MultiDimensional SolidState NMR of Proteins: Concepts

1
Multi-Dimensional Solid-State NMR of Proteins
Concepts Examples

• Chad M. Rienstra
• Department of Chemistry
• University of Illinois
• at Urbana-Champaign
• Chem590A
• 9/14/05

2
Outline

• Concepts
• Protein structure determination (Solution NMR)
• Chemical shift assignments
• Secondary structure determination
• Tertiary structure
• Solid-State NMR
• What is a solid for purposes of NMR?
• Resolution
• Sensitivity
• Applications Overview

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Structure Determination by NMR

• Choose protein(s)
• Make protein
• synthesis
• bacterial expression
• Label 15N and 13C
• Optimize sample conditions
• Acquire and interpret spectra
• (1) chemical shift
• (2) torsion angles
• (3) distances
• Compute structure

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Structures (Wüthrich)
5
The Multidimensional Paradigm

• Jeener, Ampere Summer School (1971).
• Ernst et al. (1974).(Nobel Prize 1991)
• Wüthrich (Nobel Prize 2002)

• Turn Hamiltonian termson and off as desired
• Resolve sites withchemical shifts
• Measure structural parameters in additional
  dimension(s)

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NMR for Chemistry

• H. Gutowsky, UIUC
• Applications of NMR to chemistry, biology, and
  medicine derived from work
• Based on two advances
• High resolution (site specificity)
• Chemical insight from observed frequencies

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High Resolution 13C NMR
Silverstein, Bassler, and Morrill, Spectrometric
Identification of Organic Compounds, Fig 5.2
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The Chemical Shift

• Not called NMR emission frequencies
• More usefulthan that!
• Electronicstructure

Silverstein, Bassler, and Morrill, Spectrometric
Identification of Organic Compounds, Appendix B
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Chemical Shifts in Proteins

• FromWüthrichlecture

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Unique Chemical Shifts in Proteins

• 1H and 13C shifts depend on
• 1. amino acid residue type
• 2. residue type of neighbors
• 3. conformation
• 4. hydrogen bonding

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Chemical Shift Trends

• Color coded by carbon type
• Each residue hasdistinctive pattern

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Residue Type ID

• Always clearG, A, S, T, P, I
• Usually clearL, V, D/N
• Aromaticsuse Cb-Cg todistinguish
• AmbiguousC, M, K, R, E/Q

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Sample Preparation

• Unfolded protein (top)
• Folded protein (bottom)
• Chemical shift dispersion

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High Resolution Solution NMR

• Basic ideas
• (1) site resolution
• (2) detailed chemical information at each site
• chemical shifts
• couplings
• relaxation rates
• dynamics
• function

• Gardner, Zhang, Gehring, and Kay J. Am. Chem.
  Soc. 12011738 (1998) Maltose binding protein,
  370 residues, 42 kDa complex with b-cyclodextrin

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Assignments Backbone Walk
Solid N-CO-CX CO-N-CA/CB N-CA-CX
Solution H-N-CO H-N-(CO)-CA H-N-CA H-N-CA/CO
H-N-CA/CB
- Baldus, M., et al. Mol. Phys. 95, 1197- 1207
(1998). - Takegoshi, K. et al. Chem. Phys. Lett.
344, 631-637 (2001).
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Chemical Shift Assignments

• 1. Identify amino acid type from chemical shifts
• 2. Connect to i-1 and i1 neighbors (also
  identified by amino acid type)
• 3. Iterate for entire protein until
  self-consistent solution is found

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Local NOEs Secondary Structure

• Helical region i to i3 contacts

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Long Range Distances

• Constrain fold
• NOE nuclear Overhausereffect (1/r6)

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Distance Geometry (Geography) Mapping
Molecular Structure
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What is Solid-State NMR?

• What is a solid?

