defence talk 24oct

CORRELATION SPECTROSCOPY andDISTANCE MEASUREMENTS IN PROTEINS

USING HIGH-FIELD PULSED EPR

• Electron-nuclear interactions (hyperfine)

Interactions in pulsed EPR

• Electron-electron interactions

Few MHz=Several angstrom <5 A Few MHz=2 nm<r< 8 nm

High field/high frequency

conventionalH

igh-frequency

3 cm, 0.3 T

3 mm, 3 T

High field.

AMOUNT OF SAMPLE:

50 µl

2-3 µl

Bruker X-band (wavelength~3 cm)

W-band (wavelength~3 mm)

High field.

Spectral resolution: H=gβB0SzH=gβB0Iz

0 100 200

1H31P13C2H

0 100 200

1H31P

13C2H

Nuclear frequencies at X-band (9.5 MHz)

W-band (9.5 MHz)

MW

RF

echo

π

π π/2 π

a

a

ff

|ααN> |αβN>

|βαN> |ββN>

MW RF

ENDOR- electron-nuclear double resonance

Double resonance experiments.

MW2

MWFIDπ/2

ΗΤΑ

MW

RF

echo

π

π π/2 π

a

a

ff

|ααN> |αβN>

|βαN> |ββN>

MW RF

a

a

ff

|ααN> |αβN>

|βαN> |ββN>

MW2

ELDOR- (electron-electron double resonance) – detected NMR

ENDOR- electron-nuclear double resonance

ELDOR-detected NMR and ENDOR.

ENDORELDOR-detected NMR

Depends on allowed nuclear transitions

Depends on forbidden electron transitions

Intensity

High (determined by the strength of RF

field B2 )

Low (determined by MW field strength, but

Tm limited)

Resolution

Narrow linesPreferable for broad lines 14N,55Mn,61Ni

Nuclei

ELDOR-detected NMR and ENDOR.

20 40 60 80 100 120 14020 40 60 80 100 120 140

Frequency, MHz

+ side- sideENDOR

Correlations for resolving crowded spectra.

• Multiple paramagnetic species• Multiple nuclei

118 120 122 124 126 128 130 132 134 136 138

RF, MHz

Single crystal of Cu2+ doped L-histidine.

ENDOR

ELDOR-detected NMR

MW2

RF

MWFIDπ/2

ΗΤΑ

π

TRIPLE ENDOR

THYCOS

MW

RF1

RF2

echoπ ππ/2

π

π

THYCOS: correlating ELDOR-detected NMR and ENDOR spectra

|α1α2>|α1β2>

|β1α2>|β1β2>

f

|α1α2>

|α1β2>|β1α2>

|β1β2>a

MW

|α1α2>|α1β2>

|β1α2>|β1β2>

|α1α2>|α1β2>

|β1α2>|β1β2>

a(n)|α1α2>|α1β2>

|β1α2>|β1β2>

|α1α2>|α1β2>

|β1α2>|β1β2>

RF

|α1α2>|α1β2>

|β1α2>|β1β2>

f

|α1α2>

|α1β2>|β1α2>

|β1β2>a

MW

|α1α2>|α1β2>

|β1α2>|β1β2>

|α1α2>|α1β2>

|β1α2>|β1β2>

a(n)|α1α2>|α1β2>

|β1α2>|β1β2>

|α1α2>|α1β2>

|β1α2>|β1β2>

RF

MW2

RF

MWFIDπ/2

ΗΤΑ

π

Ms |α>

Ms |β>

e

n1

n2

A1

A2


variable

-> nuclei belonging to the same paramagnetic center-> the lines from the opposite electronic manifold

