This document discusses using high-field pulsed electron paramagnetic resonance (EPR) techniques such as DEER (double electron-electron resonance) to measure distances in proteins and other biomolecules. It describes how high frequencies allow for better resolution of spectral lines and sensitivity with smaller sample volumes compared to conventional EPR. Specific experiments are discussed that use DEER at high fields to measure distances in bis-Gd3+ complexes and resolve different species in metalloproteins using THYCOS (three-HYperfine-species CORrelation SpectroscopY). The document concludes by noting both advantages and limitations of high-field DEER for distance measurements.

1. CORRELATION SPECTROSCOPY and
DISTANCE MEASUREMENTS IN PROTEINS
USING HIGH-FIELD PULSED EPR

2. Interactions in pulsed EPR
• Electron-nuclear • Electron-electron
interactions interactions
(hyperfine)
Few MHz=Several angstrom <5 A Few MHz=2 nm<r< 8 nm

3. High field/high frequency
3 cm, 0.3 T
conventional
High-frequency
3 mm, 3 T

4. High field.
AMOUNT OF SAMPLE:
50 µl
Bruker X-band (wavelength~3 cm)
W-band (wavelength~3 mm)
2-3 µl

5. High field.
Spectral resolution: H=gβB0Iz H=gβB0Sz
Nuclear frequencies at X-band (9.5 MHz)
1H
31P
13C
2H
0 100 200
W-band (9.5 MHz)
1H
31P
13C
2H
0 100 200

6. Double resonance experiments.
|βαN> |ββN>
π π/2 π a
MW echo
MW RF
f f
RF π
a
ENDOR- electron-nuclear double
resonance |ααN> |αβN>

7. ELDOR-detected NMR and ENDOR.
|βαN> |ββN>
π π/2 π a
MW echo
MW RF
f f
RF π
a
ENDOR- electron-nuclear double
resonance |ααN> |αβN>
|βαN> |ββN>
π/2 FID
MW
a
MW2 MW2
ΗΤΑ f f
a
ELDOR- (electron-electron
double resonance) – detected
NMR |ααN> |αβN>

8. ELDOR-detected NMR and ENDOR.
ELDOR-detected NMR ENDOR
Intensity Depends on forbidden Depends on allowed
electron transitions nuclear transitions
Resolution Low (determined by High (determined by
MW field strength, but the strength of RF
Tm limited) field B2 )
Nuclei Preferable for broad Narrow lines
lines 14N,55Mn,61Ni

9. Correlations for resolving crowded spectra.
• Multiple paramagnetic species
• Multiple nuclei
+ side
- side
ELDOR-detected NMR
ENDOR
20 40 60 80 100 120 140
Frequency, MHz
Single crystal of Cu2+ doped L-histidine.
118 120 122 124 126 128 130 132 134 136 138
RF, MHz
ENDOR

10. THYCOS: correlating ELDOR-detected NMR and
ENDOR spectra
TRIPLE ENDOR
π π/2 π
MW echo
RF1 π
RF2 π
THYCOS
MW2 ΗΤΑ
RF π
π/2 FID
MW

11. THYCOS: correlating ELDOR-detected NMR and
ENDOR spectra
A1 n1
MW2 ΗΤΑ e A2
variable RF π n2
π/2 FID
MW
Ms |α> |β1α2>
|β1β2>
|β1α2>
|β1β2>
|β1α2>
|β1β2>
|α1β2> |α1β2> |α1β2>
|α1α2> |α1α2> |α1α2> a(n)
MW RF
f
a
|β1β2> |β1β2> |β1β2>
|β1α2> |β1α2> |β1α2>
|α1β2> |α1β2> |α1β2>
Ms |β> |α1α2> |α1α2> |α1α2>

12. THYCOS: correlating ELDOR-detected NMR and
ENDOR spectra
-> nuclei belonging to the same paramagnetic center
-> the lines from the opposite electronic manifold n1
A1
e A2
ED-NMR excitation n2
A1 A2
νΙ1 νΙ2
α β α β
A1>0 A2>0
νI1 νΙ2
α β β α
A1>0 A2<0
νI1 νΙ2

13. Frozen solution: complex of Cu2+ L-histidine
COOH Ham
Hβ Ham
N1
Hε H im
Hβ Ha
Hδ N
N2 Cu N2
Hδ
Hε Hβ Hε
N
Ha Hβ
H im Hε
N1
H am Hβ
Hα ENDOR
H am COOH
Hα
νHTA
14N
2
H
ELDOR THYCOS with MW irradiation on nitrogen
63
Cu, 65Cu
-10 -8 -6 -4 -2 0 2 4 6 8 10
νH-νRF, MHz
-120-100 -80 -60 -40 -20 0 20 40 60 80 100 120
Frequency, MHz

