NMR in Structural Biology Exercise session 2 1. a. List 3 NMR observables that report on structure. b. Also indicate whether the information they give is short/medium or long-range, or perhaps all three? c. Also indicate whether the information they give determines the secondary or tertiary structure, or both. d. Also indicate their shortcomings/drawbacks. 2. a. List 2 NMR observables that report on dynamics. b. Which NMR parameter is related to the molecular weight and how? 3. Give two parameters that give information about the quality of an NMR structure and explain. c. After having assigned the full HNCACB spectrum of a certain protein, what can you than already tell about the protein structure? 8. Protein X is the second most important protein in the world. It interacts with protein Y, the third most important protein in the world, to form a quite essential protein complex. The two proteins and the complex are way too big to study by NMR, but luckily the interaction is between two smaller protein domains. How would you study this interaction by NMR? 9. a. Where does the abbreviation PRE stand for? b. What do you need to record PREs? c. What is the principle behind the PRE? d. How can it be used to get information about the protein structure? 10. Below are the strips out of a HNCACB experiment for 6 residues out of a hypothetical protein:... EFAGKD... Do the sequential assignment for this stretch and assign all the peaks in these strips 4. Give two types of information that you need besides the experimental data when calculating an NMR structure. 5. a. Why should we take care when using the RMSD to define the precision of an NMR structure? b. Can we use the RMSD to say something about the accuracy of an NMR structure ensemble? 6. What information can you use for predicting the structures of protein-ligand complexes? Give three examples 7. Consider the HNCACB experiment. a. Draw schematically the magnetization transfer for this experiment in the figure below. b. At which coordinates (i.e. frequencies) do you see a peak? 1 2
11. a. What information does the spectral density function provide? b. Why does only the R 2 relaxation rate depend on the spectral density function at zero frequency J(0)? c. Why is T1 relaxation more efficient (i.e. short T1) for small proteins (i.e.! c! 5 ns) than for large proteins (i.e.! c! 20 ns)? d. Explain why T2 relaxation depends linear on the correlation time, while this is not the case for T1 relaxation. 14. In the figure you see a solid-state NCO (left) and NCA (right) NMR spectrum of the following peptide sequence. 12. a. Why does conformational exchange broaden the NMR lines? b. How can we reduce the broadening by conformational exchange using a CPMG sequence? 13. a. Give two reasons why solid-state NMR (ssnmr) spectra have very broad lines when you do not apply Magic Angle Spinning (MAS). b. Why do ssnmr spectra have narrow line-widths when applying MAS? c. Why using ssnmr we prefer to observe heteronuclei (e.g. 15 N and 13 C) instead of protons 1 H? d. How can you under MAS enhance the resolution of your spectra? 3 4
Answers 1. - chemical shift short secondary only some atoms/angles - NOE intensity short-medium-long secondary-tertiary only distances < 5 Å not very precise (spin diffusion) - J-coupling short secondary only some angles, relation J-angle not unique - residual dipolar coupling short-medium-long secondary-tertiary (RDC) need special medium, protein might not like it. - PRE long tertiary need to mutate residues to attach spin-label, spin-label can be flexible 2. - T1 - linewidth /T2 -> big molecules tumble slow and have short T2 s and large linewidths. Small molecules tumble fast and have long T2 s and small linewidths. 3. - ramachandran plot statistics - no. of violations (rms. of violations) - no. of atomic bumps - energy / target function value - local geometry 4. - amino acid sequence - geometrical constraints (bond lengths,angles, planarity etc.) - vanderwaals repulsion - electrostatic attraction 5. a. The RMSD could also reflect the lack of (long-range) restraints due to dynamics b. The RMSD gives information about the precision but not the accuracy. You could have calculated a very precise but wrong structure. 6. - basically any atomic/residue level info - chemical shift perturbation (HSQC titration) - mutagenesis (when you mutate at the interface the interaction is gone) - H/D exchange (residues at interface with the ligand exchange slower) - also RDC s (global shape) c. You will know the H, N, CA and CB chemical shifts of all amino-acids. These chemical shift you can compare to random coil chemical shifts and this will give you information on the secondary structure of your protein. 8. express both domains, label one with 15N/13C, do HSQC titration => chemical shift perturbation, record intermolecular NOEs by isotope filtered exps, do H/D exchange with and without, do RDC w/o, structure calculcation/prediction. 9. PRE: paramagnetic relaxation enhancement. By attachment of a paramagnetic spinlabel (i.e. unpaired electron) to the protein (by site-directed spin-labeleling), the relaxation rates of the nuclei in close proximity to the spinlabel will have enhanced relaxation rates; this effect scales with 1/r 6. Signals will broaden and the intensity will drop. This intensity drop can be measured and used to calculate the distance to the spin-label 10. see next page. 11. a. J(") describes if a certain frequency can induce relaxation and whether it is efficient. b. R2 relaxation is dephasing of the transverse magnetization in the xy plane. No energy transitions are needed and fluctuations parallel to the magnetic field that have no associated frequency in the x/y plane (i.e. " = 0) can thus induce relaxation. c. T1 relaxation depends mainly on the J(" N) which is larger for a small protein than for a large protein. J(") " d. The T2 mainly depends on J(0) that linearly depends on! c. For T1 relaxation exchange of energy is needed so that transitions can take place to get back to Boltzmann equilibrium. Therefore you need fluctuations in the xy-plane of the local magnetic field with frequencies close to the Larmor frequency (i.e. 1/! c ~ " N). Fluctuations with lower or higher frequencies will thus be less efficient to cause relaxation. 7. a. HN -> N -> CA -> CB -> CA -> N -> HN (both to i and i-1) b. (F1, F2, F3) = (CA/CB, N, HN) => Ca-i, N-i, HN-i Cb-i, N-i, HN-i Ca-i-1, N-i, HN-i Cb-i-1, N-i, HN-i 5 6
12. a. Due to conformational exchange the chemical shift of a certain nucleus will vary in time. This enhances the dephasing of transverse magnetization and thus increases the R2 relaxation rate. The line-width is proportional to the R2 relaxation rate (line-width ~ R2 ~ 1/T2) so the lines will broaden. b. In a CPMG sequence you apply a train of 180 pulses while the magnetization is in the x/y plane to refocus the chemical shift. If you apply these pulses fast enough (i.e. faster than the exchange rate between the different conformations) you can still refocus (partially) the chemical shift, even if during the CPMG period the chemical shift has changed due to conformational exchange. 13. a. The chemical shift anisotropy (CSA) and the dipolar couplings can be large and cause that the solid-state NMR line-widths are large. b. By spinning fast enough you can average out the anisotropic interactions and obtain narrow line-widths (3-4 times the dipolar coupling). c. The density of protons in a protein is much higher than for heteronuclei, and thus each proton interacts with several other protons. These 1 H- 1 H dipolar couplings are very large and we cannot spin fast enough using MAS to obtain narrow 1 H NMR line-widths. d. By applying efficient decoupling schemes. 7 8
14. 9