Topic 3 – NMR methods for small biomolecules

Question 1

What is homonuclear NMR and what are some advantages and examples of types of molecules it can be used for

Answer 1

• ¹H detection in both dimensions
• Circumvents need for expensive and time-consuming isotopic labelling.
• Works with even only a few signals/¹Hs
• Can be used for
  
  • Peptides (short proteins)
  • Carbohydrates
  • DNA/RNA fragments

Question 2

• … … (TOCSY) 2D spectrum yields correlation map for …coupled ¹Hs.
• Coupling observed … bonds of intervening ¹H couples
• Interrupted only by small/zero …-…coupling or …atoms e.g carbonyl

Answer 2

• Total correlation (TOCSY) 2D spectrum yields correlation map for scalar coupled ¹Hs.
• Coupling observed ~6 bonds of intervening ¹H couples
• Interrupted only by small/zero ¹H-¹H coupling or hetero atoms e.g carbonyl

Question 3

What is scalar coupling?

Answer 3

• Coupling between two adjacent atoms, in which electronic information is transferred through the bonds that connect them.

Question 4

What are the limitations of TOCSY?

Answer 4

• While TOCSY good for isolating spin systems due to coupling within an amino acid side chain.
• Presence of carbonyl shuts of scalar coupling, halting the TOCSY chain
• Can assign by residue type, but if > 1 of the same AA, cannot discriminate easily in sequential assignment of chain.

Question 5

• Describe and sketch a simple ¹H-^{1 H}H spectrum

Answer 5

• Cross peaks between ¹H’s that are connected by a chain of scalar couplings form, telling us what couples to what, in an amino acid here.
• Don’t necessarily have to be directly coupled.

Question 6

Do the values given in the TOCSY example correspond to experimental chemical shift?

Answer 6

• No, they are random coil chemical shift values that give an indication of a chemical environment in our system and to detect patterns in our spectrum.

Question 7

Briefly describe the pulse program of ¹H-¹H TOCSY

Answer 7

• Equilibrium magnetization into xy plane via 90^o pulse (this is where all scalar coupling occurs)
• This if followed by an evolution period, t₁, and a spin locked period.

Question 8

What is spin locking?

Answer 8

• A method of maintaining coherence and preventing dephasing during transfer of magnetisation through scalar coupling.
• This is done by locking magnetisation along an axis using an RF (pulse) field, causing magnetisation to flip and reacquire coherence.

Question 9

• Correlations in ¹H-¹H spectra are seen through … networks as … of peaks.
• Intensity is not related to number of bonds connecting ¹Hs, due to … relationship meaning … angles result in little/no coupling for protons in same spin system.

Answer 9

• Correlations in ¹H-¹H spectra are seen through coupling networks as strips of peaks.
• Intensity is not related to number of bonds connecting ¹Hs, due to Karplus relationship meaning dihedral angles result in little/no coupling for protons in same spin system.

Question 10

The Nuclear Overhauser (NOE) effect is a form a … (…-…) relaxation, in which … information is coupled between nuclei through …(<… Å)

Answer 10

The Nuclear Overhauser (NOE) effect is a form a dipolar (spin-lattice) relaxation, in which electronic information is coupled between nuclei through space (<5 Å)

Question 11

• Outline NOE’s origin

Answer 11

• Perturbation of one nucleus (spin I) from equilibrium can cause changes in relaxation rate (R₁) and population distribution of nearby spins (S)
• Cross relaxation generates an increase or decrease in intensity on neighbouring spins (NOE).

Question 12

Outline a typical (Nuclear Overhauser Effect Spectroscopy) NOESY spectrum

Answer 12

• Similar in appearance to TOCSY off diagonal cross peak correlation map of coupled ¹Hs, but now dipolar coupled
• Intensity of through space coupling proportional to 1/r⁶

Question 13

Why is NOESY useful?

Answer 13

• Can determine ¹Hs distant in sequence but close in space
• This is key for 3D structure determination.
• Can go past C=O in peptide and transmit information between AA sidechains

Question 14

• (IMP) Describe a ¹H-¹H NOESY pulse program

Answer 14

• Three π/2/90^o pulses
• 1: Creates transverse magnetisation precessing during incremented evolution period, t₁
• 2: Creates longitudinal magnetization that mixes (population distribution changes via dipolar relaxation of excited spins) during mixing period, t_m­ which varies with size
• 3: Creates transverse magnetization from remaining longitudinal magnetization for detection.

Question 15

• Dipolar coupled ¹Hs take part in NOE if <… in space
• This NOE …-… rate, σ_ij is measured directly and internuclear …, measured.

Answer 15

• Dipolar coupled ¹Hs take part in NOE if <5 Å in space
• This NOE build-up rate, σ_ij is measured directly and internuclear distance, r_ij measured.

Question 16

How does molecular size affect NOE?

Answer 16

• Small molecules tumble rapidly (small τ_c) à +ve NOE
• Vice versa with large/highly viscous molecules, giving – ve NOEs approaching 100%
• Certain NOE’s may have zero intensity
• Can be avoided by changing T or viscosity

Question 17

How can NOESY data be used in combination to TOCSY spectra

Answer 17

• Spectra can be overlayed on to one another, the differences of which give an indication of ¹Hs that are close in space but not in bonds
• Namely, the coupling between residues in the fingerprint region, walking from one residue to another via NH-H_a dipolar coupling

Question 18

backbone walk

Answer 18

Question 19

Give a solution to the size effect problem in NOESY.

Answer 19

• Use ROESY (rotating frame Overhauser effect spectroscopy)
• Operates in same plane (XY) and through space to give map of dipolar coupled ¹Hs
• Always gives a + ve value and leads to increase in signals regardless of size

Question 20

What is a key difference in the process of NOESY vs ROESY?

Answer 20

• NOESY: spin-lattice relaxation (T₁)
• ROESY: spin-spin relaxation (T₂)

Question 21

Describe the ¹H-¹H ROESY pulse spectrum.

Answer 21

• Magnetization transfer in ROESY occurs in xy plane using spin lock sequence

Question 22

• CSI or … … … method can be used to identify … structure type e.g. α-helices, …-strands, … …
• Attained from … … … of the backbone.
• AA - ¹H_α in α-helices shifted … relative to RC values.
• AA - ¹H_α in β-sheets shifted … relative to RC values.

Answer 22

• CSI or chemical shift index method can be used to identify secondary structure type e.g. α-helices, β-strands, random coil.
• Attained from sequential peak assignment of the backbone.
• AA - ¹H_α in α-helices shifted upfield relative to RC values.
• AA - ¹H_α in β-sheets shifted downfield relative to RC values.

Question 23

What is secondary chemical shift and what does it depend on?

Answer 23

• Δ𝛿 = 𝛿obs – 𝛿RC
• Depends on the secondary chemical structure
• H_α secondary shift varies +/- 0.4 ppm
• C_α ranges form + 2.6 (helix) -1.4 ppm (sheet)
• C_β ranges from – 0.4 (helix) -2.2 ppm (sheet)

Question 24

• Assigning CSI data
  
  • If Δ𝛿 > |0.1| residue assigned +1 or -1
  • If Δ𝛿 < |0.1| residue assigned value of 0
  • Stretch of 4 or more +ve/-ve values gets assigned as a helix/sheet
  • Mixed /0 assigned as random coil
  • 75% accuracy if only H_α used
  • >90% accuracy if ¹³C considered

Question 25

Following CSI, assignment of all NOESY crosspeaks outside of fingerprint region

Answer 25