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 1.NMR & Paramagnetic NMR. 2.Possum: aim & design. 3.Possum: results. 4.Conclusion. 2/25

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

4. Paramagnetic NMR  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. Paramagnetic NMR provides restraints that are global and long range:   xx yy zz r S   xx yy zz N H

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

6. Pseudocontact shift: theory. 1H1H Tensor frame Lanthanide r Isosurface (PCS > 0) θ φ Isosurface (PCS < 0) “Magnitude” of the tensor

7. = Magnitude Back calculation of PCS Diamagnetic spectrum assigned.Paramagnetic spectrum assigned. Measurement of PCS LocationOrientationStructure THEORYTHEORY EXPERIMENTEXPERIMENT

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

9. Contents 1.NMR & Paramagnetic NMR. 2.Possum: aim & design. 3.Possum: results. 4.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 1 H NMR signals.

11. Location Magnitude 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. and Otting, G. JACS in press Structure ? ?

12. 1.Protein studied: subunit ε from E. coli DNA polymerase III. 2.Lanthanide used: –Dysprosium (Dy) –Ytterbium (Yb) –Lanthanum (La) 3.Protein is 13 C selectively labelled: 4.Residue recorded: –Methionine, Threonine, Alanine. (1 methyl group class) –Valine, Isoleucine, Leucine. (2 methyl groups class) The program Possum (paramagnetic) (diamagnetic) 13 C-    ( 13 C-Leu)   

13. Multidimensional assignment problem.  n! possible assignments.  Solved in a polynomial time. A a1a1 … aiai … B b1b1 … bjbj … ij anan bnbn q i,j 2D assignment problem  (n!) 2 possible assignments.  Non Polynomial problem. A a1a1 … aiai … B … bjbj … anan bnbn C c1c1 … ckck … cncn b1b1 3D assignment problem

14. Using one paramagnetic lanthanide. para δ j dia δ k PCS calc i Using two paramagnetic lanthanides. para δ 1 j dia δ l l 1 l 2 i para δ 2 k PCS calc Possum for one-methyl-group residues:

15. γ2γ2 γ1γ1 γ2γ2 γ1γ1 Possum for two-methyl-group residues: Methyl connectivity and methyl specificity are additional information. γ2γ2 γ1γ1 γ2γ2 γ1γ1

16. Methyl connectivity on. Methyl specificity on. para δ m 1 m 2 dia δPCS calc m 1 m 2 Methyl connectivity on. Methyl specificity off. para δ m 1 m 2 dia δPCS calc m? m?m? m? m 1 m 2 Methyl connectivity off. Methyl specificity on. para δ m 1 m 2 dia δPCS calc m1m1 m 1 m 2 para δ m2m2 Possum for two-methyl-group residues:

17. Contents 1.NMR & Paramagnetic NMR. 2.Possum: aim & design. 3.Possum: results. 4.Conclusion. 18/25

18. Possum: data sets. Simulated data set. 1.Perfect ”synthetic data set”. 2.Methyl group randomly shifted according to the “0.33  Å” distribution. 3.Methyl group randomly shifted according to the “0.66 Å” distribution. 4.Exchange experiment provide the correct assignment. Experimental data set. Angstrom 1.Is possum noise resistant? 2.Is the approach valid? (90%) 3.What amount of information is necessary? –Multiple lanthanides? –Methyl specificity? –Methyl connectivity? Methyl C z -EXSY experiment. Maxwell-Boltzmann distribution

19. Possum for one methyl group residue: results.

20. 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 Possum for two methyl groups residue: 8 cases. 2*2*2 = 8 Methyl connectivity? Methyl specificity? γ2γ2 γ1γ1 γ2γ2 γ1γ1 γ2γ2 γ1γ1

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)