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The NMR Fingerprints of Proteins: What you can see in simple spectra. typical chemical shifts observed in proteins interpreting simple 1D spectra interpreting 15 N- 1 H 2D correlation (HSQC) spectra using changes in HSQC spectra to measure binding events . Chemical shift.
What you can see in simple spectra
not all nuclei in a protein will have the same resonance frequency (the spectrum would be pretty uninformative if they did!)
a reference frequency of
0 ppm is defined by the signal
from an internal standard
such as DSS or TMSP
x axis is the chemical
shift d, in units of
parts per million (ppm)
of the B0 field strength
so why don’t
all the nuclei
chemical shift dispersion is very small (~12 ppm) compared to the B0 field strength (like ripples on an ocean surface). At 500 MHz, 12 ppm is a 6 KHz range. This makes it easy to pulse in the center of the 1H spectrum, around 4.5-5 ppm, and excite all resonances nearly evenly.
electrons surrounding the nucleus create a magnetic field which opposes the B0 field and reduces the effective field felt by the nucleus. This is called diamagnetic shielding.
fr. Macomber p. 69
The amount of shielding the nucleus experiences will vary with the density of the surrounding electron cloud
If a 1H nucleus is bound to a more electronegative atom
e.g. N or O as opposed to C, the density of the electron cloud will be lower and it will be less shielded or “deshielded”. These considerations extend beyond what is directly bonded to the H atom as well.
higher resonance frequency
lower resonance frequency
most HN nuclei come between 6-11 ppm while most
HC nuclei come between -1 and 6 ppm.
Groups such as carbonyls and aromatic rings have associated anisotropic fields or “shielding cones” that will either shield or deshield nearby 1H nuclei depending upon where the 1H nuclei are located relative to the shielding cone.
aromatic region (6-8 ppm)
amide region (7-10 ppm)
One consequence of these effects is that aromatic protons, which are attached to aromatic rings, are deshielded relative to other HC protons. In fact, aromatic ring protons overlap with the amide (HN) region.
a protein will resonate at different frequencies based on simple chemical considerations. For instance, Ha protons will resonate in a region centered around the relatively high shift of 4.4 ppm, based on the fact that they are adjacent to a carbonyl and an amine group, both of which withdraw electron density. But not all Ha protons resonate at 4.4 ppm: They are dispersed as low as ~3 and as high as ~5.5. Why?
One reason for this dispersion is
that the side chains of the 20 amino
acids are different, and these differences will have some effect on the Ha shift.
The table at right shows “typical” values observed for different protons in the 20 amino acids. These were measured in unstructured peptides to mimic the environment experienced by the proton averaged over essentially all possible conformations. These are sometimes called “random coil” shift values.
Note that the Ha shifts range from ~4-4.8, but Ha shifts in proteins range from ~3 to 5.5. So this cannot entirely explain the observed dispersion.
note: the shifts are somewhat different from the
previous page because they are measured on the free amino
acids, not on amino acids within peptides
A simple reason for the increased shift dispersion is that the environment experienced by 1H nuclei in a folded protein (B) is not the same as in a unfolded, extended protein or “random coil” (A).
shift of particular proton in unfolded protein is averaged over many fluctuating structures
will be near
shift of particular proton in folded protein influenced by
groups nearby in space,
conformation of the backbone, etc. Not averaged among many structures because there is only one folded structure.
So, some protons in folded proteins will experience very particular environments and will stray far from the average.
chains in folded proteins
Picture shows the side chain packing in the hydrophobic core of a protein--the side chains are packed in a very specific manner, somewhat like a jigsaw puzzle
a consequence of this packing is that some protons may be positioned within the shielding cone of an aromatic ring such as Phe 51. Such protons will exhibit unusually low resonance frequencies (see picture at left). Note that such effects depend upon precise positioning of side chains within folded proteins
of protein spectrum
so you can tell if your protein is folded or not by looking at the 1D spectrum...
shifts above 9
and below 7
notice alpha protons
with shifts above 5
notice all these methyl peaks with
chemical shifts around zero or even
Linewidths get broader with larger particle size, due to faster transverse relaxation rates. We’ll learn the physical basis for the faster relaxation later. Broader than expected linewidths can indicate that the protein is aggregated. It can also indicate that the protein has conformational flexibility, i.e. that its structure is fluctuating between several slightly different forms. We’ll learn why this is when we cover the effect of protein dynamics on NMR spectra. Conformational flexibility also tends to reduce dispersion by averaging the environment experienced by a nucleus.
Hill & DeGrado used measurements of chemical shift dispersion and line broadening in the methyl region of 1D spectra to gauge the effect of mutations at position 7 on the conformational flexibility of a2D protein
leucine and valine mutants have poor
dispersion and broad lines, despite being very stably folded
and not aggregated (circular dichroism and analytical ultra- centrifugation measurements). These mutants are folded but flexible.
Hill & DeGrado (2000) Structure 8: 471-9.
In general, 1D NMR provides only qualitative information about your protein:
Does it have a stable, specific folded structure under the NMR conditions?
Does it seem to be aggregated?
Spectra of even small proteins (e.g. 6 kD), unlike the spectra of small organic molecules and short peptides, are just too complex to be studied by 1D methods.
There are many overlapping resonances in 1D protein spectra. One way to remove this overlap is to label your protein with 15N and/or 13C and correlatethe chemical shift of each 1H nucleus with the chemical shift of the 15N or 13C atom to which it is directly attached.
This is done by transferring the magnetization between the two atoms using the large one-bond 13C-1H or 15N-1H scalar coupling
J = ~89-95 Hz
J = ~110-160 Hz
1D 1H spectrum of same region
peaks occur where the chemical shift of a 1H and the chemical shift of the attached 15N atom intersect
note less overlap
the ability to spread the
spectrum out into a second
dimension, thus achieving
better resolution of the resonances, is a major reason for isotopic labelling
aromatics in 1D e.g. * not visible in HSQC due to isotope editing--they aren’t bound to 15N!
dispersion in both
Since each amino acid
residue (except proline)
has an amide proton, we
might expect about the
same number of peaks
as residues. This is roughly
true, but some side chains,
like glutamine, asparagine
and arginine have amides too, so there will be more peaks than residues.
The glutamine and asparagine peaks are especially recognizable--they are pairs of 1H shifts correlated to a single nitrogen in the upper right portion of the spectrum
This is about a 60-residue protein-->small
move, some don’t--
binding to specific region
because there is
about one peak per
residue, a 15N-1H HSQC is
something of an NMR
fingerprint of a protein.
HSQCs are very commonly used to detect ligand binding--if the fingerprint changes (peaks move) it indicates that binding is occurring. This is the basis of “SAR by NMR”, about which you read in Shuker et al.
HSQC in absence (magenta) and
presence (black) of ligand
approach whereby two ligands are sought
which bind to two different places with micromolar affinity. They are then linked together to produce a single ligand with nanomolar/picomolar affinity. One can tell that the two ligands bind in different places because different peaks move, or because one will bind in the presence of the other (no competitive inhibition).
Why don’t they just screen for a single ligand with nanomolar/picomolar affinity in the first place?
Structural interpretation of changes in HSQC upon binding requires resonance assignment
Notice that the peaks which move upon binding have been labelled with the residue names and numbers--if they want to know where on the protein the ligand is bound they need to know which peak corresponds to which amino acid residue. How is this determined? This is the fundamental problem of resonance assignment, which we will cover a few lectures later.
in this region