14. Frozen solution: complex of Cu2+ L-histidine
COOH Ham
Hβ Ham
N1
Hε H im
Hβ Ha
Hδ N
N2 Cu N2
Hδ
Hε Hβ Hε
N
Ha Hβ
H im Hε
N1
H am Hβ
Hα ENDOR
H am COOH
Hα
14N
2
H
THYCOS with MW irradiation on nitrogen
63
Cu, 65Cu
-10 -8 -6 -4 -2 0 2 4 6 8 10
νH-νRF, MHz
-120-100 -80 -60 -40 -20 0 20 40 60 80 100 120
Frequency, MHz Α(α-proton) > 0, therefore A(14N)>0

15. THYCOS: correlating ELDOR-detected NMR and
ENDOR spectra
Disadvantages:
•Lack of resolution of ELDOR-dimension
•Limited sensitivity.
•Cavity bandwidth
Advantages:
•Experiment is suitable for samples
with pronounced forbidden transitions.
•Does not require large power.
•Sensitivity may be considerably
gained by reducing the resolution.
•No subtraction

16. Frozen solution: ascorbate oxydase
Type 1
Type 2
β-protons S
3.00 3.05 3.10 3.15 3.20 3.25 3.30 3.35
Magnetic field, T

17. Frozen solution: ascorbate oxydase
B0=3300 mT
Axx,Ayy,Azz=[30 30 36] MHz and
Euler angles) (α,β,γ)=(0, 50, 0)
B0=3200 mT B0,, mT
B0=3360 mT
B0=3075 mT 3360
3000 3100 3200 3300 3400 3330
Magnetic field, mT
3200
3075
-20 -15 -10 -5 0 5 10 15 20
νRF-v1H, MHz

18. Frozen solution: ascorbate oxydase
14N1 sq
B0,, mT
3360 Lines at ~10,~30 and ~20 MHz
να,β=|A/2±νI|
3330 νI =20 MHz
14N2, sq
3200
14N1 dq
3075
-60 -40 -20 0 20 40 60
∆ν , MHz

19. Frozen solution: ascorbate oxydase
HYSCORE
14N1 sq
t1 t2
B0,, mT
3360 Lines at ~10,~30 and ~20 MHz
να,β=|A/2±νI|
sq
sq
dq ωβ νI =20 MHz
3330
14N2, sq
3200
14N1 dq
3075
sq
sq
dq ωα
-60 -40 -20 0 20 40 60
∆ν , MHz

20. Frozen solution: ascorbate oxydase
14N1 sq
B0,, mT
3360
A1=23 MHz
A2=40 MHz
3330
14N2, sq
3200
14N1 dq sq-dq sq-sq
3075 ν2, MHZ
sq-sq
-60 -40 -20 0 20 40 60
∆ν , MHz ν1, MHZ

21. Frozen solution: ascorbate oxydase
ELDOR-detected NMR
14N
-60 -50 -40 -30 -20 -10 0 10 20 30 40 50 60
∆ν, MHz
(A)
ENDOR
x20
THYCOS
(B)
A(β-proton)>0 A(14N)>0 or
A(β-proton)<0 A(14N)<0
120 126 132 138 144 150 156 162
RF, MHz

22. Frozen solution: ascorbate oxydase
•Using THYCOS experiment the lines
are assigned as: Aiso=23 MHz – type
1; Aiso~40 MHz – type 2
•Resolving species using
spectroscopy only.

23. Distance measurements in bis-Gd3+ complex using DEER at high-
field
π/2 π π
Refocused echo
MW Spins A
π
MW2
Spins B
pulsed ELDOR, DEER

24. Distance measurements in bis-Gd3+ complex using DEER at high-
field
π/2 π π
Refocused echo
MW Spins A
π
MW2
Spins B
Bloc1 Bloc2
V(t)
∆φ~ωddt
t

25. Distance measurements in bis-Gd3+ complex using DEER at high-
field
V(t)
SUM V(t)
t
t

26. DEER data analysis.
V(t)
t Unknown distance distribution
inverse problem
inverse problem
Tikhonov regularization
Tikhonov regularization
Measured trace
Kernel for ideal pulses:

27. Distance measurements in bis-Gd3+ complex using DEER at high-
field
O O
O O
O O
N N
O O
(H2O)2 Gd N N Gd (OH2)2
O O
N N
O O
O O
2.2126 nm (DFT)
O O
Better sensitivity compared to nitroxide spin label
X-band 0.1 mM 50 ul ~12-24 h
W-band 0.1 mM 2-3 ul ~12-24 h
In collaboration with:
A. Raitsimring
D. Milstein

