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Protein assignment strategies in the solid-state
102
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Sample preparation
103
Types of sample:
• Microcrystalline
• Fibrillar
• Membrane
• Other
Considerations:
• Linewidth
• Salt concentration
• Hydration
• Temperature
Lyophilized
Fast evaporation from MeOH/H2O
Slow evaporation from MeOH/H2O
Changes in Antamanide spectra as a function of sample preparation
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Acquisition conditions
104
Temperature
• Ensure heating from spinning/rf pulses adequately compensated
• Optimal dynamics for sensitivity/resolution
Spinning speed
• Volume of rotor (can you get 15mg’s sample into it)
• Avoid rotational resonance conditions
• Sample heating
Decoupling
• Higher decoupling fields typically give best results (heating)
• Ensure no interference with spinning, pulse sequences applied to low- nuclei
• Can you manage without?
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Overview
105
Identify spin-systems
Correlate spin-systems with amino acids
Identify sequential
connectivities
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Identification of spin systems
106
Identify all connected 13C atoms
• PDSD/DARR
• Double quantum expt’s
– SQ/SQ
– DQ/SQ
• Considerations
– Efficiency
– Adjacency (multiple bond transfers)
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Single Quantum/Single Quantum
107
Range of expts:
• Proton driven spin diffusion
• Dipolar assisted rotary resonance
• RFDR/C7/MELODRAMA etc ….
• J-coupled transfers TOBSY
TOBSY spectrum of antamanide (r=20 kHz)
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Double quantum recoupling experiments
108
• Sequence of transfer encoded in sign of cross-peak
• Efficiency of transfers high
• Can filter diagonal
However
• Application at high fields difficult
2D DREAM spectrum of antamanide (r=20 kHz)
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DQ/SQ Correlation Spectra
109
• Different chemical shift dispersion to SQ/SQ expts.
• Maybe helps resolve ambiguities
DQ/SQ Spectrum of Crh (10kDa) acquired at 600 MHzDouble Quantum recoupling achieved with SPC-5 (C7 variant that works at higher spinning speeds), r=11kHz
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Identification of amino acids
110
• Connectivity's
– Work through side chain resonance
• Chemical shifts
– Identify characteristic chemical shift patterns for given amino acids
– For random-coil shifts of amino acids see additional handout.
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Assignment of the individual amino acids
111
• Can now assign peaks to individual classes of amino acids
• Side chains enable us to link C/CO resonances to amino acid types
• How do we work out which order they are in?
R i-2
N
O
NN
NN
O
O
O
O H
O
H
R i-1
R i
R i+1
R i+2
H
H H
H
H
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Nitrogen the link
112
• Nitrogen can be correlated to i and i-1 residues
• Enables us to make assignments sequential
• How do we assign the nitrogen spectrum?
Ri-2
N
O
NN
NN
O
O
O
OH
O
H
Ri-1
Ri
Ri+1
Ri+2
H
H H
H
H
C Cii-1
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Assigning nitrogen spectra - CP
113
1H
/2)ySpinLock
Decouple
13C
15N
DecoupleDecouple
Ri-2
N
O
NN
NN
O
O
O
OH
O
H
Ri-1
Ri
Ri+1
Ri+2
H
H H
H
H
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Assigning nitrogen spectra - CP
114
Ri-2
N
O
NN
NN
O
O
O
OH
O
H
Ri-1
Ri
Ri+1
Ri+2
H
H H
H
H
1H
/2)ySpinLock
Decouple
X
Y
r
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Nitrogen/Carbon Correlation
115
1H
/2)ySpinLock
Decouple
13C
15N
DecoupleDecouple• Magnetisation transfers in both
directions• Can assign nitrogen resonances
through 13C shifts
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Directed transfers – making CP specific
116
1H-13C
15N-13C
1) Hartmann-Hahn condition
small for 15N/13C correlations
2) Application of weak rf fields
for cross-polarisation
Advantages
• Sensitivity (magnetization transferred in one direction)
• Alleviates problems with broad-banded transfers
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Directed transfers – specific transfers
117
1H
/2)ySpinLock
Decouple
13C
15N
DecoupleDecouple
Set carrier toCO or C freq.
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NCA expt (15N to Ci)
118
NCA Expt.
