NMR spectrum description

[vanklik]

Being a biochemistry student I have done NMR workshop using Lysozyme; now I need to describe the spectrum. I wasn't taught how to do it on the lectures and have no idea how describe the spectrum. It would be really helpful if someone could give me some guidance on what to include in this description.

 

Thank you!

[Jens]

(On a one-dimensional NMR spectrum of a complete protein you cannot really see much, because it is completly overcrowded.)

 

To discuss it you should proceed as follows:

• Search for the standard shift values of the amino acid side chains (you should have this in your books or received as input, or use google scholar)
• What you actually see is quite different in some cases. Think about it and make some proposals here. Once you have done this, I will point you to more answers. (Since for good reasons I am not allowed to do your homework in this thread

The parameters to consider are everything you can see in this spectrum:

- ppm value of the peak (mainly)

- heigth of the peak

- width of the peak

- potential artifacts (H2O)

 

What does the spectrum tell you about the protein?

Under which conditions it has been recorderd? (what do you expect to see under this conditions?)

How would the spectrum look like, if the protein had been denatured?

Edited November 25, 2012 by Jens

[Jens]

generic info (to be applied to your case, to be combined with your chemical know-how):

 

- all H atoms are visible (also those in H₂O). Deuterium does not give any signals.

- broader peaks indicate that the H atom(s) move less than narrow peaks

- multiple H atoms at the exactly same shift value (ppm) sum up to big peaks

- the shift values of the H atoms can deviate from standard values depending on their environment in space. In practise especially neighboring aromatic rings are the source of strong changes of the shift values. The strongest shift changes are produced by Tryptophane (Trp, W).

- the 3 H atoms in methyl groups always produce only one peak, since they rotate too quick (even in the center of proteins) so that all of them always "see" the same neighborhood.

 

There is a small story to tell on your spectrum, but you have to find out (or at least make the first steps).

 

hope this helps more.

 

...I will not tell more until you make a proposal....

[Jens]

I assume it is too late now for your homework.

But for the other readers, here is what I see out of your spectrum:

• The huge peak of H₂O between 4 and 5 ppm is completly missing -> This means your spectrum has been taken in D₂O instead of H₂O (and multiple cycles of lyophilisation to completly exchange any H by D). Deuterium shows no signals. --> Since D2O quickly exchanges with any NH groups, you have no signals for NH groups under such conditions either. --> "amides (backbone HN)" and "sidechain HN" of your legend are not visible. --> This means all signals in that area are coming from aromatic sidechains (or some stronger than usual shifted H_alpha.)
• The spectrum shows a lot of broad peaks and at ppm values far from standard values --> This means the protein is well folded and not denatured.
• The peaks at 0 ppm and below are all coming from aliphatic H atoms that are directly near an aromatic ring in the 3D folding of the protein. The sharp thin single peaks are from methyl groups. The broader peaks from CH₂ groups (which rotate less quickly).
• At many ppm values (especially between 0.7 and 4.7 ppm) signals from different H atoms overlap. Structural relationships therefore cannot be deducted out of this spectrum.
• Aliphatic side chains H atoms show the highest peaks because there are more of them than of the aromatic H atoms and the H_alpha.
• Sharpest and therefore highest individual signals are typically coming from the methyl groups in Met, since they rotate freely as all other methyl groups and show no splitting because of neighboring covalent H-atoms (since the next atom is an S-atom with no H-atoms bound to it). However, individual signals in that area are not necessarily resolved in this spectrum.