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Spin-labeling
An introduction to Electron Spin Resonance (ESR), November 7, 2007
E h g H
where:
Relaxation
Evolution of a spin system is described
by Bloch equations:
Mx, My Mz magnetization components in the
rotating frame
0= eH0 the Larmor Frequency
e 1.76 10 7 rad /( s G )
ESR linewidth:
T2
1
1 2T22 ( H H 0 ) 2
T2
1
exp( 2T22 ( H H 0 ) 2 ) = inhomogeneous broadening
2
2
1
k e H
T2'
1
1
1
T2' T2 2T1
k=1
Lorentz
k ln 2 Gauss
proton
ratio
Rest mass
me =9.1094*10-28 g
mp =1.6726*10-24 g
5.446*10-4
Charge
e=-4.80286*10-10ESU
e=4.80286*10-10ESU
-1
Angular momentum
h/4
h/4
Magnetic dipole
moment
S=-ge eS
ge= 2.002322
S=-gN NS
gN= 5.5856
me=eh/4mec =
mN=eh/4mNc =
9.274*10-21 erg/G
5.0504*10-24 erg/G
1836.12
Sensitivity : Factor 1 000 000 better than in NMR !! (1nM instead of 1mM )
An ideal case, though
An introduction to Electron Spin Resonance (ESR), November 7, 2007
Circulator
electromagnet
Modulation coils
Detector
Resonator (cavity)
Field modulation
Commercial 95GHz
Spectrometer (3T)
The g-factor:
E = h = gH
The field at each spin influenced by local magnetic fields, not just the external field :
g-value
Flavin semiquinone, ubiquinone,
ascorbate, etc
2.0030 2.0050
2.0020 2.0090
2.02 - 2.06
1.94
Cu2+
2.0 - 2.4
1.4 - 3.1
2.0 - 10
I=1/2, 2I+1=2
I=1, 2I+1=3
Pyrazine anion
Observation of the
1:8:28:56:70:56:28:8:1
spectrum shows that eight
protons are equivalent
K+ is the counterion
Anisotropy in g and A
Many measurements are made in the solid state in EPR spectroscopy.
The ability of EPR to obtain useful information from amorphous (glassy) and
polycrystalline (powders) as well as from single crystal materials has attracted
much biology and biochemistry research
Usually : gX, gY, gZ are not all equal, so g is anisotropic. Same for AX, AY, AZ.
For EPR the local symmetry at an unpaired electron center is
categorised as :
Cubic. If x = y = z is cubic (cubal, octahedral, tetrahedral) No
anisotropy in g and A.
Uniaxial (Axial). If x = y, and z is unique.
Linear rotation symmetry (at least 3-fold). Two principal values
each for g and A. For an arbitrary orientation:
2
2
2
g 2 g XX
sin 2 cos 2 gYY
sin 2 sin 2 g ZZ
cos 2
Hr
h
h 2
The given solid angle is defined to be the ratio of the surface area A to the
total surface area on the sphere: A/4r2
sin
d2r2sind4r2sind
P ( H )dH sin d P ( H )
1
P( H )
h H r3 ( g II2 g 2 ) cos
An introduction to Electron Spin Resonance (ESR), November 7, 2007
dH / d
Axial Lineshape
Rhombic Lineshape
EPR Middle-Earth
Human hair
EPR dosimetry:
5000
10000
15000 G
The rotational angular momentum,
which is quenched in the liquid or
solid phases couples strongly to the
electronic spin and orbital angular
momenta
CH3
CH3
N
O
H3C
H3C
O
N
O
9.4 GHz
component I=0
is spread over hg/4
component -1
is spread over A+ hg/4
not overlap
170 GHz
Nitroxyl Lineshapes
As the tumbling correlation time
decreases, the extent of averaging of
anisotropic features increases and the
spectrum approaches the 3-line signal
that is characteristic of rapid tumbling.