Easier to ask what isnt a solid?
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SSNMR A Functional Definition

• For purposes of NMR, a solid is any material that
  does not tumble faster than the NMR time scale
  (ms to s)

ubiquitin tc 100 ps
lipid bilayer tc 1 s
micelle tc 10-100 ns
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Dipolar Averaging

• Solution State
• rapid tumbling tc lt 10-20 ns
• molecular weight limit 50-100 kDa
• Solid State
• global motion frozen
• three approaches
• static powder
• macroscopically oriented
• magic-angle spinning

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Types of Solids for NMR

• Chemical context
• Precipitated globular proteins
• Peptide aggregates
• Enzyme-substrate complexes
• Amyloid or synuclein fibrils
• Membrane proteins
• Trapped intermediates
• Physical appearance
• Powdered crystals (dry)
• Microcrystals (wet)
• Goopy precipitates
• Viscous solutions (slowly frozen)
• Membranes jelly or jello-like

?-amyloid fibrils, Tycko et al., Current Opinion
in Chemical Biology (2000).
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Membrane Proteins gt30 of Sequenced Genomes

• PDB 26,811 structures (8/17/04)www.pdb.org
• 143 membrane protein structures (82
  unique)blanco.biomol.uci.edu/Membrane_Proteins_x
  tal.html

22,886 by X-ray 3,925 by NMR

• GPCRs
• Drug design
• Bioenergetics
• Cell recognition
• Fundamental biophysics

new folds
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Next several slides are courtesy Stan Opella,
UCSD
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1H - 1H spins in water in gypsum. 1H are dilute
by space.
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1H - 15N spins in peptide plane. 15N is dilute
by space. 1H is abundant in large bath.
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Structure determination of membrane proteins
Indirect mapping through "residual" effects.
Direct mapping through "static" effects.
15N Shift (ppm)
1H-15N Cplg (kHz)
15N Shift (ppm)
residue number
1H Shift (ppm)
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Bicelle samples for structure determination of
membrane proteins.

• Native membrane environment.
• Phospholipids.
• Bilayers.
• Planar.
• Fully hydrated.
• Liquid crystalline phase.
• 37 oC
• Advantages over bilayers on glass plates.
• Sealed liquid samples.
• Solenoid RF coil
• No loss in filling factor.

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15N NMR spectra of aligned MerFt (2TM, 60
residues).
Bilayers on plates
Flipped bicelles
Unflipped bicelles
Tyr (3 sites)
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PISEMA spectra of membrane proteins in bicelles.
Pf1 coat Vpu TM domain
MerFt PISA Wheels 1TM, 46
residues 1TM, 36 residues 2TM,
60 residues
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Hydrophobic mismatchLength of helix vs.
thickness of hydrophobic center of bilayers.
White and Wimley, Annu. Rev. Biophys. Biomol.
Struct. 28, 319 (1999)
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Tilt angle compensates for hydrophobic
mismatch.No change in rotation angle.
T
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Spectra of membrane proteins in bicelles have
little or no isotropic resonance intensity from
motionally-averaged sites.
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Effect of flipping on MerFt spectra.
-NH3
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Structures of membrane proteins determined by
solid-state NMR of aligned samples.
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Assigned spectrum and preliminary
three-dimensional structure of MerFt in bicelles.
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Magic-Angle Spinning Methods

• 80 of proteins cant be studied by X-ray or
  solution NMR

membrane proteins (e.g., G-protein coupled
receptors)
microcrystalline globular proteins
peptide aggregates and fibrils
all of these proteins can be examined at high
resolution by employing 2D/3D magic-angle
spinning methods
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Progress with Small Peptides
f-MLF-OH 2002
12 Structures RMSD 0.02 Å (backbone)
Rienstra, C. M. et al. Proc Natl Acad Sci USA 99,
10260-5 (2002).
Lansbury, P. T. et al. Nature Structural Biology
2, 990-998 (1995).
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Amyloid Peptide Structures

• b-Amyloid
• Alzheimers disease
• Tycko (NIH)
• Griffin (MIT)
• Botto (Argonne)
• Transthyretin (TTR)
• senile dementia
• Jaroniec Griffin (MIT)

Petkova, A. T. et al. J. Mol. Biol. 335, 247-260
(2004). Jaroniec, C. P. et al. Proc. Natl. Acad.
Sci. USA 101, 711-716 (2004).
41
GB1

• 56 residues
• known structure
• rehearsal molecule

a-Synuclein

• 140 residues
• no known structure
• Parkinsons disease

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Typical Experiment Late 1990s

• 13C-Asp retinal labeled in bacteriorhodopsin
• Slow, labor-intensive, myopic

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Distance Geometry (Geography) Mapping
Molecular Structure
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Ultra-High Resolution SSNMR

• 2,000 resolved peaks in one 2D spectrum

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Outline

• Concepts
• Protein Structure Determination
• Solid-State NMR
• Application 1
• GB1
• Known protein structure
• Develop and demonstrate methods
• Application 2
• Synuclein
• Parkinsons disease
• Impossible to study by solution NMR or X-ray