28. Distance measurements in bis-Gd3+ complex using DEER at high-
field
D2
FWHH ~
gβ H 0
pump pump
1.0 1.0
W-Band
Ka-Band
0.8 0.8
Normalized echo intensity
Normalized echo intensity
observe.
0.6 0.6 observe
0.4 0.4
10 K 25 K
0.2
0.2
0.0
1.10 1.15 1.20 1.25 1.30 1.35 0.0
3.34 3.36 3.38 3.40 3.42 3.44 3.46 3.48
Magnetic field, T
Magnetic field, T
Ka-band (26-40 GHz) W-band(95 GHz)

29. Distance measurements in bis-Gd3+ complex using DEER at high-
field
g1 g 2 β 2 µ0
ν DD (θ , r ) = (3 cos2 θ − 1)
4πhr 3
1.00
0.99
0.98
2.01 nm
0.97
0.96
0.95
0.94
0 200 400 600 800 1000
t1
2 3 4 5
r,nm

30. Distance measurements in bis-Gd3+ complex using DEER at high-
field
Ms=7/2
5/2
3/2 M s geβeH 0
pM s ~ exp( − )
1/2
kT
-1/2 At W-band TZ~4.6 K
-3/2 Chance to find a pair~(p1/2+p-1/2)2
-5/2
-7/2

31. Distance measurements in bis-Gd3+ complex using DEER at high-
field
Contra’s:
Contra’s:
•Smaller modulation depth
•Smaller modulation depth
•Sensitivity to field drifts
Pro’s:
•High repetition rate
•Good sensitivity in terms of sample
amount
•No need for large power

32. Distance measurements in bis-Gd3+ complex using DEER at high-
field
Evaluated distance distribution
0.00
0.0016
0.0012
Ln normalized anplitude
-0.01
0.0008
-0.02
0.0004
-0.03 0.0000
15 20 25 30 35 40 45 50
-0.04 In collaboration with:
400 600 800 1000 1200 1400 1600
t/ns A. Raitsimring
T. Meade

33. Distance measurements in proteins using DEER at high-field
Cystein
+
4-Mercaptomethyl-
dipicolinic acid
P75 neurotrophin receptor
Gd3+
In collaboration with:
G. Otting Su et al JACS 2008, 130, 10486-10487.

34. Distance measurements in proteins using DEER at high-field
0.000
-0.002
-0.004
ln(V(t))
-0.006
-0.008 2 3 4 5
nm
-0.010
-200 0 200 400 600 800
t1, ns

35. Nitroxide spin label at high-magnetic field: orientation selection
sample: p75 labelled with MTSSL
DEER Field: 3378.2 mT
Dataset: a23060901_B
field = 3380.3 mT File: p75_summary.opj
∆ν
field = 3378.2 mT observe pump
field = 3383 mT
1.00
normalized echo intensity
field = 3388.3 mT
0.98
0.96 3370 3375 3380 3385 3390 3395 3400
magnetic field, mT
0.94
0.92
0.90
0.88
-200 0 200 400 600 800 1000 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0
sample: p75 labelled with MTSSL r, nm
t1, ns
DEER Field: 3380.3 mT
Dataset: alldeers_B
File: p75_summary.opj

36. Nitroxide spin label at high-magnetic field: orientation
selection
Domains IVa and V of the
τ subunit of DNA
polymerase
Su et al, Nucleic Acids Res,
2007, 35, 2825 observe pump
1.05
1.00 3386.5 mT
normalized echo intensity
3365 3370 3375 3380 3385 3390 3395 3400 3405
3385.5 mT
magnetic field, mT
0.95 3383 mT
3380.4 mT
3378.7 mT
0.90
0.85
0.80
0.75
0.70
0.65
-500 0 500 1000 1500 2000
t1, ns
1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0
r, nm

37. Distance measurements in bis-Gd3+ complex using DEER at high-
field
raw data
background corrected
normalized echo intensity
1.000
fit by two gaussian functions
Tikhonov regularization
0.995
0.990
2 3 4 5 6
0.0 0.5 1.0 1.5 2.0 2.5
r (nm)
t1 (µs)

38. Summary and outlook
•Correlation spectroscopy at high
field
- THYCOS, HYSCORE
•Distance measurements at high field
- DEER

39. Acknowledgements
EPR team at Weizmann
Gd3+:
David Milstein
Gottfried Otting
Thomas Meade
Arnold Raitsimring – Gd3+
MW bridge – Yakov Lipkin, Yehoshua Gorodetski ,Koby Zibzner
Boris Epel - SpecMan