NMR 3 Flashcards
How can you tell what the oligomerisation state of the protein
- Self‐association: broadens almost all signals
- Conformational exchange e.g. monomer/dimer or free/bound: broadens only signals from residues affected (e.g. at interaction site)
- But, can be difficult to distinguish self-association from conformational exchange
How can you distinguish self-association from conformational exchange
- Use additional method(s) e.g.
- size exclusion chromatography-multi‐angle light scattering (SEC-MALS)
- dynamic light scattering (DLS)
- analytical ultracentrifugation (AUC)
What other parameters affect NMR spectra?
- degradation or unfolding
- increasing magnetic field strength – better sensitivity and resolution
- Temperature: raising temp increases tumbling rate so signals are sharper, but some proteins less stable plus exchange rate of labile protons is increased
Why is Solution NMR of proteins not a molecular imaging technique
- wavelengths involved (1 mm to 10 km, cf 0.01-10 nm for X-rays) are too long
How is Structure determination carried out by solution NMR
- collect pieces of indirect evidence of structural features (structural restraints)
- Structural restraints include distance-, angle-, and bond orientation-based restraints
- Aim: calculate a structure consistent with the experimental data (i.e. the structural restraints)
What are advantages of solution NMR
- can measure more than static image of molecule
2. Study molecules in solution (including in cells)
What is the workflow for protein structure determination using NMR
- Make protein samples (can be a bottleneck) – often need multiple samples, depending on protein stability
- Acquire NMR data sets
- Signal assignments (sequential resonance assignment): assign each peak to a particular nucleus in a particular amino acid
- Simulated annealing with NMR-base restraints
- Combine different types of NMR data to:
- figure out the locations in the amino acid sequence of the secondary (2∘) structure elements (mainly a-helices and b-strands)
- determine the tertiary (3∘) structure (includes the spatial relationships between the secondary structure elements)
- Validation of Precision and accuracy of the 3D structure you’ve determined
What can you use to figure out the locations in the amino acid sequence of the secondary (2∘) structure elements
- scalar coupling values
2. secondary chemical shift values
What is Scalar or J coupling
- Nuclei are scalar coupled via bonding electrons
- Measurable only if nuclei linked by small number of bonds, including H bonds
- Intra-molecular
- Indicates which peaks in an NMR spectrum are from atoms covalently bonded to one another – important for sequential resonance assignment
- Strength of scalar/J coupling (symbol J) reflects angles (dihedral angles/torsion angles) about chemical bonds
- Scalar couplings provide backbone and side chain structural information
What do different scalar coupling frequencies indicate for NH group
- 3JHNHa ≥ 9 Hz indicates NH group is in a b-strand
2. 3JHNHa ≤ 4 Hz indicates NH group is in an a-helix
What is needed for tertiary structure determination of a protein
- Need to establish the spatial relationships between the 2∘ structure elements (mainly a-helices and b-strands)
- Dipolar coupling is important here, because it depends on nuclei being close in space, whether or not they are close in the amino acid sequence of the protein
What is Dipole-dipole (dipolar) coupling (through space coupling)
- Magnetic field generated by nucleus j at site of nucleus k, and vice versa: dipolar interaction is mutual
What is Dipole-dipole (dipolar) coupling particularly important for
- Particularly important between protons as
- 1Hs stick out,
- there’s lots of 1Hs,
- 1Hs are sensitive (have big g)
- Each 1H can sense other 1Hs up to about 6 Å (0.6 nm) away
What is dipole-dipole coupling between Hs measured by
- This interaction is measured in NMR spectra as a 1H-1H nuclear Overhauser effect (NOE)
- Closer 1Hs = stronger NOE peak
- NOEs can be inter-molecular- unlike scalar couplings
Why is dipolar coupling important
- NOEs are important for protein structure determination
- NOEs between protons in different amino acids indicate those protons are spatially close to one another
- These NOEs (100s-1000s of them) are entered into an algorithm together with other types of structural restraints – algorithm finds minimum energy 3D protein structure compatible with all the NOEs (and other structural restraints)
