NMR 5

Question 1

What is the Linked fragment approach- SAR (structure activity relationship) by NMR

Answer 1

1. 15N labelled and assigned protein- already assigned peaks in NMR to specific nuclei from amino acids in protein
2. Screen for first ligand-
3. Optimise first ligand- improve its fit by adding functional groups- improve affinity
4. Screen for second ligand
5. Optimise second ligand
6. Link ligands- chemical link has to be such that ligands can continue optimal interaction with binding sites

Question 2

Describe use of NMR to study protein dynamics

Answer 2

1. Dynamics is essential for protein function e.g. interactions, catalysis, folding
2. NMR - most powerful technique for study of dynamics?
3. NMR can be used to probe molecular motions over a wide range of timescales
4. Fast motions (ps-ns): information obtained via 1H-15N HSQC-like NMR spectra
5. Slower (ms-ms) motions: studied by NMR method called relaxation dispersion
6. Even slower motions can be accessed via H-D exchange, for example
7. NMR provides dynamics information at level of individual groups in a protein
8. e.g. backbone NH groups, methyl groups, guanidino groups in Arg side chains

Question 3

Describe the energy landscape of enzyme catalysis of a chemical reaction

Answer 3

1. Steps: substrate binding (1↔2), protein conformational changes (2↔3) and (4↔5), the chemical step (3↔4), and product dissociation (5↔6).
2. Landscape defined by individual free energy differences between ground states (ΔG) and the individual activation free energies (ΔG‡) dictating microscopic rate constants (k1, k−1, k2, etc).

Question 4

What can be used as an example of protein dynamics

Answer 4

1. adenylate kinase (Adk) from a mesophile (E. coli; mesoAdk) and a hyperthermophile (Aquifex aeolicus; thermoAdk)

Question 5

Why is Adk chosen

Answer 5

1. Catalyses reversible conversion of ATP and AMP into two ADP molecules, allowing measurement of enzyme dynamics during turnover at equilibrium
2. Many crystal structures available, both free and with substrates and inhibitors
3. Adk has two ‘substrate lids’ that close upon substrate binding as a requirement for phosphoryl transfer
4. MesoAdk and thermoAdk are monomeric (23 kDa) and produce high quality NMR spectra

Question 6

NMR characterisation of Adk dynamics during catalysis

Answer 6

1. Relaxation dispersion technique can be used to measure characteristics of individual NH/CH groups in a protein and map those dynamics onto structure to show which parts are more or less dynamic
2. Relaxation curves measured during turnover provide evidence for a global conformational exchange with a single rate constant: kex = 1660 ± 90 s-1
3. This and other evidence indicates that opening and closing of the substrate lids dominate the relaxation dispersion curves
4. Measuring relaxation dispersion at two magnetic field strengths (e.g. 14 T and 18.8 T) permits determination of kopen and kclose, where kex = kopen + kclose
5. The lid opening rates measured during turnover coincide quantitatively with steady state turnover numbers determined independently by enzyme assays:
6. Conclusion: the rate limiting step is lid opening, not phosphotransfer

Question 7

How does evidence from XRC and MD support conclusion from NMR about Adk

Answer 7

1. Support that lid opening and lid closing are key conformational changes in Adk catalysis
2. Three conformations observed in XRC structure of ligand-free Aquifex Adk lie along trajectory towards closed conformation observed in Zn2+-bound Adk
3. MD simulation: lids are most mobile parts of Adk

Question 8

How does evidence from FRET and NMR PRE support conclusion from NMR relaxation dispersion data that lid opening and closing are key conformational changes in Adk catalysis

Answer 8

1. Single-molecule FRET on substrate-free thermoAdk is consistent with transition between fully open and closed states at a rate in good agreement with NMR data.
2. NMR paramagnetic relaxation enhancement (PRE), an effective measure of distance, confirmed sampling of a closed state by substrate-free thermoAdk:
3. substantial line broadening was seen for residues far from the paramagnetic label in the open state but close to the paramagnetic label in the closed state.
4. Tag is incorporated -paramagnetic NO- bleach out signals from nuclei that come within a certain distance to it

Question 9

What are the conclusions of adenylate kinase study

Answer 9

1. Product release (i.e. lid opening) is the rate limiting step for overall catalysis.
2. Conformational dynamics are slower in thermoAdk at ambient temperature.
3. Chemical reactions in the active site are fast.- The limitation of the enzyme’s catalytic power is its ability to move.
4. Dynamics on the ms-ms timescale involve a large scale transition from a closed conformation required for catalysis to an open conformation required for substrate access to active site and for product release.
5. Fast (ps-ns) atomic fluctuations are the physical origin of the slower (micros-ms), larger amplitude lid motions: fast and slow motions are linked.

Question 10

How can you characterise protein dynamics by combining bulk and single-molecule techniques

Answer 10

1. NMR or XRC to determine atomic resolution structure of domains
2. NMR (e.g. PRE) and/or other methods to define spatial relationship between domains
3. Apply methods such as:
4. CXMS (chemical cross-linking coupled with mass spectrometry)
5. SAXS (small angle X-ray scattering)
6. smFRET (single molecule FRET)
   Able to find percentage at any one time the conformation – open closed etc
7. Combination of data from multiple techniques permits determination of the ensemble structures of a multidomain protein to high precision and accuracy

Question 11

What does CXMS tell you

Answer 11

1. CXMS (chemical cross-linking coupled with mass spectrometry)
2. Cross links that apparently exceed the distance limit of the cross linker reflect transient switching of protein to alternative conformation(s)

Question 12

What does SAXS tell you

Answer 12

1. Provides low resolution structural information akin to a “molecular envelope”.
2. For a dynamic protein, the SAXS profile corresponds to the population-weighted average of all SAXS profiles for the constituent conformational states.
3. Algorithms exist to resolve conformational dynamics from SAXS measurements

Question 13

What does smFRET tell you

Answer 13

1. Monitors the time-dependent fluorescence fluctuations of donor and acceptor fluorophores conjugated to a single protein molecule.
2. Not ensemble-averaged - can discern constituent conformational states