• Good spectral dispersion in C
750MHz, r=12 kHz, 13C/15N cross polarization B115N=30 kHz: B1
13N=15 kHz (centred on 45 ppm)
NN
N
O
O
H
Ri-1
Ri
H
H
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NCO expt (15N to COi-1)
119
NCO Expt.
• Poor spectral resolution in CO
750MHz, r=12 kHz, 13C/15N cross polarization B115N=30 kHz: B1
13N=15 kHz (centred on 180 ppm)
NN
N
O
O
H
Ri-1
Ri
H
H
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Spectral crowding in the CO region
120
Problem
• Poor resolution in the carbonyl region, particularly acute in -helical proteins
Solution:
• Make assignments relying solely on C
i and Ci-1
• Transfer magnetization from NCOC
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NCOC: A solid state N(CO)CA
121
1H
/2 SpinLock
Decouple
13C
15N
DecoupleDecouple
13C/13Ctransfer
Ri-2
N
O
NN
NN
O
O
O
OH
O
H
Ri-1
Ri
Ri+1
Ri+2
H
H H
H
H
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Sidechains already assigned
122
Identified spin systems which we can correlate to particular amino acids
We know C/CO shifts for given amino acid type
TOBSY spectrum of antamanide (r=20 kHz)
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N(CO)CA - Antamanide
123
600 MHz, r=20 kHz, 13C/15N cross polarization B115N=30 kHz: B1
13N=50 kHz (centred on 180 ppm)Directionality of transfer determined by direction of sweep of rf field in NC cross polarisation
• Walk through assignment
• 15N degeneracy?
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Implementation of the N(CO)CA expt.
124
Considerations:
• Transfer efficiency (CP, 13C/13C mixing)
• Sensitivity – optimize transfers
– NC transfer
• Double cross-polarization• TEDOR (hardware dependent)
– CC Homonuclear recoupling
• Directed/Coherent transfers RR-tickling (80% efficiency)• PDSD/DARR – weak correlations to side chains
Decouple
1H
/2 SpinLock
Decouple
13C
15N
Decouple
13C/13Ctransfer
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Assignment of protons
125
• Resolution in proton spectra under MAS poor
• Homonuclear decoupling methods for acquiring proton spectra in direct dimension exist but resolution poor
• Typically proton chemical shifts obtained through correlation with carbon/nitrogen
• Implemented as a HETCOR experiment, with homonuclear decoupling in the indirect dimension
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1H/X Correlation Spectroscopy
126
• Spin lock typically shift to prevent spin-diffusion
• Spinning speeds typically low to aid LG decouplinig
1H
/2)yLee-GoldbergDecoupling
Decouple
X
t1
t2
SpinLock
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Lee-Goldberg Decoupling
127
• Protons irradiated off-resonance by 1/√2
• Precess at magic-angle, averaging homo-nuclear interactions
• Scaling other interactions e.g. chemical shift
• Typically implemented as a phase ramp (as opposed to frequency jump)
Iz
Ix
1
1
I
I
I 1/√2
54.7º
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1H/X Correlation Spectroscopy
128
• Resolution, primarily from low- nuclei
• 1H Spectra more sensitive to changes in environment
– H-bonding
– Ring current effects
1H/15N HETCOR Spectrum of SH3750 MHz, r=8 kHz, 750s cross-polarization van Rossum et al. 2003 Journal of Biomolecular NMR 25: 217-223
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So what is stopping us!
129
Identify spin systems
Correlate them to particular amino acids
Link them together
sequentially
We currently rely on:
• Unambiguous assignment of 15N spectrum (no overlap)
• Ability to resolve all resonances in the C/CO region
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Target proteins
130
• Some proteins more amenable than others!
• To date many of the larger proteins assigned by solid-state NMR have been -sheet structures (convenient for amyloid based studies)
• What about all the -helical proteins (membrane proteins)
Spera & Bax 1991 JACS
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Challenging systems
131
• Higher fields
– As inhomogeneous broadening is no longer the problem, improvements in resolution are realised at higher field
• Better decoupling (J-decoupling)
– Refocus J couplings during evolution periods
• Progress to 3D/4D experiments
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15N resolved 13C/13C spectra
132
1H
/2 SpinLock
Decouple
13C
15N
DecoupleDecouple
13C/13Ctransfer
Ri-2
N
O
NN
NN
O
O
O
OH
O
H
Ri-1
Ri
Ri+1
Ri+2
H
H H
H
H
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3D correlation spectra of DsbB
133
NCACX CAN(CO)CX
N(CO)CX CON(CA)CX
Li et al. 2007 Protein Science 17: 199-204DsbB, 4 transmembrane domain (20kDa), data acquired at 750 MHz.Transfers made using CP and DARR. Decoupling 75 kHz TPPM, 90kHz CW during CP.
