Topic 4

Question 1

Why may experimentalists need heteronuclear 2D NMR to characterise a biomolecule fully

Answer 1

• Above 3-4 kDa, homonuclear ¹H NMR becomes very crowded, leading to large amount of overlap in spectra.
• Instead a heteroatom X can be used as a second dimension
• E.g. ¹³C or ¹⁵N

Question 2

Heteronuclear 2D NMR requires isotopic labelling, what are two methods in which this can be done?

Answer 2

• Synthetic peptide preparation: labels added to specific AA’s during peptide synthesis, very expensive
• Uniform labelling with bacteria: use feedstocks of E.coli to label proteins. Handles ¹⁵N well, but ¹³C gives a lower yield.

Question 3

• In ¹H-¹⁵N HSQC, every …-bearing … yields a peak (now labelled …)
• Every AA in backbone + additional AA’s with …-bearing …
• Polarisation transfer via … (through …) from ¹H spins (high γ_H) to spins with low γ_x
• Enhances signal from … … …nuclei.

Answer 3

• In ¹H-¹⁵N HSQC, every H-bearing N yields a peak (now labelled ¹⁵N)
• Every AA in backbone + additional AA’s with N-bearing sidechains
• Polarisation transfer via J-coupling (through bonds) from ¹H spins (high γ_H) to spins with low γ_x
• Enhances signal from low-sensitivity heteronuclei.

Question 4

What is INEPT?

Answer 4

• INEPT or Insensitive nuclei enhanced by polarisation transfer describes the transfer of polarisation of ¹H to ¹⁵N back to ¹H in 2D hetero NMR.
• This is done by pulsing the nuclei until their spins are antiphase to one another, aligned with the magnetic field (z-axis), inverting population from H to N
• Incremented evolution period follows, before a reverse INEPT sequence, before acquiring our FID.

Question 5

What are the key regions of a ¹H-¹⁵N HSQC spectrum?

Answer 5

• Will have fewer peaks than homonuclear cross peak data as only observing N-H interactions
• Generally, peaks are at a high ppm (6-11) as N’s bearing protons are deshielded.

Question 6

When would ¹H-¹³C HSQC be used?

Answer 6

• For biomolecules with few N atoms, otherwise use ¹H-¹⁵N
• Isotopic enrichment with ¹³C can lead to lower yields.

Question 7

What are hybrid techniques?

Answer 7

• Once a good HSQC is obtained, it can be used in combination with a homonuclear 2D method (e.g. TOCSY) as an additional filter
• For example, combine with a ¹H-¹H TOCSY spectrum to see only peaks corresponding to that N chemical shift (CS), through addition of a 3^rd dimension
• HSQC can form hybrids before or after TOCSY/NOESY experiments.

Question 8

Describe an assignment strategy for a ¹H/¹⁵N only system.

Answer 8

• Acquire ¹H-¹⁵N HSQC, HSQC-TOCSY, and HSQC-NOESY
• In HSQC-TOCSY, obtain a ¹H-¹H TOCSY at each ¹⁵N shift of interest, using the strips of peaks gained from each different ppm to find patterns in data.
• Use HSQC-NOESY (¹H-¹H NOESY at each ¹⁵N shift) to walk down protein backbone, collecting data at same ppm as previous to overlay differences (same as before but now with a nitrogen filter)
• Plot each assignment back on original ¹H-¹⁵N as you go.

Question 9

What can we infer from HSQC data prior to assignment?

Answer 9

• Protein is well folded if the chemical shift dispersion (CSD) in H-N doesn’t indicate peaks are all on top of each other
• Can also count peaks to ensure we get out what we expect.

Question 10

• … … is sensitive to nuclear environment, for example a binding interaction will cause the protein … site’s … … to change in response.

Answer 10

• Chemical shift is sensitive to nuclear environment, for example a binding interaction will cause the protein binding site’s chemical shift to change in response.

Question 11

What sort of interactions cause a change in chemical shift (CS)?

Answer 11

• Changes caused by covalent (bonds forming) and non-covalent (H-bonding, vdW) interactions are sensitive probes for folding, ligand binding.

Question 12

What sort of chemical shift perturbations might we see in regions of our spectrum’s peaks in a ligand binding event?

Answer 12

• No change (no binding)
• Peak shift (binding, fast exchange)
• Peak splitting (new conformers formed)
• Peaks appear-disappear (binding; slow exchange)

Question 13

How would we interpret chemical shift perturbations in a spectrum?

Answer 13

• As [ligand] increases, chemical shifts of nuclei at/near binding site affected
• Given as a weighted change Δ_total
• More smeared peak from blue to red show CS with increased concentration, indicating direct involvement with binding, or indirect conformational change due to binding.

Question 14

How can we use chemical shift changes in perturbation to locate a binding site in a protein?

Answer 14

• Once NMR peaks assigned, can map out largest chemical shift changes throughout sequence
• A threshold can be set above noise level, and map the largest remaining changes on to a protein structure a propose the binding site.

Question 15

Discuss the drawbacks of using Chemical shift perturbation, and what types of interactions it is best suited for.

Answer 15

• No direct way to know if interaction if due to direct binding or structural rearrangement upon complex formation
• Spread of data gives an indication of which it is (tight –> binding site, diffuse –> conformational change)
• This approach is best suited to weak interactions in low mM range showing clear fast exchange, where we know the old and new peak location and how they are related.

Question 16

What is Deuterium exchange NMR and how can it be used in HSQC?

Answer 16

• In a high pH solution of D₂O, an undeuterated protein will undergo rapid H/D exchange.
• Only H’s participating in H-bonds (amide hydrogen) and those in the centre of the fold (protected) will not exchange
• Deuterium does not show up in proton spectra so in ¹H-¹⁵N HSQC will see surface H’s peaks vanishing
• Great chemical probe of exposed surface of protein to see what AA’s are available