In the motional narrowing region, the
dependence of the width of an
individual hyperfine line on the nuclear
spin state (mI) can be expressed as
B (mI ) A B mI C m I
Rotation correlation times between
10-11 and 10-6 are detectable by ESR
X-band
An introduction to Electron Spin Resonance (ESR), November 7, 2007
I(0)
I ( 0)
I( 1)
I(1)
1
C
2
I(0)
I(0)
2
I(1)
I(1)
2 b
1
2 4Bo b 1
and C
B
3
g
15
o
o
Bo
3g o
Where
1
g o (g x g y g z )
3
( g z 0.5( g x g y ))
Bo
g o
2
(A z 0.5(A x A y ))
3
Sample Calculation
OH
N
O
(arbitrary units)
Bo = 3.52 Gauss
= 9.274x10-21 erg/G
= 9.2449x109 s-1
h=6.626x10-27 erg s
= 2.1x10-9 s from B or = 2.3x10-9 s from C
The disagreement is an indication of the approximate nature of this
calculation.
Determination of microviscosity:
V
kT
(Stocks-Einstein)
N
O
Toluene
2.00986
Water/glyc 2.00878
Aiso
g-resolved spectra
NO
O
OH
NO
C
O
OH
O
C O
NO
X-rotation
Averaging effective tensor
components
gxx,, (gyy+ gzz)/2, (gyy+ gzz)/2,
Axx, (Ayy+ Azz)/2, (Ayy+ Azz)/2
Y-rotation
Averaging effective tensor
components
(gxx+ gzz)/2, gyy, (gxx+ gzz)/2,
(Axx+ Azz)/2, Ayy, (Axx+ Azz)/2
16-PC
CH3
_
CH2
CH2
CH2
ym
zR
_
O
ym
CH3
H3C
CH2
CH3
CH3
C
CSL
xm
+N
O
P
CH2
zm
xm
CH2
Side view
zm
O
N
O
Upper view
O
CH
CH2
CH2
zd
O
N
O
zd
ym
xm
zR
N
O
Z-ordering
X-ordering
Y-ordering
Aligned membrane
Vesicles
4 5
9 0
M O M D
6 0 5 0 0
6 0 6 0 0
6 0 7 0 0
6 0 8 0 0
6 0 9 0 0
6 1 0 0 0
, G
6 1 1 0 0
N
O
9 GHz
170 GHz
OH
NO
NO
O
R
OH
DCC
HO
N O
O
O
Protein
SH
NO
NO
CH3
SO2
CH2
Protein
CH2
Multifrequency Analysis
Time scale
R (s-1)
Note: R = (6R)-1
An introduction to Electron Spin Resonance (ESR), November 7, 2007
Oxygen Accessibility
Oxygen accessibility and
probe mobility were measured
as a function of sequence
number for spin labels
attached to T4 lysozyme (T4L)
and cellular retinol binding
protein (CRBP).
The correlation between the
two parameters indicates that
the most mobile sites are also
the most oxygen accessible.
The repeat period of about 3.6
for T4L is consistent with the
-helical structure of this
segment of the protein.
W. L. Hubbell, H. S. Mchaourab, C. Altenbach, and M. A. Lietzow,
Structure 4, 779-783 (1996).
An introduction to Electron Spin Resonance (ESR), November 7, 2007
S S 3( S j rjk )( Sk rjk )
g j g j ( j 3 k
)
rjk
rjk5
2
B
H DD
1 2 3( 1 r )( 2 r )
3
r
r5
1 2 2
2
g
(
3
cos
1)
e
B
3
rjk
Some modern pulse EPR techniques (DQC, DEER) can cancel out all interactions
resulting in an EPR spectrum except the dipole interaction in spin pairs
Example: spin labeled Gramicidin A
2 e / r 3
5.2 10 4
dipolar [ MHz ] / 2
3
r []
Interspin distance=
30.9
dipolar, MHz,
DQC-EPR ruler:
10
20
30
40
50
60
70
80
90
100
DQC RULER