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Summary
134
Progress critically dependent on
• Sensitivity !!!!!
• Resolution (sample, magnetic field)
• Chemical shift dispersion
• Careful optimization of transfer steps
• Sample stability
However
• Tools for assignment exist
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Use of assignments
135
• Local electrostatic environment
– Hydrogen bonding
– Ring current effects
• Conformation
– Backbone chemical shifts (torsion angles)
• Subsequent structural studies
– Structural studies (direct determination of distances/torsion angles etc).
• Next seminar
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Structures from solid-state NMR: Oriented samples
136
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Oriented samples
137
Cys192/193
Field (B0)
C3
C3’
Orientation
0°
90°
Necessary to introduce macroscopic alignment:1) Crystallization2) Oriented membranes3) Fibres (Silk/DNA)
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Orientation constraints from multiply labelled proteins
For proteins and peptides
• Need resolution
• Characterise backbone orientation
Solution
• Exploit 15N chemical shielding anisotropy
• 1H-15N dipolar coupling
• Characterise orientation of peptide plane
138
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PISEMA spectraPolarization inversion spin exchange at the magic angle
• 15N chemical shielding anisotropy
• 15N-1H dipolar interaction
Good scaling factor (0.82) and can be implemented in 3/4D experiments to improve resolution
1H
X
/2)X
-Y Decouple
35.5º-X
Y+LG -Y-LG
X X -X
m
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PISEMA spectra of Fd coat protein
/2)X 35.5º-X
1H
X
-Y DecoupleY+LG -Y-LG
X X -X
m
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Tilt of helices from PISA wheelsPISA
Polarity Index Slant Angle
Position of wheels in PISEMA spectra give
orientation of helices in samples
141
TMD 30º with respect to
bilayer
Amphipathic helix on bilayer surface
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Assignment of PISEMA spectra
PISEMA Spectra of amino acid selectively labelled Fd cost protein (Marrassi, 2002)
Position of PISA wheel gives helix
tilt
Amino acid selective labelling provides
assignment
Assignment gives rotation axis of
peptide
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Extracting structure: dipolar wavesDipolar waves
• dipolar coupling verses residue
• periodicity arises from repeating structure (e.g. -helix)
•enables comparisons to be made with rdc’s in solution
•disruption in ideal nature of secondary structure readily apparent
143
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144
Dipolar waves: Fd coat protein
Breaks in wave indicate:
• Start of new secondary structure
• Deformation in secondary structure (kinks in helices)144
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Structures from solid-state NMR: Magic angle spinning
145
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Structures from MAS data
146
Assignment
• Spin systems• Sequential assignment
Chemical Shifts
• Secondary shifts• Dihedral angle prediction
Structural Constraints
• Proton driven spin diffusion• Torsion angle measurements• Specific distance constraints
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Conformation dependent shift
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• C, CO and C chemical shifts are sensitive to backbone conformation
Spera & Bax 1991 JACS
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Secondary chemical shifts
Can compare chemical shifts with random coil to extract structure
• >0 helical conformation
• <0 sheet conformation
Typically sheet resonances are more negative than helical resonances are positive
Convenient does not require assignment of all CO’s
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)()()()( rcobsrcobs CCCCCC Luca, S. et al. J. Biomol. NMR 20: 325-331
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Secondary chemical shifts
Quick check on secondary structure e.g. analysis of Het-S prion
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Ritter, C. et al. 2005 Nature 435:844-848
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Chemical shifts as structural constraints
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• Comparing the observed assignments with a database of structures whose assignment is known allows the backbone torsion angles to be determined
• Several programs now available:
– TALOS (http://spin.niddk.nih.gov/NMRPipe/talos/)
– DANGLE (part of ccpn)
• Typically give range of dihedral angles consistent with chemical shifts
• Can be used as restraints in structure refinement
