Paramagnetic Effects
BCMB/CHEM 8190
2012
References
Expanding the utility of NMR restraints with paramagnetic compounds:
Background and practical aspects, Koehler J and Meiler J,
Prog. NMR Spect. 59: 360-389 (2011)
Paramagnetic tagging for protein structure and dynamics analysis,
Keizers PM and Ubbink M, Prog. NMR Spect. 58: 88-96 (2011)
Exploring sparsely populated states of macromolecules by diamagnetic
and paramagnetic NMR relaxation, Clore GM, Prot. Sci. 20: 229-246
(2011)
Lanthanide-tagged proteins - an illuminating partnership, Allen KN and
Imperiali B, Curr. Opin. Chem. Biol. 14: 247-254 (2010)
Protein NMR Using Paramagnetic Ions, Otting G, Ann. Rev. Biophys,
39: 387-405 (2010)
Paramagnetic labelling of proteins and oligonucleotides for NMR, Su
X-C and Otting G, J. Biomol. NMR, 46: 101-112 (2010)
Paramagnetic Dipolar Spin Relaxation Effects
τc
3τc
6τc
2 μo 2 γ2H(geμB)2S(S+1)
R1p = ( )
+
+
[
]
6
2 2
2 2
2 2
r
15 4π
1+(ωH −ωS) τc 1+ ωHτc 1+(ωH + ωS) τc
3τc
τc
1 μo 2 γ2H(geμB)2S(S +1)
R2p = ( )
[4τc +
+
6
2 2
r
15 4π
1+ (ωH − ωS ) τc 1+ ωH2 τc2
6τc
6τc
]
+
+
2 2
2 2
1+ (ωH + ωS ) τc 1+ (ωS ) τc
• τc-1 = τm-1 + τe-1 where τm and τe are molecular
tumbling and electron spin correlation times
• Shortest term dominates, τe can add field dependence
• ωS is large at 11.7T (2x1012)
•τc of 10-9, ωH, τc terms dominates, R1 < R2
Relaxation Enhancement by Free Radicals
(Nitroxides) can Identify Interaction Sites.
Example: Galectin Interacting with LacNAc
Synthesis of a Spin-Labeled N-acetyllactosamine
O
OH
N
O N
O
.
HO
HO
O
OH
O
HO
OH
O
AcNH
HO
HO
N
O
.
THF, DCC
OH
OH
O
Dhbt-OH
N
O
N
DMF, DIPEA
CH3
CH3
NH
O
O
OH
O
HO
CH3
N
CH O
3
.
OH
O
AcNH
NH2
Intensities in HSQC Experiments are Measures of R2
(transverse proton magnetization during 2τ period is major loss)
1H
1H(I)
90-x
τ
τ = 1/4J
τ
180y
15N
90y
15N(S)
90x
Battiste and Wagner (2000) Biochemistry 39:5355-5365
Change in 15N HSQC spectrum (800 MHz)of
Galectin-3 upon addition of LacNac-TEMPO
0 mM
10 mM
Distances from R2 Equation
Residue
X-Ray model
(Å)a
Spin Label method (Å)b
τc = 6 ns τc = 8 ns τc = 10 ns
182
14.0
14.9
15.6
16.2
184
11.9
10.7
11.2
11.6
185
14.2
14.9
15.5
16.1
186
14.9
13.7
14.4
14.9
187
17.2
17.2
18.0
18.6
162
18.9
17.4
18.2
18.9
164
19.1
19.5
20.4
21.1
X-Ray crystal structure of Galectin-3
(Seetharamana et al. 1998)
E184
E165
R186
K227
A245
There are some anomalies – control looking at TEMPO alone
Proteins Can Also Be
Tagged
Membrane-Bound
myr-yARF1-GTP
Membrane model:
DHPC/DMPC bicelles.