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Model System GB1

• Expresses well (100 mg/L)
• Easy purification (heat, one column)
• High quality crystal (1pga) and NMR (1gb1)
  structures available
• Thermostable (Tm 85 oC)

1 MET GLN TYR LYS LEU ILE LEU ASN GLY LYS 11
THR LEU LYS GLY GLU THR THR THR GLU ALA 21 VAL
ASP ALA ALA THR ALA GLU LYS VAL PHE 31 LYS GLN
TYR ALA ASN ASP ASN GLY VAL ASP 41 GLY GLU TRP
THR TYR ASP ASP ALA THR LYS 51 THR PHE THR VAL
THR GLU
47
Crystal Growth Rate v. Quality
Weeks to months
Seconds to minutes
Martin, R. W. Zilm, K. W. J. Magn. Reson. 165,
162-174 (2003).
Hours to days
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GB1 Sample Preparation

• Start with (concentrated) solution conditions
• 5 mM (30 mg/mL)
• pH 5.5 phosphate buffer, 50 mM
• Add precipitant (2-methylpentane-2,4-diol
  isopropanol, 21)
• Nanocrystals grow in seconds to minutes
• Pellet into NMR rotor

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Kinetics of Crystal Growth

• Single crystal
• (0.1 mm)3 cube -gt 1 x 10-4 m on each side
• 1 month -gt 2.6 x 106 s
• 4 x 10-11 m / s (presuming linear, constant)
• In reality, crystal growth slows as
  concentration of solution decreases
• Nanocrystal (in this example)
• Growth time ltlt15 minutes 103 s
• Linear growth gives major dimension 4 x 10-8 m
• 400 Å on each side (10 times protein size)
• Lower bound (kinetics are in fact not zeroth
  order)

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Assay with 1D 15N Spectra

• 30/g 15NH4Cl
• 1 mmol 15 min 200 nmol 3-4 hrs.

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GB1 13C 1D Spectrum (5 min.)

• 13C,15N
• 12 mg
• 128 scans
• No LB
• 11 kHzMAS
• 70 kHzTPPM

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The NMR Hamiltonian Magnitude of Interactions

• Technical challenges
• Peak rf fields corona discharge (high voltage
  breakdown) of air gap in coil
• Peak MAS rates speed of sound atsurface of
  rotor, material hardness

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Magic-Angle Spinning
maximum structural information
maximum resolution and sensitivity
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Resolution Technical Challenges

• Static CP spectrum of 10 mg protein (GB1)
• 128 scans (4 minutes)
• Scale adjusted (10x) relative to subsequent

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Magic-Angle Spinning (1960s)

• 11 kHz MAS
• No 1H decoupling
• 128 scans
• Andrew, E. R. Bradbury, A. Eades, R. G.,
  "Nuclear magnetic resonance spectra from a
  crystal rotated at high speed", Nature (London)
  1958, 182, 1659.
• Lowe, I. J., "Free induction decays of rotating
  solids", Phys. Rev. Lett. 1959, 2, 285-287.

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CW Decoupling (1975)

• 75 kHz 1H decoupling field (CW)
• 11 kHz MAS
• Same 4 minute acquisition time
• Schaefer, J. Stejskal, E. O., "13C-NMR of
  Polymers Spinning at the Magic Angle", J. Am.
  Chem. Soc. 1976, 98, 1031.

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TPPM Decoupling (1995)

• 75 kHz 1H field with phase modulation
• 11 kHz MAS
• 4 minutes
• Bennett, A. E. Rienstra, C. M. Auger, M.
  Lakshmi, K. V. Griffin, R. G., "Heteronuclear
  decoupling in rotating solids", J. Chem. Phys.
  1995, 103, 6951-6958.

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Why We Bother The Two Limits
single methyl signal
all aliphatic intensity
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Dipolar Recoupling

• MAS averages couplings to zero
• Multiple pulse sequence restores dipolar
  couplings

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Recoupling Example 15N-13C Cross Polarization

• Non-zero time-averaged coupling

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Assignments Backbone Walk
Solid N-CO-CX CO-N-CA/CB N-CA-CX
Solution H-N-CO H-N-(CO)-CA H-N-CA H-N-CA/CO
H-N-CA/CB
- Baldus, M., et al. Mol. Phys. 95, 1197- 1207
(1998). - Takegoshi, K. et al. Chem. Phys. Lett.
344, 631-637 (2001).
62
Residue Type ID

• Always clearG, A, S, T, P, I
• Usually clearL, V, D/N
• Aromaticsuse Cb-Cg todistinguish
• AmbiguousC, M, K, R, E/Q

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An NMR Crossword Puzzle
15N
Franks et al., J.. Am. Chem. Soc. (submitted)
64
Comparison with Solution NMR

• Excellent agreement with solution
• General trends consistent with secondary
  structure
• How precise is this approach?

