ReferencesExpanding 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
])(1
61
3)(1
[)1()()4
(152
2222226
22H2
1cSH
c
cH
c
cSH
cBeop r
SSgRτωω
ττω
ττωω
τμγπμ
+++
++
−++
=
])(1
6)(1
61
3)(1
4[)1()()4
(151
2222
22226
22H2
2
cS
c
cSH
c
cH
c
cSH
cc
Beop r
SSgR
τωτ
τωωτ
τωτ
τωωτ
τμγ
πμ
++
+++
++
−++
+=
• 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
NO.
OHONN
N
O
NO.
OO
Dhbt-OH
THF, DCC DMF, DIPEA
O
AcNHHOO
OH
O
OHHO
HO OH
NH2
O
AcNHHOO
OH
O
OHHO
HO OH
NHO N
CH3
CH3CH3
O.CH3
Synthesis of a Spin-Labeled N-acetyllactosamine
15N1H
Intensities in HSQC Experiments are Measures of R2(transverse proton magnetization during 2τ period is major loss)
1H(I)
15N(S)
Battiste and Wagner (2000) Biochemistry 39:5355-5365
90-x 180y 90y
90x
τ ττ = 1/4J
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
R186
E165
A245
K227
There are some anomalies – control looking at TEMPO alone
Bicelle
Proteins Can Also Be Tagged
Membrane-Bound myr-yARF1-GTP
Membrane model: DHPC/DMPC bicelles.
Complex ~ 70kDA
PREs Give Long-Range Distance Constraints
∑∑= =
−− =∝e mN
i
N
jij
me
rNN
rPRE1 1
66 1
+
N O
15 N-1 H
r
Fitting PREs Required 3-State Averaging for N-terminal Helix
Ensemble Averaging:XPLOR-NIHover 3 nitroxide conformersover 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
Metal Ions Have useful Paramagnetic PropertiesBertini, 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
)1
34()2(
)1()(Br1)
4(
51
222
2242H
2o
62
rH
rr
B
BJoPRE
TkJJg
τωτ
τμγ
πμ
λ+
++
=
• 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 crosspeakattenuation at high and low protein concentration. Ratio of effects givens protection factor. Red are protected
Pseudo Contact Shifts and Molecular AlignmentBertini, I., et al. (2002). Concepts in Magnetic Resonance 14: 259-286.
]2cossin)1cos3([B
1201
]2cossin)1cos3([12
1
223
22o
2
223
ΦΘΔ−−ΘΔ=
Δ−−Δ=
rhaxBNH
NH
rhax
TkrS
RDC
rPCS
χχγγ
π
ϕθχθχπ
h
• These effects depend on anisotropic susceptibilities• Size of Induced dipoles depends on molecular orientation
Hence, rotational averaging does not reduce dipolar field tozero 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-3Zhuang T, Lee H-S, Imperiali B et al., Prot. Sci. 17:1220-1231 (2008)
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