NMR of Proteins

NMR of Proteins
Determining Protein Structures by NMR
• the process of determining a solution structure by NMR is one
of measuring many (hundreds/thousands) of short protonproton distances and angles, and restraining the protein structure
with these computationally
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d
H …. H
1H
NMR Spectra of Proteins
• 1D, 1H NMR spectra of even small proteins are impossible
to interpret in any comprehensive manner
-normally, only gross statements about secondary ubiquitin (76 amino acids, 8.5 kDa)
structure, tertiary structure, etc. can be made
simple 1D 1H experiment
2D 1H “COSY” experiment
90
COSY
90
t1
t2
• for even moderate sized proteins, addition of a second
dimension still does not alleviate spectral crowding
and overlap in 1H spectra
cytochrome c, 12.5 kDa
nD, heteronuclear NMR Spectra of Proteins
• Modern NMR spectroscopic studies of proteins rely on multidimensional experiments
involving 1H, 13C, and 15N nuclei in isotopically labeled proteins
• These methods provide for signal selection (selectivity) and a means to reduce signal overlap
ubiquitin (76 amino acids, 8.5 kDa)
simple 2D 1H, 15N “HSQC” experiment
• In order to measure the distances between protons, we need to find out what protons give
rise to the signals in the spectra, I.e. we have to “assign” the protein (figure out the
chemical shifts for all of the protons)
• The methods used are based on heteronuclear spectra
Triple Resonance Approach
• applicable to uniformly isotopically enriched proteins
-uniform 13C and 15N labeling: spin 1/2
-three nuclei (1H, 15N, and 13C) are involved
• based on magnetization transfer via (mostly) one bond J couplings
-most of these couplings are large compared to linewidths for
moderate sized proteins (~20 kDa)
-magnetization transfer is efficient
-indirect (1H) detection
• provides selective chemical shift correlation
-spectral degeneracy minimized
Uniform Isotopic Labeling of Proteins
• Proteins can be uniformly isotopically labeled by recombinant
expression using defined media
-bacterial expression most common
-also yeast, and cell-free systems are being developed
-minimal media using 13C6 glucose as the sole carbon source
and 15NH4Cl (or -SO4) as the sole nitrogen source
-normally >98% atom excess
-also labeled “rich” media ($$)
-for larger proteins, uniform or fractional 2H labeling also used
-2H, 13C glucose and D2O
1J
and 2J Couplings in Proteins
- these 1J and 2J couplings are uniform throughout polypeptides/proteins
- these 1J and 2J couplings are virtually conformation independent
Prototypical Triple Resonance Experiment: HNCA
• correlates the chemical shifts
of 1HN, 15N, 13Cαi and 13Cαi-1
Prototypical Triple Resonance Experiment: HNCA
• both 13Cαi and 13Cαi-1 chemical shifts are correlated
-the peak for the intra-residue correlation is usually more
intense (11 Hz 1JNCα coupling vs 7 Hz 2JNCα coupling)
1H, 15N-HSQC
HNCA
Triple Resonance Approach: A Simple Example
2D HNCA projection
3D HNCA
Triple Resonance Approach: A Simple Example
Triple Resonance Approach: A Simple Example
• link the correlated shifts numerically….
