slide1 l.
Skip this Video
Loading SlideShow in 5 Seconds..
The NMR Fingerprints of Proteins: What you can see in simple spectra PowerPoint Presentation
Download Presentation
The NMR Fingerprints of Proteins: What you can see in simple spectra

Loading in 2 Seconds...

play fullscreen
1 / 23

The NMR Fingerprints of Proteins: What you can see in simple spectra - PowerPoint PPT Presentation

  • Uploaded on

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.

I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
Download Presentation

PowerPoint Slideshow about 'The NMR Fingerprints of Proteins: What you can see in simple spectra' - senta

An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.

- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript

The NMR Fingerprints of Proteins:

What you can see in simple spectra

  • typical chemical shifts observed in proteins
  • interpreting simple 1D spectra
  • interpreting 15N-1H 2D correlation (HSQC) spectra
  • using changes in HSQC spectra to measure binding events

Chemical shift

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

have the

same frequency?

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


Simple shielding effects--electronegativity

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.

more electron


less shielded

less electron


more shielded






Simple shielding effects--electronegativity

less shielded

higher resonance frequency

more shielded

lower resonance frequency

aliphatic/alpha/beta etc.(HC)

amides (HN)

most HN nuclei come between 6-11 ppm while most

HC nuclei come between -1 and 6 ppm.

more complex shielding effects
More complex shielding effects

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.




are deshielded









More complex shielding effects:Aromatic protons

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.


It should now be apparent to you that different types of proton in

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?

“Ha region”


“Average” or “random coil” chemical shifts in proteins

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.


Amino acid structures and chemical shifts

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

random coil


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.


Example: shielding by aromatic side

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

shielded methyl


methyl region

of protein spectrum


so you can tell if your protein is folded or not by looking at the 1D spectrum...


dispersed methyls


dispersed amides


dispersed alphas


dispersed aromatics



very shielded




what specifically to look for in a nicely folded protein
What specifically to look for in a nicely folded protein



protons with

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 in 1D spectra: aggregation and

conformational flexibility

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.


An example of analyzing linewidths and dispersion:

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.


Limitations of 1D NMR

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.


Adding a second dimension: Isotopic labelling

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


HSQC: Simple 2D Heteronuclear Correlation Spectrum





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

in 2D!

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!


Appearance of 15N-1H Correlation Spectra (HSQC)

glutamine and

asparagine peaks

good shift

dispersion in both

dimensions--folded protein

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


15N-1HHSQC spectra as “protein fingerprints”

some peaks

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


Notice that SAR by NMR uses an

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.

compound binds

in this region