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Distance constraints in proteins• 13C-13C spectra show high number of
correlations
• Transfer dipolar in origin – through space interaction
• Should have a number of through space constraints analogous to a 2D NOE experiment
The problems
• Dipolar truncation effects
• Long distances and weak couplings between 13C atoms
151Castellani, F. et al. 2002 Nature 420:98-102
SH3
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Dipolar truncation effects
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23 1Strong coupling between
adjacent 13C atoms
Weak coupling to remote spin
Magnetization trapped
Structurally interesting long range distances lost
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Partial and selective labelling• Partial and selective labelling
– 1,3-13C Glycerol
– 2-13C Glycerol
• Introduces 13C at only selective sites
• Reduces strong couplings
• Increases resolution
– Less peaks
– Fewer J-couplings
• Prevents relayed transfers
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Hong, M. and Jakes, K. 1999 j. Biomol. NMR 14: 71-74
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Partial and selective labelling
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[1,3-13C]-Glycerol[2-13C]-Glycerol
NB/ Care with fractional labelling different isoptomers present, not just a percentage incorporation at a given site
Castellani, F. et al. 2002 Nature 420:98-102
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Partial and selective labelling of SH3
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[2-13C]-Glycerol [1,3-13C]-Glycerol
500 ms proton driven spin diffusion data on SH3 (Castellani, F. et al. 2002 Nature 420:98-102)Long range couplings observed
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Distances from PDSD data• Measure pdsd at 50, 100, 200 and 500 ms mixing time
• Calibrate on conformation independent distances
• Cross peaks grouped into four catorgaries (in all cases lower limit 2.5Å)
– 2.53.8Å peaks between sequential C’s appear in first 50ms
– ~4.6Å (sequential C’s and C’s) and 4.65.4Å interstrand C’s and C’s) within first 100ms
– ~5.8Å (sequential C’s)
– All other interactions <7.5Å
• Grouping used as a basis for structure calculations
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Structure of SH3
157Castellani, F. et al. 2002 Nature 420:98-102
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Is partial labelling necessary
• Interactions between 1H and low nuclei remain
• Transfer driven by:
– heteronuclear coupling between 1H and low nuclei
– 1H-1H homonuclear interactions not averaged by MAS158
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Build-up of cross peak intensity
159Grommek et al. 2006 Chem. Phys. Letts 427:404-409
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Increasing the distances probed
• 13C/13C interactions – limited by strength of dipolar couplings
• 1H spectra difficult to manipulate
• Acquire low- resolved 1H/1H spectra
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1H
/2)ySpinLock
Decouple
13C
Decouple
CP CP
H
H
C
C
r12
t1 t2
m
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CHHC Spectra
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• Long range distances (red)
• Good agreement between distance and cross peak intensities
Gardiennet et al. 2008 J. Biomol. NMR 40:239-250
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Summary
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Assignment
• Spin systems• Sequential assignment
Chemical Shifts
• Secondary shifts• Dihedral angle prediction
Structural Constraints
• Proton driven spin diffusion• Long distance constraints (CHHC)
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Best results to date!
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Backbone rmsd 0.4ÅHeavy atom rmsd 1.0Å
Wasmer et al. 2008 Science 319:1523-1526
Type of restraints
Total 1H-1H 13C-13C Intra-molecular
Distance Total 134 44 90 30
Short range 12 12 - -
Medium 9 2 7 3
Register 68 13 55 14
Long-range 45 17 28 13
Total Intra-molecular
Inter-molecular
Ambiguous
H-bond 23 7 7 9
Periodicity 206 - 206 -
TALOS 74
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References1. Spin Dynamics: Basics of Nuclear Magnetic Resonance, Malcolm Levitt2. Biomolecular NMR, Jeremy Evans3. Principles of Magnetic Resonance, C.P. Slichter
Proton driven spin diffusion1. Distance information from proton-driven spin diffusion under MAS, Andreas
Grommek, Beat H. Meier, Matthias Ernst , Chemical Physics Letters 427 (2006) 404–409
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Appendix 1
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2sincos3
2coscos12
sin23
3
2sinsin32
2cos3cossin32
02
2202
01
01
S
C
S
C