…. or visually
Triple Resonance Approach: HNCA/HN(CO)CA Example
• problems:
13Cα chemical shift degeneracy in proteins
13Cα linewidths/resolution
-these preclude complete linkage via 13Cα alone
-the same is true for 13Cβ, 13C′
The Nuclear Overhauser Effect
• The Nuclear Overhauser Effect or Nuclear Overhauser Enhancement is the change
(enhancement) of the signal intensity from a given nucleus as a result of exciting
or saturating the resonance frequency of another nucleus
• It is based on through-space interactions
• The magnitude of the effect is dependent on distance
-enhancement depends on 1/r6, where r is the internuclear distance
-thus, the effect is limited to distances of approximately 5Å or less
• This provides a means to determine if any two protons in a protein are < 5Å apart
• The basic experiment used for proteins is called a NOESY
cytochrome c, 12.5 kDa
90
2D NOESY
90
t1
90
τm
t2
• Even for relatively small proteins, the 2D NOESY
spectrum is hampered by severe spectral overlap
3D NOE Experiments for Distance Restraints
-pulse sequences: combine 2D sequences to get 3D sequences
-get increased dimensionality and increased resolution
without an increase in the number of signals (peaks)
90
NOESY
90
90
t2
90
180
τm
t1
τ
1H
HSQC
90
180
τ
90
1H
t1
τm
180
τ
15N
90
τ
180
decouple
180
90
τ
90
90
t2/2
t2/2
t2
180
t1/2
τ
180
NOESY-HSQC
90
t1/2
90
90
180
90
τ
15N
90
180
τ
t3
180
decouple
3D NOE Experiments for Distance Restraints
Left: 2D NOESY
Far left: 2D plane of 3D NOESYHMQC (1H, 13C)
-1H signals resolved by 13C
chemical shifts of bound
13C atoms
Structure Calculations
• the primary structural restraint information for high resolution protein structures are the
• NOE-based distance restraints
-additional restraints include angle restraints based on coupling constants, long range
restraints based on dipolar couplings, hydrogen bond restraints
• calculation of structures involves satisfying structural restraints using simulated annealing/
• restrained molecular dynamics
-a target/energy function including terms for covalent geometry (known bond lengths
and bond angles) and experimental restraints is minimized
-the molecule is computationally heated/cooled to attempt to find a global minimum
• the number of restraints is an important indicator of the quality of final structures
Practical Aspects of Protein Structure
Determination using NMR
Introduction
• NMR vs X-ray crystallography for protein structure determination
• in an x-ray diffraction pattern, each datum (reflection) contains
information about each atom in the asymmetric unit
-each atom contributes information that contributes to the
intensity of each reflection
• in an NMR spectrum, each datum (peak) contains information
about only a single interatomic distance or angle
-the process of determining a solution structure by NMR
is one of measuring many small distances and angles
“one at a time”
• Why use NMR?
• can’t get a crystal / want to work in solution
• want to look at binding to other proteins/molecules
• want to understand stability
• want to measure fast dynamics processes
Introduction
• Some things you should know before you talk to an NMR
spectroscopist about determining a structure
• Protein production
• Protein purity
• Isotopic labeling
• NMR samples / conditions / tubes
• Simple spectra / evaluation (stability, tertiary structure)
• Protein size / magnet size
Protein production
• protein sample(s)
• in theory, as little as a few mg of protein is sufficient
-best if the protein sample for NMR is > 1 mM
• in practice, tens of milligrams (or more) are usually necessary,
as are multiple samples
-multiple samples if your protein is not stable
-multiple samples with different isotopic labeling schemes
• many very good bacterial expression vectors/cell strains are
available for expression in bacteria
-good track record, easily automated
• expression in eukaryotic cells more complicated (yeast, insect,
human cells)
• are cell-free systems currently available (i.e. Roche “Rapid
Translation System (RTS))….good for specific isotope labeling,
not good for uniform isotopic labeling
Protein purity
• protein samples should always be as pure as possible
• in practice, for small proteins, small amounts of high
molecular weight contaminants are OK
SDS-PAGE
molecular weight markers
sample
Isotopic labeling of protein for NMR
• protein must be uniformly isotopically labeled with 13C and 15N
• not difficult these days: bacterial expression
cell strains grow well on minimal media
(D-glucose (U-13C6, 98-99%) and 15NH4Cl
(98-99%) as sole carbon and nitrogen
sources, respectively)
• for 1L medium, $300 for glucose (2 g @ $150/g), and
$30 for 15NH4Cl (1 g @ $30/g)
• there are also alternatives to minimal media (i.e. isotopically
labeled rich media), but they are much more expensive
Isotopic labeling of protein for NMR
• in practice, often multiple samples with other isotopic labeling
schemes are necessary
• 15N only for certain angle (φ, ψ) measurements
-also, usually used for initial evaluation of sample/spectra
• 13C, 15N plus partial or uniform deuteration for large proteins
-requires growth in minimal media in D2O
-cells must be adapted to growth on D2O
• samples with only specific amino acid types
labeled assist in NMR resonance assignment
-cells grown on medium with all unlabeled amino acids
except for the one of interest
-more common for larger proteins
• samples made with a mixture of 10% U-13C6 glucose and 90%
unlabeled glucose are used for stereospecific -CH3 assignment
-proR methyl groups of Valine and Leucine
Isotopic labeling of protein for NMR
• isotopic labeling to identify specific amino acid types, groups, or
to assign stereospecificity
15N-Gly
only
labeled protein
uniformly 15N
labeled protein
ε13CΗ3-Met only
labeled protein
uniformly 13C
labeled protein
Left: sample prepared by growth
on 100% uniformly 13C-labeled
glucose
Right: sample prepared by growth
on 10% uniformly 13C-labeled
glucose and 90% unlabeled
glucose
The NMR sample
• buffer: no C- or N-bound protons
• phosphate is the best….
