solid-state nmr: principles - tifriupab/madhu3.pdfsolid-state nmr: principles p. k. madhu department...
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Solid-State NMR: Principles
P. K. MadhuDepartment of Chemical Sciences
Tata Institute of Fundamental ResearchHomi Bhabha Road
ColabaMumbai 400 005, India
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Solid-State NMR
Matter
Gas Liquid Solid
Isotropic Anisotropic
Ordered Disordered
MembraneCrystals Fibrils Glasses
Biological materials
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Targets for SSNMR
Membrane proteins
Rhodopsin, Gramicidin, …….
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Targets for SSNMR
β-Amyloid fibrils
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Targets for SSNMR: Biology
• Lipid bilayers
• Membranes reconstituted with different additives such as cholesterol, drugs or peptides
• Structure analysis of membrane-active peptides, ion channels, and receptors
• Amyloid fibrils, silk, and elastic proteins
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Difficulties
•Restricted or no internal motion, unlike solution-state
•All interactions present in toto
•Interactions are anisotropic leading to broadeningof spectral lines
•Plethora of information present, leading to a completecharacterisation of materials
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Reality
Simple 1D solution-state spectrum1H spectrum of a protein
Simple 1D solid-state spectrum13C spectrum of glycine
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Remedies
• Mimick the inherent averaging processes in solution-state to obtain high-resolution, isotropic information
• Goal #1:(Resolution and Sensitivity): Remove anisotropic parts and retain only isotropic parts: Decoupling
• Goal #2:(Let us have the cake and eat it as well) Get backthe anisotropic parts for elucidation of geometry parameters: Recoupling
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Remedies
Anisotropic Part
Spatial Part
Spin Part
Independent:Can be individuallymanipulated
Mechanical manipulation
RF manipulation
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Hamiltonians and their Manipulation
[ ] isotropiccanisotropiSPINSPACETOTAL Η+Η⊗Η=Η
•Rotating the crystallites in a given powder •Sample spinning: Mechanical manipulation
•Easier to visualise•Difficult to implement
Spatial Part: Manipulation Spin Part: Manipulation
•Rotating the spins in a given powder •Spins rotation: Manipulation by RF pulses•Easier to Implement•Difficult to visualise
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Which Angle to Roate at?
Spatial part of the anisotropic Hamiltonian
)1cos3(2/1
)(cos
2
2
−θ
θP
0 100 200 300-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
P2(cosθ)=0 for θ=54.70
54.7
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B0
SAMPLEROTATION
Average out the chemical shift anisotropy, to achieve good
sensitivity and resolution
Magic-Angle Spinning (MAS)
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Averages out the chemical shift anisotropy, to achieve good
sensitivity and resolution
Magic-Angle Spinning (MAS)
θ=54.7
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13C spectra of [13C2]-glycine
no spinning
with MAS at 12 kHz
+H3NC
H H
O
O-
Resolution and Sensitivity Enhancement by MAS
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Magic-Angle Spinning Spectra: Resolution Enhancement
Glycine
The powder pattern breaks up into a centreband and sidebands spaced at integer multiples of the rotor frequency
14 kHz
8 kHz
5 kHz
3 kHz
1181 Hz
Static
Chemical Shift δ/ (ppm)50 0100150200250
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MAS Rotor TypesMAS Rotor Types
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Rotor caps
ZrOZrO22 MacorMacor BNBN KelKel--FF VespelVespel
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Standard Bore MAS ProbeStandard Bore MAS Probe
stator flip mechanism
bearing gas inlet
RF electronics
BN stator
proton trap
RF coil
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Abundant and Rare Nuclear Spins
Rare spins experience weaker homonuclear dipolar couplings, hence, the resolution limiting aspect is the heteronuclear dipolar coupling to the abundant 1H
13C, 15N…. 1H, 19F….
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Heteronuclear Dipolar Decoupling
MAS RF
Decoupling
Homonuclear dipolarcoupling
Heteronuclear dipolarcoupling
Abundant spins1H
Rare spins13C, 15N
Typical 1H-13C coupling= -25 kHz
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Heteronuclear Dipolar Decoupling
Decoupling
(π/2)y
S
I1H
13C
MAS RF
Decoupling
S spin detection
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15202530354045505560 ppm
MAS + Heteronuclear Dipolar Decoupling
Static
Static+Decoupling
MAS
MAS+Decoupling
13C spectra of adamantane
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MAS + Heteronuclear Dipolar Decoupling
2-13C Glycine5 kHz broadening
334 Hz broadening
80 Hz broadening
CW decoupling at 150 kHzMAS at 30 kHz
Only decoupling
Only MAS
MAS+Decoupling
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Cross Polarisation, CP
ωI ωS
½
-½
-½
½
Laboratory frame
ωI ωS
½
-½ -½
½
Doubly rotating frame
Energy levels of both nuclei are matched in the doubly rotating frame.A spin-lock RF field is equivalent to producing a rotating-frame transformation.Hence, we need a continuous spin-lock RF field on both the nuclei for CP.
