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
• 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
1H, 15N-HSQC
HNCA
Triple Resonance Approach: A Simple Example
…. or visually
Triple Resonance Approach: HNCA/HN(CO)CA Example
• problems:
13Cα chemical shift degeneracy in proteins
13Cα linewidths/resolution
• 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 90 90
t2
2D NOESY t1 τm
90 90 90
t2
NOESY t1 τm
Left: 2D NOESY
• 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
• 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
∫
x
F(ω ) = f (t)e iωt dt
−x
1
∫
x −iωt
f (t) = F(ω )e dω
2π −x
• 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
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
S2
residue
END