• deuterated Tris, deuterated acetate, deuterated imidazole, etc.,
are OK (can be expensive)
• salts
• K+, Na+, Cl-, SO42-, etc., all OK (no protons)
• too much salt leads to decreased S/N
• pH: neutral or lower is best
• must minimize the rate of exchange
of amide protons with solvent
• solvent
• 90% H2O, 10% D2O (for instrumental lock)
The NMR sample
• temperature
• sample dependent: usually 25 to 35 °C
• bacteriostatic agents
• sodium azide used widely
• put it all together in a good quality, clean NMR
tube
• “standard” NMR tube is 5 mm diameter
(for use in a 5 mm NMR probe)….volume
of sample is ~500 - 700 uL (~1 mM protein)
• magnetic susceptibility matched tubes,
“Shigemi” tubes, permit lower volume
samples to be used (i.e. less sample, or more
concentrated sample), usually without
deleterious effects (but the tubes are
complicated)…volume of sample is
200-300 uL
Initial NMR spectra / evaluation
• 1D 1H NMR spectrum of a small organic compound
F(ω ) =
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1
2π
∫
f (t) =
x
−x
f (t)e iωt dt
x
−iωt
F(
ω
)e
dω
−x
• 1D 1H NMR spectrum of a small protein
ubiquitin (76 amino acids, 8.5 kDa)
• for even small proteins, 1D spectra
are complicated and cannot be
analyzed comprehensively
• 1D spectra can be useful, however,
for evaluating the suitability/stability
of a protein sample
Initial NMR spectra / evaluation
• 1D 1H NMR spectrum of a small protein
• for properly folded small proteins
-peaks should be sharp
-peaks should show good
chemical shift dispersion (i.e.
tertiary structure intact)
acid-unfolded ubiquitin
ubiquitin, neutral pH
• for unfolded proteins
-peaks are usually broad (many
protons in each peak)
-chemical shift dispersion poor
(leading to the broad peaks)
Initial NMR spectra / evaluation
• sample stability
• can takes weeks of instrument time to
acquire all data for structure determination
• sample has to be stable for the amount of time necessary to
acquire all of the data (at the data acquisition temperature), plus
the time between experiments (all data is rarely acquired all at
once)
protein x, t = 0
protein x, t = 2 weeks
(at room temperature)
properly folded
partially unfolded
Initial NMR spectra / evaluation
• simple 2D 1H, 15N correlation NMR spectra of proteins
• reduce complex spectra to simple ones based on isotope editing
• reduce/eliminate spectral overlap/spectral degeneracy
• correlate amide 1H-15N pairs
ubiquitin (76 amino acids, 8.5 kDa)
simple 2D 1H, 15N “HSQC” experiment
• this spectrum demonstrates 1). that you can express your protein,
2). That you can isotopically label your protein, 3). That your
protein is pure (1 peak per amino acid), 4) that your protein
is folded (tertiary structure / good chemical shift dispersion), 5). etc.
• granting agencies need to see this spectrum or similar (akin to
a crystal and a diffraction pattern)
Initial NMR spectra / evaluation
• tertiary structure and sample stability
• chemical shift dispersion
and peakwidths reflect
tertiary structure
c: apomyoglobin
b and a: acid unfolded apomyoglobin
protein x, t = 0
properly folded
protein x, t = 2 weeks
(at room temperature)
partially unfolded
Size (of the protein) matters
• the rotational correlation time (τc) scales with protein size
• larger τc: peak broadening and decreased S/N
• larger proteins have more atoms, therefore more peaks in the spectra
• more peaks: increased peak overlap/chemical shift degeneracy
ubiquitin (76 amino acids, 8.5 kDa)
AlgH (189 amino acids, 20.2 kDa)
EPSP synthase (427 amino acids, 46.2 kDa)
Size (of the magnet) matters
• 1H, 15N-HSQC (TROSY) spectra of EPSP synthase (46 kDa)
at 600 and 800 MHz
• higher field means higher sensitivity (increased S/N), increased
resolution (decreased peak overlap), and a bonus increase in
S/N in TROSY experiments
600 MHz
800 MHz
NMR: beyond structure
• ligand binding
• slow dynamics / local and global
stability (hydrogen exchange)
residue
• fast dynamics (ps/ns)
via nuclear relaxation
• mutational affects
S2
residue
END