NMR 4

Question 1

How do you determine structure by NMR

Answer 1

1. Multiple repeats of calculation where using list of structural restraints
2. Each time starting from random coil version of polypeptide
3. Compare resulting structures by overlaying them
4. Highly flexible regions of proteins can access multiple conformations of too short duration to produce structural restraints:
5. their conformation therefore differs in each member of the ensemble.
6. Such regions are usually excluded from the rmsd calculation.

Question 2

What are Limitations of NMR

Answer 2

1. Limitations largely relate to resolution and sensitivity
2. Low sensitivity due to small DE – hence small population difference between energy levels
3. Bigger molecule – more nuclei – more peaks/signals
4. Bigger molecule – peaks/signals become broader

Question 3

Use of NMR to study protein interactions

Answer 3

1. mapping of binding site(s)
2. binding-induced folding
3. drug discovery

Question 4

Types of complexes that can be studied by NMR

Answer 4

1. Protein-protein
2. Protein-DNA/RNA
3. Protein-ligand (e.g. -drug), protein-carbohydrate, protein-lipid
4. DNA-drug and RNA-drug
5. Cellular machines/assemblies (e.g. chaperones, proteasomes)

Question 5

When is NMR preferred over XRC

Answer 5

1. Some complexes may not crystallise, or not crystallise in a biologically relevant conformation e.g. when interactions are weak due to the cell’s need for transient, reversible binding
2. Crystal packing forces may distort molecular interactions
3. NMR is very well suited to, but not limited to, studying complexes involving weak interactions (Kd up to 10-2 M)
4. Ideal: combine techniques

Question 6

NMR methods to study interactions

Answer 6

1. Differential isotope labelling: for example, only one component of a bimolecular complex is labelled with 15N.
2. Combine differential isotope labelling with one or more of approaches like:
3. Intermolecular NOEs: NMR peaks that result from two molecules being close together. Using both intramolecular and intermolecular NOEs, plus other restraints, detailed 3D structure of a complex can be determined.
4. Chemical shift changes: usually indicate direct involvement of nuclei in binding and can be used to map interface between two molecules.
5. Caveat: structure (and therefore chemical shifts) may change beyond the immediate vicinity of the binding site – reflects cooperative nature of proteins (groups of amino acids acting as networks).
6. Measurements of motion: measure motional changes upon interaction.

Question 7

Differential isotope labelling

Answer 7

1. The two (or more) components of a complex are prepared separately.
2. One component uniformly isotope labelled (e.g. 15N);
3. other component contains natural abundance isotopes (e.g. 99.6% 14N / 0.4% 15N).
4. The labelled and unlabelled components are mixed.
5. In an NMR experiment that depends on scalar coupling between 15N and 1H, only the component with 15N nuclei (the “labelled” component) will produce signals in the NMR spectrum. The 0.4% 15N in the “unlabelled” component is too low to detect – the “unlabelled” component is “NMR silent”.
6. Could equally use carbon, for example replace 12C by 13C in one component.
7. If desired, it is also possible to “filter” or “edit” out the isotope labelled component and detect only the unlabelled component.
8. Ligand is NMR silent

Question 8

Chemical shift perturbation mapping

Answer 8

1. Monitor 1H-15N HSQC when unlabelled/differently labelled molecule added
2. 1H-15N HSQC: every residue type (except Pro) contains a highly sensitive probe to changes in local environment
3. 1H-13C HSQC: information on sidechain-mediated interactions, but signal overlap is an issue in proteins > 25 kDa
4. Binding of ligand/macromolecule changes environment of nuclei at molecular interface – hence chemical shifts change (and/or peaks get weaker)
5. Effect may propagate beyond binding site as proteins are “cooperative” materials

Question 9

Linked fragment approach to drug discovery

Answer 9

1. Fragments (MW < 300 Da, high ligand efficiency) that bind to proximal sites on protein are linked to form one compound with enhanced affinity
2. High ligand efficiency- good binding
3. Theoretical binding energy of a linked molecule, DG(AB), composed of two components A and B, is represented by
4. DG(AB) = DG(A) + DG(B) + DG(L)
5. DG(A) and DG(B) = binding energies of the unlinked components
6. DG(L) includes contributions to binding energy due to linking
7. LE = ligand efficiency (deltaGbinding per heavy atom)

Question 10

What is advantage of NMR to discover fragment hits

Answer 10

1. Chemical shift (or intensity) perturbation to discover fragment hits
2. Advantage: NMR can detect very weak interactions (Kd up to 10-2 M)
3. Hence, NMR can detect weak binding fragments (initial lead compounds

Question 11

Chemical shift perturbation mapping: -compound cocktails

Answer 11

1. Reduce peaks as only specific amino acids are labeled

2. Looks to see if any of these compounds are involved in interaction with protein

Question 12

What are some other fragment screening methods

Answer 12

1. Thermal shift: increase in melting temperature of protein if fragment binds.
2. Mass spectrometry (MS): detect a protein:fragment complex directly.
3. NMR (WaterLOGSY): unbound ligand signals (green spectrum) and inversion in the presence of target (red).
4. Surface plasmon resonance (SPR): sensorgrams as function of concentration - binding kinetics.
5. X-ray crystallography (XRC): unambiguous determination of fragment binding mode, essential for fragment development.
6. In silico (docking): ensemble of ranked, docked conformations.