102

Protein assignment strategies in the solid-state

102

103

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

104

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?

105

Overview

105

Identify spin-systems

Correlate spin-systems with amino acids

Identify sequential

connectivities

106

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)

107

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)

108

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)

109

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

110

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.

111

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

112

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

113

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

114

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

115

Nitrogen/Carbon Correlation

115

1H

/2)ySpinLock

Decouple

13C

15N

DecoupleDecouple• Magnetisation transfers in both

directions• Can assign nitrogen resonances

through 13C shifts

116

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

117

Directed transfers – specific transfers

117

1H

/2)ySpinLock

Decouple

13C

15N

DecoupleDecouple

Set carrier toCO or C freq.

118

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

119

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

120

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

121

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

122

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)

123

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?

124

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

125

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

126

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

127

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

128

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

129

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

130

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

131

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

132

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

133

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.

134

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

135

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

136

Structures from solid-state NMR: Oriented samples

136

137

Oriented samples

137

Cys192/193

Field (B0)

C3

C3’

Orientation

0°

90°

Necessary to introduce macroscopic alignment:1) Crystallization2) Oriented membranes3) Fibres (Silk/DNA)

138

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

139

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

140

PISEMA spectra of Fd coat protein

/2)X 35.5º-X

1H

X

-Y DecoupleY+LG -Y-LG

X X -X

m

141

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

142

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

143

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

144

Dipolar waves: Fd coat protein

Breaks in wave indicate:

• Start of new secondary structure

• Deformation in secondary structure (kinks in helices)144

145

Structures from solid-state NMR: Magic angle spinning

145

146

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

147

Conformation dependent shift

147

• C, CO and C chemical shifts are sensitive to backbone conformation

Spera & Bax 1991 JACS

148

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

148

)()()()( rcobsrcobs CCCCCC Luca, S. et al. J. Biomol. NMR 20: 325-331

149

Secondary chemical shifts

Quick check on secondary structure e.g. analysis of Het-S prion

149

Ritter, C. et al. 2005 Nature 435:844-848

150

Chemical shifts as structural constraints

150

• 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

151

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

152

Dipolar truncation effects

152

23 1Strong coupling between

adjacent 13C atoms

Weak coupling to remote spin

Magnetization trapped

Structurally interesting long range distances lost

153

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

153

Hong, M. and Jakes, K. 1999 j. Biomol. NMR 14: 71-74

154

Partial and selective labelling

154

[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

155

Partial and selective labelling of SH3

155

[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

156

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

156

157

Structure of SH3

157Castellani, F. et al. 2002 Nature 420:98-102

158

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

159

Build-up of cross peak intensity

159Grommek et al. 2006 Chem. Phys. Letts 427:404-409

160

Increasing the distances probed

• 13C/13C interactions – limited by strength of dipolar couplings

• 1H spectra difficult to manipulate

• Acquire low- resolved 1H/1H spectra

160

1H

/2)ySpinLock

Decouple

13C

Decouple

CP CP

H

H

C

C

r12

t1 t2

m

161

CHHC Spectra

161

• Long range distances (red)

• Good agreement between distance and cross peak intensities

Gardiennet et al. 2008 J. Biomol. NMR 40:239-250

162

Summary

162

Assignment

• Spin systems• Sequential assignment

Chemical Shifts

• Secondary shifts• Dihedral angle prediction

Structural Constraints

• Proton driven spin diffusion• Long distance constraints (CHHC)

163

Best results to date!

163

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

164

164

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

165

Appendix 1

165

2sincos3

2coscos12

sin23

3

2sinsin32

2cos3cossin32

02

2202

01

01

S

C

S

C