νΙ1 νΙ2

A1 A2

ED-NMR excitation

νI1 νΙ2

A1>0 A2>0α β α β

νI1 νΙ2

A1>0 A2<0α β β α


e

n1

n2

A1

A2

Hα

Hε

Hα

HεHβ

-10 -8 -6 -4 -2 0 2 4 6 8 10-10 -8 -6 -4 -2 0 2 4 6 8 10

νH-νRF, MHz

ENDOR

THYCOS with MW irradiation on nitrogen

Frozen solution: complex of Cu2+ L-histidine

-120-100 -80 -60 -40 -20 0 20 40 60 80 100 120

Frequency, MHz

νHTA

63Cu, 65Cu

2H

N

N2

Hδ

Him

Hβ

Hβ

COOH

Ha

N1

Ham

Ham

Hε

Cu

N

N2

Hδ

Him

Hβ

Hβ

COOH

Ha

N1

Ham

Ham

Hε

14N

ELDOR

Hα

Hε

Hα

HεHβ

-10 -8 -6 -4 -2 0 2 4 6 8 10-10 -8 -6 -4 -2 0 2 4 6 8 10

νH-νRF, MHzΑ(α-proton) > 0, therefore A(14N)>0

ENDOR

THYCOS with MW irradiation on nitrogen

Frozen solution: complex of Cu2+ L-histidine

-120-100 -80 -60 -40 -20 0 20 40 60 80 100 120

Frequency, MHz

63Cu, 65Cu

2H

N

N2

Hδ

Him

Hβ

Hβ

COOH

Ha

N1

Ham

Ham

Hε

Cu

N

N2

Hδ

Him

Hβ

Hβ

COOH

Ha

N1

Ham

Ham

Hε

14N

Advantages:•Experiment is suitable for sampleswith pronounced forbidden transitions.•Does not require large power.•Sensitivity may be considerably gained by reducing the resolution.•No subtraction

Disadvantages:•Lack of resolution of ELDOR-dimension•Limited sensitivity.•Cavity bandwidth


3.00 3.05 3.10 3.15 3.20 3.25 3.30 3.35

Magnetic field, T

Type 1

β-protons

Frozen solution: ascorbate oxydase

Type 2

S

-20 -15 -10 -5 0 5 10 15 20

3000 3100 3200 3300 3400

Magnetic field, mT

B0=3360 mT

B0=3300 mT

B0=3200 mT

B0=3075 mT

3000 3100 3200 3300 3400

Magnetic field, mT

B0=3360 mT

B0=3300 mT

B0=3200 mT

B0=3075 mT

B0=3360 mT

B0=3300 mT

B0=3200 mT

B0=3075 mT


νRF-v1H, MHz

Axx,Ayy,Azz=[30 30 36] MHz and Euler angles) (α,β,γ)=(0, 50, 0)

3360

3330

3200

3075

B0,, mT


-60 -40 -20 0 20 40 60∆ν, MHz

14N1 sq

14N2, sq

14N1 dq

3360

3330

3200

3075

B0,, mT

να,β=|A/2±νI|νI =20 MHz

Lines at ~10,~30 and ~20 MHz

-60 -40 -20 0 20 40 60∆ν, MHz

14N1 sq

14N2, sq

14N1 dq

3360

3330

3200

3075

B0,, mT

HYSCORE

t1 t2t1 t2

ωαsqsq

dq

ωβsqsq

dq

ωαsqsq

dq

ωβsqsq

dq


να,β=|A/2±νI|νI =20 MHz

Lines at ~10,~30 and ~20 MHz


3200

-60 -40 -20 0 20 40 60∆ν, MHz

14N1 sq

14N2, sq

14N1 dq

3360

3330

3075

B0,, mT

A1=23 MHzA2=40 MHz

sq-dq

sq-sq

sq-sqsq-dq

sq-sq

sq-sq

ν1, MHZ

ν 2, M

HZ

-60 -50 -40 -30 -20 -10 0 10 20 30 40 50 60

∆ν, MHz

A(β-proton)>0 A(14N)>0 orA(β-proton)<0 A(14N)<0

14N

ELDOR-detected NMR


120 126 132 138 144 150 156 162RF, MHz

x20

(A)

(B)

120 126 132 138 144 150 156 162RF, MHz

x20

(A)

(B)THYCOS

ENDOR


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

MW

MW2

Refocused echoπ/2 ππ

π

Spins A

Spins B

pulsed ELDOR, DEER

Distance measurements in bis-Gd3+ complex using DEER at high-field

MW

MW2

Refocused echoπ/2 ππ

π

Spins A

Spins B


Bloc1 Bloc2

∆φ~ωddt

t

V(t)


t

V(t)

t

V(t)SUM

DEER data analysis.

Measured traceKernel for ideal pulses:

Unknown distance distribution

inverse problemTikhonov regularization

inverse problemTikhonov regularization

t

V(t)

N

N

N

O

OO

O

O

OO

Gd

O

N

N

N

O

OO

O

O

OO

Gd

O

(OH2)2(H2O)2

2.2126 nm (DFT)

Better sensitivity compared to nitroxide spin label


W-band 0.1 mM 2-3 ul ~12-24 h

X-band 0.1 mM 50 ul ~12-24 h

In collaboration with:A. RaitsimringD. Milstein

1.10 1.15 1.20 1.25 1.30 1.350.0

0.2

0.4

0.6

0.8

1.0

observe.

