Automatic NMR resonance assignment of methyl groups using paramagnetic lanthanides.

1. Automatic NMR resonance assignment of methyl groups using paramagnetic lanthanides. Christophe Schmitz School of Molecular & Microbial Sciences University of Queensland Supervised by Thomas Huber & Gottfried Otting East Coast Protein Meeting. 12-14 July 2007

2. Contents • NMR & Paramagnetic NMR. • Possum: aim & design. • Possum: results. • Conclusion. 2/25

3. “Classical” NMR Classical NMR provides restraints that are localandshort range: • NOE effect tell us about distances between two spins. • J-coupling effect tell us about dihedral angles. d < 0.8 nm

4. Paramagnetic NMR Paramagnetic NMR provides restraints that are globalandlong range: • Pseudocontact shift (PCS) tell us about the location of a spin with respect to an internal frame. • Residual Dipolar Coupling tell us about the orientation of N-H bond with respect to an internal frame. cz S q r cx j cy cz Q H N cx cy F

5. Ala45 PCS(45) Pseudocontact shift: experiment. Diamagnetic metal ion (Lanthanum) Paramagnetic metal ion (paramagnetic lanthanide) 2D 15N-HSQC “diamagnetic spectrum”. 2D 15N-HSQC “paramagnetic spectrum”.

6. Pseudocontact shift: theory. 1H θ r Isosurface (PCS > 0) Tensor frame Isosurface (PCS < 0) Lanthanide φ “Magnitude” of the tensor

7. T H E O R Y Magnitude Location Orientation Structure Back calculation of PCS = Measurement of PCS E X P E R I M E N T Diamagnetic spectrum assigned. Paramagnetic spectrum assigned.

8. What PCS is used for? Structure refinement Protein-ligand complexes Protein-protein complexes Allegrozzi, M.; Bertini, I.; Janik, M. B. L.; Lee, Y. M.; Lin, G. H.; Luchinat, C. JACS 2000, 122, (17), 4154-4161. John, M.; Pintacuda, G.; Park, A. Y.; Dixon, N. E.; Otting, G., JACS 2006, 128, (39), 12910-12916. Pintacuda, G.; Park, A. Y.; Keniry, M. A.; Dixon, N. E.; Otting, G. JACS 2006, 128, (11), 3696-3702. But we need to assign the diamagnetic and paramagnetic spectrum first.

9. Contents • NMR & Paramagnetic NMR. • Possum: aim & design. • Possum: results. • Conclusion. 9/25

10. The program Possum: objective. Automatic assignment of methyl groups: • Methyl groups are probes for the study of proteins: • On the surface: report on protein-protein interaction. • Buried: report on the packing of the side chains. • Favorable relaxation properties. • Intense 1H NMR signals.

11. Location Magnitude Structure Orientation The program Possum. ? ? Sequence- and stereospecific assignment of methyl groups using paramagnetic lanthanides. John, M., Schmitz, C., Park, A.Y., Dixon, N.E., Huber, T. andOtting, G. JACS in press

12. The program Possum • Protein studied: subunitε from E. coli DNA polymerase III. • Lanthanide used: • Dysprosium (Dy) • Ytterbium (Yb) • Lanthanum (La) • Protein is 13C selectively labelled: • Residue recorded: • Methionine, Threonine, Alanine. (1 methyl group class) • Valine, Isoleucine, Leucine. (2 methyl groups class) (paramagnetic) (diamagnetic) (13C-Leu)e1-186/q 13C-e1-186/q

13. 3D assignment problem A B C a1 b1 c1 … … … • (n!)2 possible assignments. • Non Polynomial problem. ai bj ck … … … an bn cn Multidimensional assignment problem. A B qi,j a1 b1 … … i j ai bj … … an bn • n! possible assignments. • Solved in a polynomial time. 2D assignment problem

14. Using two paramagnetic lanthanides. PCScalc l1l2 paraδ1 paraδ2 diaδ i i j k l Possum for one-methyl-group residues: Using one paramagnetic lanthanide. PCScalc paraδ diaδ i j k

15. γ1 γ1 γ1 γ1 γ2 γ2 γ2 γ2 Possum for two-methyl-group residues: Methyl connectivity and methyl specificity are additional information.

16. Methyl connectivityon. Methyl specificityoff. PCScalc paraδ diaδ m1m2 m1m2 m1m2 PCScalc paraδ paraδ diaδ m1m2 m1 m2 m1m2 PCScalc paraδ diaδ m1m2 m?m? m1m2 Methyl connectivityoff. Methyl specificityon. Possum for two-methyl-group residues: Methyl connectivityon. Methyl specificityon.

17. Contents • NMR & Paramagnetic NMR. • Possum: aim & design. • Possum: results. • Conclusion. 18/25

18. Possum: data sets. Maxwell-Boltzmann distribution • Perfect ”synthetic data set”. • Methyl group randomly shifted according to the “0.33 Å” distribution. • Methyl group randomly shifted according to the “0.66 Å” distribution. • Exchange experiment provide the correct assignment. Simulated data set. Angstrom Experimental data set. • Is possum noise resistant? • Is the approach valid? (90%) • What amount of information is necessary? • Multiple lanthanides? • Methyl specificity? • Methyl connectivity? Methyl Cz-EXSY experiment.

19. Possum for one methyl group residue:results.

20. γ1 γ2 γ2 γ1 γ2 γ1 Possum for two methyl groups residue: 8 cases. • Methyl specificity available in the diamagnetic state? Y / N • Methyl specificity available in the paramagnetic state? Y / N • Methyl connectivity available in the paramagnetic state? Y / N 2*2*2 = 8 Methyl specificity? Methyl connectivity?

21. Possum for two methyl groups residue:“worst case”results.

22. Possum for two methyl groups residue:“best case”results. 64.7% 86.5% 78.0%

23. Conclusion for Possum. • Noise resistant. • Crossing two experiments with two different paramagnetic lanthanides improve drastically the assignment. • Next step: apply to Fluorine labelled protein. • Quantify the accuracy of an assignment.

24. Conclusion • The “global” and “long range” characteristic of PCS => attractive subject. • Paramagnetic NMR / PCS provides useful restraints. • Assignment of NMR spectrum is the key to access PCS. • Possum assigns methyl groups automatically.

25. Acknowledgments. • Thomas Huber (UQ) • Gottfried Otting (ANU) • Michael John (GAU) • Ah Young Park (ANU) • Nick Dixon (UOW) • Don A. Grundel (UFL)