band) (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
gxx mS = -½
gyy
O gxx
gyy
gzz
X-Band G-Band
Field dependence of spectra
Distringuishes field dependent and field independent parameters
Nitroxid spectra as a
function of magnetic field
Instrumentation: 180 GHz Spectrometer
Pulsed EPR
Hahn Echo sequence
Refocusing technique eliminates

inhomogeneous linewidth
t
3
t
0
t
1
t
π/2 π
2
microwave
FID ECHO
t 0
t 1
t
2 t time
3
ESEEM Spectroscopy
Small Hyperfine couplings
Measure of the echo amplitude as a function of T
Echo Amplitude
H
O
Me O 4 C H3
3 5
2 6
1
Me O H 0 2 4 6 8 1 0 1 2 1 4 1 6 1 8
T im e (µ s )
CH3 n
O
FFT
H
F F T A m p l i t u d e ( a. u. )
The semiquinone interacts

with 14N nitrogen
0
0 2 4 6 8
F r e q u e n c y ( M H z )
Electron Nuclear Double Resonance (ENDOR)
Anisotropic Hyperfine interactions
m S
m
I
“NMR detected by EPR”
Simultaneous irradiation of the sample

with microwave and radio frequencies
Enhanced spectral resolution
Simplification of hyperfine spectra

Electron Nuclear Double Resonance (ENDOR)
ENDOR spectra
-3
x 10
6
N H 17O P CH3
4
-2
-100 -80 -60 -40 -20 0 20 40 60 80 100
0.3
0.2
0.1
-0.1
10 20 30 40 50 60 70 80 90 100
0.3
0.2
0.1
-0.1
20 40 60 80 100 120 140 160 180 200
Pulsed Electron Double Resonance (PELDOR)
Dipolare coupling between paramagnetic molecules
distances of r AB between 10Å - 50Å
rAB = 29.1Å
O
O
N
O O
N
O O
1.2
1.2
1.1 S-Band (3.6 GHz)

X-Band (9.7 GHz) 1
Echoamplitude [a.u.]
1
0.8
0.9
0.6
0.8
0.4
ωDip = 2.1 MHz
0.7
0.2
0.6 r AB = 29.2 Å
0
0.5 0 5 10 15
0 1 2 3 4 5
Frequency [MHz]
Pump Pulse Position [µs]
Instrumentation: Pulsed X-band EPR/ENDOR
The
Theunpaired
unpairedelectron
electronas
asaalocal
localprobe
probe
Puls-ESR,PELDOR
ENDOR, ESEEM
cw-ESR
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
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
GHzGHz
QB
95 GHz
95 GHz
Structure
Structureofofthe
thechormophores
chormophoresininPSI
PSIby
byEPR
EPR
Abstand und Orientierung

P700 der Chromophore zueinander
und zur Membranebene
2.5 nm
27°
A1
1.48 nm
Fe/S
Semiquinone radical QA in bRC
Dynamics of protein bound molecules
echo intensity
echo detected
spectrum ϕ
O
O
T [µs]
1.6
relaxation
1.2 time 190 K
θ
2
0.8
ϕ
O
0.4 120 K
O
magnet field B 0
P21ras · MnII+ · GDP protein complex
§ Molecular switch for signal transduction
"active state"
Hydrolysis
GDP
exchange- Effector
k k
factor (GEF) d is s c a t
/ 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
C
active (GDP) state N GppNHp
loop L2
T35
loop L4
p21 --Mn2+
2+--GTP
p21ras
ras Mn GTPprotein
proteinnucleotide
nucleotidecomplex
complex
Konformationsänderung bei -> Hydrolyse
loop L2
H
loop L2 H Thr 35 O
O NH
Thr 35 H 2O H 2O
Ser 17 H2 O Ser 17
NH Mn Mn
H 2O
O H2O O O H 2O O O
"Nukleophiler
O Pγ O Pβ O Pα O Pβ O Pα
Angriff" H2O
O O O
- Pi O O
Lys 16 Lys 16
NH
loop L4
GTP NH
loop L4
Mulitfrequency EPR of P21ras · MnII+ · GDP protein complex
δ f
180 GHz
x 10
δ f 1. Mn
95 GHz Hyperfine
line
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 Mg GDP
Asp57 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 50
relative magnetic field B [G]
HF- EPR of P21ras · MnII+ · GDP protein complex
wt G12V
x5 n=5
n=4
n=4
n=3
n=2 n=3
T35S T35A
n=4 n=5
n=3
n=4
n=2 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 33800

M a g n e t fe ld [ G a u s s ]
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
His A403
H2O Mn
H. Käß, F. MacMillan, B. Ludwig, T. Prisner Biochemistry 104, 5362-5371 (2000)

Structure determination by EPR spectroscopy
15 2D-ESEEM Experimental
14
N (HYSCORE) 1
H
10
Spectra
F2 [MHz]
Experiment
5
a Bestimmung der
1 4 1
N und H
0
Wechselwirkungen
-5
-10
2 4 6 8 10 12 14 16
QM-Simulations
F1 [MHz]
Parameters
MO-Calculations
Molecular
Structure
Electron
ElectronParamagnetic
ParamagneticResonance
Resonance(EPR)
(EPR)
Multifrequency
Multifrequencycw-EPR:
cw-EPR:
Identification
Identificationand
andCharacterisation
CharacterisationofofRadicals
Radicals
PULSE-EPR
PULSE-EPRand andENDOR:
ENDOR:
Structural
StructuralInformation:
Information: Identification
IdentificationofofLigand
LigandSphere
Sphere(<
(<0.8
0.8nm)
nm)
PELDOR:
PELDOR:
Distance
Distancebetween
betweenParamagnetic
ParamagneticCentres
Centres(<
(<6nm)
6nm)
Time
TimeResolved-
Resolved-and
andFT-EPR:
FT-EPR:
Photoinduced
PhotoinducedElectron-Transfer
Electron-TransferKinetics
Kinetics
Dynamic Pulsed-High-Field-EPR:
DynamicInformation:
Information: 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

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Elektron-Paramagnetic Resonance Spectroscopy

Basics & Applications to Biological Systems

§ Basics (What is EPR ?)

EPR is a spectroscopical technique that detects:

EPR is nondestructive, needs 100 µl sample (or less!),

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

Magnetic Resonance Condition

Under resonance conditons

1897: Pieter Zeeman Line splitting in external magnetic field

Frequency: Factor 1000 larger in EPR ! (GHz instead of MHz)

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

Reflects symmetry of the electronic orbital of unpaired electron

Electron spin density at nucleus: isotropic a

Dipolare coupling to distant nucleus: anisotropic A

0.1 0.3 1 3.4 6.4 12.8 Magnetfeld

3 9 35 95 180 360 Mikro-

Resolution of G-anisotropy: Orientation selection

Distringuishes field dependent and field independent parameters

Refocusing technique eliminates

Measure of the echo amplitude as a function of T

The semiquinone interacts

“NMR detected by EPR”

Simultaneous irradiation of the sample

Enhanced spectral resolution

Simplification of hyperfine spectra

distances of r AB between 10Å - 50Å

1.1 S-Band (3.6 GHz)

Microwave transmission detection: sensitivity >1014 spins

(BChl)2+ BPh QA- QB

(BChl)2+ BPh QA QB-

Abstand und Orientierung

§ Molecular switch for signal transduction

Oncogenic mutation at glycin12 position

Konformationsänderung bei -> Hydrolyse

9.5 GHz Two states distinguishable

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

11800 12000 12200 12400

33200 33400 33600 33800

Distance and orientation between binuclear

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

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