Lecture 6: Multinuclear NMR

Question 1

List 2 stable isotopes that are often needed to be enriched in the protein sample in a Multinuclear NMR experiment. Why do they need to be enriched?

Answer 1

• The solution to this would be to add more NMR active nuclei to the molecule enriched with stable isotopes 13C and 15N, since there natural abundance is low (1.1 for C and 0.37 for N).
• They must be enriched with these isotopes due to high spectral overlap. This allows for unique identification of sites within molecules.

Question 2

List the four parts that a 2D 1H-15N HSQC pulse sequence is composed of:

Answer 2

1. INEPT (Insensitive Nuclear Enhancement by Polarization Transfer.
   
   • Magnetization transfer from 1H to 15N. Note that spin-echo is used.
2. Magnetization of 15N is frequency labeled during t1.
3. Reversed INEPT.
   
   • Magnetization transfer from 15N back to 1H.
4. Detection of FID by the receiver during t2.
   
   • Decoupling pulse is applied to 15N.

Question 3

What information do the 3D HNCACB and CBCA(CO)NH experiments provide? Describe how to achieve protein backbone chemical shift assignment using the combination of these two NMR spectra.

Answer 3

-HNCACB is an NMR experiment that correlates the backbone NH groups to the a and b carbons within the same amino acid and of the preceding amino acid.
~provides inter and intra residue connectivity.

-CBCA(CO)NH is an NMR experiment that correlates the backbone NH group only to the a and b carbons of the preceding amino acid.
~provides only inter residue connectivity.

Question 4

What is the primary advantage of Multinuclear NMR over 2D NMR?

Answer 4

• Due to spectral overlaps, it is difficult to obtain full assignments from 2D NMR particularly for >100 amino acids.
• 3D pulse sequences that identify specific correlations between nuclei of the polypeptide backbone and side chains are used to make sequence-specific assignments.

Question 5

What information does a 2D HSQC (heteronuclear single quantum correlation) NMR spectrum provide?

Answer 5

This permits to obtain a 2D heteronuclear chemical shift correlation map between directly bonded 1H and X-heteronuclear (commonly 13C and 15N).

It is widely used because it is based on proton detection and offers high sensitivity.

Question 6

Describe what NMR experiments are generally used for chemical shift assignments of protein side chains:

Answer 6

1. HCCH-TOCSY: connect all 13C and 1H nuclei in the side chain of each residue.
2. CBCA(CO)NH: connect all 1Ha and 1Hb to backbone NH.
3. H(CCO)NH: connect all side chain 1H to backbone NH.
4. (H)C(CO)NH: connect all side chain 13C to backbone NH.