Magnetic field, T

Nor

mal

ized

ech

o in

tens

ity

pump

Ka-Band

3.34 3.36 3.38 3.40 3.42 3.44 3.46 3.480.0

0.2

0.4

0.6

0.8

1.0

Magnetic field, T

W-Band

25 K10 K

pump

observe

Nor

mal

ized

ech

o in

tens

ity

0

2

~HgDFWHHβ


Ka-band (26-40 GHz) W-band(95 GHz)

0 200 400 600 800 10000.94

0.95

0.96

0.97

0.98

0.99

1.00

t1

)1cos3(4

),( 23

02

21 −= θπ

µβθνhr

ggrDD


2 3 4 5r,nm

2.01 nm

Ms=7/2

5/2

3/2

1/2

-1/2

-3/2

-5/2

-7/2

)exp(~ 0

kTHgMp ees

M s

β−

At W-band TZ~4.6 K

Chance to find a pair~(p1/2+p-1/2)2


•Smaller modulation depth•Sensitivity to field drifts

Contra’s:

•High repetition rate•Good sensitivity in terms of sample amount•No need for large power

•Smaller modulation depth

Contra’s:

Pro’s:



Evaluated distance distribution

In collaboration with: A. Raitsimring T. Meade

400 600 800 1000 1200 1400 1600-0.04

-0.03

-0.02

-0.01

0.00

Lnno

rmal

ized

anp

litud

e

t/ns400 600 800 1000 1200 1400 1600

-0.04

-0.03

-0.02

-0.01

0.00

Lnno

rmal

ized

anp

litud

e

t/ns

15 20 25 30 35 40 45 500.0000

0.0004

0.0008

0.0012

0.0016

Distance measurements in proteins using DEER at high-field

+

4-Mercaptomethyl-dipicolinic acid

Cystein

Gd3+

Su et al JACS 2008, 130, 10486-10487.

P75 neurotrophin receptor

In collaboration with: G. Otting

2 3 4 5nm

-200 0 200 400 600 800-0.010

-0.008

-0.006

-0.004

-0.002

0.000

ln(V

(t))

t1, ns

Distance measurements in proteins using DEER at high-field

Nitroxide spin label at high-magnetic field: orientation selection

1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0r, nm

3370 3375 3380 3385 3390 3395 3400

sample: p75 labelled with MTSSLDEER Field: 3378.2 mTDataset: a23060901_BFile: p75_summary.opj

observe

magnetic field, mT

∆ν

pump

-200 0 200 400 600 800 10000.88

0.90

0.92

0.94

0.96

0.98

1.00

sample: p75 labelled with MTSSLDEER Field: 3380.3 mTDataset: alldeers_BFile: p75_summary.opj

norm

aliz

ed e

cho

inte

nsity

t1, ns

field = 3380.3 mT field = 3378.2 mT field = 3383 mT field = 3388.3 mT

-500 0 500 1000 1500 20000.650.700.750.800.850.900.951.001.05

3386.5 mT 3385.5 mT 3383 mT 3380.4 mT 3378.7 mT

norm

aliz

ed e

cho

inte

nsity

t1, ns

1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0r, nm

3365 3370 3375 3380 3385 3390 3395 3400 3405

pump

magnetic field, mT

observe

Nitroxide spin label at high-magnetic field: orientation selection

Domains IVa and V of theτ subunit of DNA polymeraseSu et al, Nucleic Acids Res, 2007, 35, 2825


0.0 0.5 1.0 1.5 2.0 2.5

0.990

0.995

1.000

norm

aliz

ed e

cho

inte

nsity

t1 (µs)

raw data background corrected

2 3 4 5 6r (nm)

fit by two gaussian functions Tikhonov regularization

Summary and outlook

•Correlation spectroscopy at high field - THYCOS, HYSCORE

•Distance measurements at high field- DEER

EPR team at Weizmann

Boris Epel - SpecMan

MW bridge – Yakov Lipkin, Yehoshua Gorodetski ,Koby Zibzner

Arnold Raitsimring – Gd3+

Acknowledgements

Gd3+:David MilsteinGottfried OttingThomas Meade

defence talk 24oct

Technology

magnetic field

ninucleieldordetected

nmrendor electron

mmhigh field

thigh field

electron double resonance

mw field strength

endoreldordetected nmrdepends