Complex ~ 70kDA
Bicelle
PREs Give Long-Range Distance Constraints
+
N O
r
1H
5
1 N
PRE ∝ r
−6
1
=
Ne Nm
Ne Nm
−6
r
∑∑ ij
i =1 j =1
PRE Data using 1H-15N HSQC Attenuations
S62C-MTSL
D67
W66
Fitting PREs Required 3-State Averaging
for N-terminal Helix
Ensemble Averaging:
XPLOR-NIH
over 3 nitroxide conformers
over 3 protein conformers
Liu Y, Kahn RA and
Prestegard JH (2010).
Nature Struct Mol Biol.
17:876-81.
Iwahara,Schwieters,and Clore
(2004) JACS, 126, 5879-96
Myr-yARF-GTP – FAPP1-PH Interactions
FAPP paramagnetic perturbations from T56C ARF
E80 A78
180
G67
N58
W15
FAPP‐ARF docked models using PREs
180
Membrane
90
Metal Ions Have useful Paramagnetic Properties
Bertini, Luchinat, Parigi, (2001) “Solution NMR of Paramagnetic Molecules”
Short Electron Spin Lifetimes: Contact Shifts,
Pseudo-Contact Shifts and Field Alignment
(Bertini, 2001)
Curie Spin Relaxation Still Occurs for Short τe
λPRE
3τ r
1 μ o 2 1 B2o γ 2H (g J μ B ) 4 J 2 ( J + 1) 2
= ( ) 6
(4τ r +
)
2
2 2
( 2k B T )
1 + ωH τ r
5 4π r
• Comes from excess population of β spin state –
there is a net average magnetic moment
• Note field squared dependence – lose signals at
10Å at 18T
• Best to use an ion with low J
Identifying a Dimer Interface with (Gd-DTPA)
Lee H-W, Wylie G, Bansal S; et al. Prot. Sci. 19:1673-1685 (2010)
Comparison of crosspeak
attenuation at high and
low protein concentration.
Ratio of effects givens
protection factor. Red are
protected
Pseudo Contact Shifts and Molecular Alignment
Bertini, I., et al. (2002). Concepts in Magnetic Resonance 14: 259-286.
1
2
2
PCS =
χ
θ
χ
θ cos 2ϕ]
[
Δ
(
3
cos
−
1
)
−
Δ
sin
ax
rh
3
12πr
2
2
1 Bo γ H γ N hS
2
2
RDC =
χ
χ
[
Δ
(
3
cos
Θ
−
1
)
−
Δ
sin
Θ cos 2Φ ]
ax
rh
2
3
rNH k B T
120π
• These effects depend on anisotropic susceptibilities
• Size of Induced dipoles depends on molecular orientation
Hence, rotational averaging does not reduce dipolar field to
zero in PCS case
• Difference in interaction with B0 for different orientations
results in field induced alignment and measureable RDCs
Metal binding peptide
sequences (EF-hand)
can be added to protein
constructs.
Pseudo-contact shifts
and RDCs
provide validation of
structure and
positioning of ligands
Lanthanide –Tagged Galectin-3
Zhuang T, Lee H-S, Imperiali B et
al., Prot. Sci. 17:1220-1231 (2008)
PCSs of Galectin-3-LBT
Lu3+ - Black
Dy3+ - Green
15N-1H
RDCs and PCSs of Galectin-3-LBT(Dy3+)
(Agreement with crystal structure 1A3K)
Agreement of RDCs and PCSs suggest a rigid model can be used
PCSs for 2.5mM Lactose with 0.5mM Galectin-3 at 1H frequency 800MHz
PCSobs(Hz)
Glc(α)
Gal
PCSbound(Hz)
Back-cal(Hz)
H1
-10±1
-48±5
-45
H2
-116±1
-53±5
-55
H3
-8±1
-42±5
-46
H5
-10±1
-51±5
-53
H1
-10±3
-50±15
-49
H2
-9±3
--45±15
-49
H3
-9±3
45±15
-43
H4
-11±3
-55±15
-51
H5
-11±3
-55±15
-59