Mpi Lecture
-
Upload
agopicha000 -
Category
Documents
-
view
214 -
download
0
Transcript of Mpi Lecture
-
8/14/2019 Mpi Lecture
1/40
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
-
8/14/2019 Mpi Lecture
2/40
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 l sample (or less!),concentrations of >100 molar paramagnetic species
-
8/14/2019 Mpi Lecture
3/40
Elektron-Paramagnetic Resonance Spectroscopy
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
-
8/14/2019 Mpi Lecture
4/40
-
8/14/2019 Mpi Lecture
5/40
Elektron-Paramagnetic Resonance Spectroscopy
Instrumentation, Basic Principle
EPR Experiment
-
8/14/2019 Mpi Lecture
6/40
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, Khler, Groenen, Borzyskowski
First single molecule EPR experiment
-
8/14/2019 Mpi Lecture
7/40
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)
amuch higher techniqual requirements !!
Sensitivity : Factor 1 000 000 better than in NMR !!
(1nM instead of 1mM )
-
8/14/2019 Mpi Lecture
8/40
Elektron-Paramagnetic Resonance Spectroscopy
EPR Parameters : G-Tensor
Sphericalsymmetric orbital
Cylindersymmetricalorbitalre
Lower symmetryorbital
Reflects symmetry of the electronic orbital of unpaired electron
-
8/14/2019 Mpi Lecture
9/40
Elektron-Paramagnetic Resonance Spectroscopy
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
-
8/14/2019 Mpi Lecture
10/40
Elektron-Paramagnetic Resonance Spectroscopy
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
-
8/14/2019 Mpi Lecture
11/40
Elektron-Paramagnetic Resonance Spectroscopy
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
-
8/14/2019 Mpi Lecture
12/40
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
-
8/14/2019 Mpi Lecture
13/40
Elektron-Paramagnetic Resonance Spectroscopy
Field dependence of spectra
Distringuishes field dependent and field independent parameters
Nitroxid spectra as afunction of magnetic field
-
8/14/2019 Mpi Lecture
14/40
Elektron-Paramagnetic Resonance Spectroscopy
Instrumentation: 180 GHz Spectrometer
-
8/14/2019 Mpi Lecture
15/40
t0
t1
t1
t2
t2
t3
t3
time
microwave /2
ECHOFID
t0
Pulsed EPRHahn Echo sequence
Refocusing technique eliminatesinhomogeneous linewidth
-
8/14/2019 Mpi Lecture
16/40
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
-
8/14/2019 Mpi Lecture
17/40
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
-
8/14/2019 Mpi Lecture
18/40
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
-
8/14/2019 Mpi Lecture
19/40
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
-
8/14/2019 Mpi Lecture
20/40
Elektron-Paramagnetic Resonance Spectroscopy
Instrumentation: Pulsed X-band EPR/ENDOR
-
8/14/2019 Mpi Lecture
21/40
The unpaired electron as a local probeThe unpaired electron as a local probe
cw-ESR
ENDOR, ESEEM
Puls-ESR,PELDOR
-
8/14/2019 Mpi Lecture
22/40
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 spinsConfocal microscope fluoreszenz: sensitivity >1 spins
-
8/14/2019 Mpi Lecture
23/40
Elektron-Paramagnetic Resonance Spectroscopy
Applications to Biological Systems
G-Protein complex
Photosynthesis
Cytochrome coxidase
-
8/14/2019 Mpi Lecture
24/40
(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
-
8/14/2019 Mpi Lecture
25/40
High-Field-EPR measurements on bRCHigh-Field-EPR measurements on bRC
QB
9 GHz330 GHz
95 GHz95 GHz
-
8/14/2019 Mpi Lecture
26/40
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
-
8/14/2019 Mpi Lecture
27/40
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
-
8/14/2019 Mpi Lecture
28/40
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 !
-
8/14/2019 Mpi Lecture
29/40
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
-
8/14/2019 Mpi Lecture
30/40
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
-
8/14/2019 Mpi Lecture
31/40
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
-
8/14/2019 Mpi Lecture
32/40
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 !
-
8/14/2019 Mpi Lecture
33/40
-50 -25 0 25 50relative magnetic field B [G]
HF- EPR of P21rasMnII+ GDP protein complex
4 H2O17 ligands
3 H2O17 ligands
-
8/14/2019 Mpi Lecture
34/40
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 !!
-
8/14/2019 Mpi Lecture
35/40
Cytochrom cOxidase of Paraccocus denitrificansCytochrom cOxidase of Paraccocus denitrificans
-
8/14/2019 Mpi Lecture
36/40
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
-
8/14/2019 Mpi Lecture
37/40
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
-
8/14/2019 Mpi Lecture
38/40
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
-
8/14/2019 Mpi Lecture
39/40
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
-
8/14/2019 Mpi Lecture
40/40
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