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Chemical Shifts Solution v. Solid
0.58 0.52 ppm
0.22 1.11 ppm
0.09 0.56 ppm
0.87 2.00 ppm
66
Comparison with X-Ray Diffraction

• TALOS semi-empirical chemical shift analyssis
• 39 f / y pairs converged to good tolerance in
  TALOS
• 71 / 78 agree within error with crystal
  structure

X-Ray Solid-State NMR
f
y

• Cornilescu, G. Delaglio, F. Bax, A., "Protein
  backbone angle restraints from searching a
  database for chemical shift and sequence
  homology", J. Biomol. NMR 1999, 13, 289-302.

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SPECIFIC CP for NC Distances

• - Baldus, M., et al. Mol. Phys. 95, 1197-1207
  (1998).

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Band Selective 15N to 13C TransferMethyl Only
Recoupling
filtered

• Strong couplingsalong backbone avoided
• Polarization transfer primarily to 13CH3signals

unfiltered
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Three Comparable Couplings NAV
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All Distances Within 6.5 Å
Leu5 N (6.2 Å)
Leu7 N (5.9 Å)
1pga reference structure (X-ray)
Ile6 N (4.3 Å)
Ile6 Cd1
Phe52 N (6.4 Å)
Thr53 N (6.4 Å)
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GB1 gt5 Å Distances

• Distanceto Ile6 Cd1

60 ms
20 ms
40 ms
60 ms (w/ LB)

• Thr53 N(6.4 Å)

• Ile6 N(4.3 Å)

• Leu5/7 N(6.2/5.9 Å)

• Phe52 N(6.4 Å)

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REDOR

• 19F-REDOR Shift NMR Spectroscopy
• 1H-19F-13C-15N 3.2 mm MAS probe
• 11.1 kHz, 0 or 512 p pulses on 19F

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FRESH 15N-13C 2D DS DataDepends on 19F-15N
Distance

• K31
• T53
• V54
• I6
• E27, K28, V29
• L5, Q2, T17
• A23, A24

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19F-15N Distances in 1pga
53
54
6
5
F
31
27
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Tying Together Domains

• Connects b3 with a-helix b1 b4
• Potential for 13C 1. Higher g 2. Located
  on side chains
• (esp. CH3)

F
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a-Synuclein

• Associated with neural plasticity
• Abundant and highly conserved
• Relatively small (140 residues)
• Natively unfolded
• Known to form fibrils
• Implicated in neurodegenerative disease
  (Parkinsons, Alzheimers, etc.)
• Collaboration with Julia George,Dept. of
  Molecular and Integrative Physiology

79
a-Synuclein and Neuropathology

• Synuclein aggregates (Lewy bodies) are the
  pathological hallmark of Parkinsons disease
• Mutations in a-synuclein can cause familial,
  early onset Parkinsons disease
• A53T
• A30P
• extra copies of gene
• a-synuclein inclusions are also observed in
• Alzheimers
• Dementia with Lewy bodies
• Downs syndrome
• Multiple system atrophy

Spillantini et al (1997) Nature 388839
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Neurodegenerative Diseases with Intracellular
Filamentous Lesions
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a-Synuclein Hydrophobicity Plot

• Alternating domains of hydrophobicity and
  hydrophilicity
• Periodicity of 11
• Detects an underlying 11-mer repeat
• Suggests amphipathic helix

repeats
acidic tail
NH3
COO-
82
a-Synuclein Fibrils

• Empirical diagnostic
• Dependent on dye affinity
• No known relationships to atomic-resolution
  structure

Conway, K. A., Harper, J. D. Lansbury, P. T.
Fibrils formed in vitro from alpha-synuclein and
two mutant forms linked to Parkinson's disease
are typical amyloid. Biochem. 39, 2552-2563
(2000).
83
SSNMR Spectra of aS Fibrils

• Incubate at 37 oC,50 mM phosphate,1 month
• Sub-ppm line widths
• CP dynamicsMotion near termini
• Kathy Kloepper
• Donghua Zhou

84
aS Fibrils 13C-13C 2D Spectrum