A match of the energy levels is produced when the nutationfrequencies of both the spins along the effective RF field direction are the same: B1I=B1S or in other words γIωI=γSωS
Hartman-Hahn condition
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CP Pulse Sequence+Decoupling
Decoupling
S
I1H
13C
90x y
CP
CP
CPMAS, basic pulse block in solid-state NMR for both sensitivity and resolution
MAS and heteronuclear decoupling lead to resolutionCP leads to sensitivity
CP contact time
RF fields adjustedfor Hartman-Hahn condition
Magnetisation transfer
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CPMAS Spectrum
*Enhanced signal,~γI/γS
*T1 of abundant high-γ nucleishorter than that of the rare low-γ nuclei
*Spatial proximity
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Decoupling
S
I1H
13C
90x y
CP
CP
ΔωnutS=2−3ωr
B0
SAMPLEROTATION
CPMAS
The routine way towards high-resolution and sensitivityin solid-state NMR experiments
θ=54.7
Stejskal, Schaefer, Waugh, JMR, 18,560,1975Stejskal, Schaefer, Waugh, JMR, 28,105,1977
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SOLID STATE NMR
Selection rules may be generated at will
RF Field
Sample Rotation
B , Static Magnetic Field0
Electromagnetic irradiation >> internal coupling strengthsSpatial+Spin External Dynamic
Symmetry
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Recoupling under Magic-Angle Spinning:Retrieving Lost Interactions
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What is Recoupling and Why Recoupling
Solid-state NMR Solution-state NMR
Anisotropic interactionswith geometry information
CSA: Local chemical environmentDD: Distances and anglesQuad: Local environment, asymmetry,
distribution
Only isotropic information areinherently present
Geometry information availableindirectly via relaxation experiments
Direct manifestation of geometry parametersProblem: High-resolution schemes kill
the anisotropy and geometry information
Question: Can the lost anisotropic interactionsretrieved whilst retaining the isotropic resolution?
Having the cake and eat it too!
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Recoupling in Solution State
Recoupling is done in solution-state NMR, NOE for example
Rapid molecular motions modulate the dipole-dipole interactionsand the fluctuating dipolar fields can drive magnetisation exchange(or cross relaxation) between spins over a wide range of chemicalshifts
This mechanism fails in solid state due to the restricted molecularmotions that cannot supply the energy differences necessary formolecular motions (also resolution and sensitivity considerations play a role)
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Recoupling of Interactions
Rigid Solid MASRF Irradiation:Recouplinginteractions
Resolution, butInformation sacrificed!
Both resolutionand geometryinformation
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Hetero/Homonuclear Recoupling
Pay a price: A scale factor!
Couplings may be reintroducedfor certain periods of the experiment
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Sequence Zoo
DRAMA
MELODRAMA
RFDR
SEDRA
SEDOR
REDOR
CNnν
RNnν
USEME
RIL
R2
R3HORROR
DARR
BABA
DRAWS
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ωr = 5000 Hzωiso= 5000 Hz
0 2500-2500
-2500 0 2500
Rotational Resonance
ωr = 3000 Hzωiso= 5000 Hz
Raleigh et al., Chem. Phys. Lett. 146, 71, 1988
The splitting indicatesthe strength of the dipolar couplingbetween the two spins
Rotational Resonance Condition:Δωiso=n ωr
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Correlation Spectrum :RFDR
[U-13C-15N]achatin-II16 π pulsesMAS 10 kHz
Being used currently in biomolecules for 13C-13C correlation towards assignments
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RFDR: SH3 Domain Protein
Pauli et al., J. Magn. Reson. 143, 411, 2000
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Secondary Structure Elements
Backbone conformation by correlating two anisotropic interactionssuch as CSA or DD
Interactions measured from 2D experiments:Recoupling, double-quantum methods
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Structure by Solid-State NMR: Schematic
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Assignments in Solid-State NMR: Example
α-spectrin SH3 domain
NCACB
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Correlation Experiments: HNCA in Solid-State
15N-α-Spectrin SH3 domain 1H-15N HSQC (dipolar). 17.6 T, MAS 8 kHz
1H-15N-13C HNCA 9.4 T, MAS 8 kHz J. Biomol. NMR, 25, 217, 2003
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Sample Preparation Issues
Martin and Zilm, J. Magn. Res., 165, 162, 2003
Ubiquitin
polycrystalline
nanocrystalline
lyophilised
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Sample Preparation Issues
Pauli,….,Oschkinat, J. Biomol. NMR, 25, 217, 2003
Lypholised from aqueous low-salt buffer
Lypholised from aqueous low-salt buffer+adding water
Lypholised from a solution also havingPEG and sucrose
Precipitated from a rich ammoniumsulphate solution
SH3 domain protein
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Proteins in Solid-State NMR
Membrane proteinsAmyloid fibrilsSH3 domain proteinUbiquitinBacillus Subtilis protein Crhα-Synuclein
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Sequential Assignment and Conformational Analysis
2*10.4 kDa dimeric form ofThe Bacillus Subtilis protein Crh
Bockmann,….,Baldus, J. Biomol. NMR, 27, 323, 2003
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Sequential Assignment and Conformational Analysis
Bockmann,….,Baldus, J. Biomol. NMR, 27, 323, 2003
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Conclusions
•Solid-state NMR has come of age
•A rich pasture for spin gymnastics and choreography
•A judicious combination of MAS and RF very vitalfor most experiments
•Selective manipulation of each spin interaction possible
•The methods in vogue now are being used to develop toolsto do solution-state kind of experiments for assignmentsin biomolecules
• Remarkable progress already made in various biomolecularsystems