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NESG NMR Conference Call Applications of RDCs and Use of Lanthanide Tags

NESG NMR Conference Call Applications of RDCs and Use of Lanthanide Tags. Presenter: J. Prestegard, UGA 9/26/05. Residual Dipolar Couplings in Structure Determination – Recent Reviews. Prestegard, A-Hashimi & Tolman, Quart. Reviews Biophys. 33 , 371-424 (2000).

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NESG NMR Conference Call Applications of RDCs and Use of Lanthanide Tags

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  1. NESG NMR Conference CallApplications of RDCs andUse of Lanthanide Tags Presenter: J. Prestegard, UGA 9/26/05

  2. Residual Dipolar Couplings in Structure Determination – Recent Reviews • Prestegard, A-Hashimi & Tolman, Quart. Reviews Biophys. 33, 371-424 (2000). • Bax, Kontaxis & Tjandra, Methods in Enzymology, 339, 127-174 (2001) • Prestegard, Bougault & Kishore, Chemical Reviews, 104, 3519-3540 (2004) • Lipsitz & Tjandra, Ann. Rev. Biophys. Biomol. Struct., 33, 387-413 (2004) • Fushman et al., Prog. NMR Spect. 44, 189-214 (2004)

  3. B0 1H r 15N RDCs Come From the Dipolar Interaction Between Two Spins Brackets denote averaging – goes to zero without partial orientation

  4. B0 Inducing Order Using Liquid Crystalline MediaRestores Dipole Interaction in Solution Most versatile medium: C12E5:octanol

  5. -1/4(J + D) bb ab E 1/4(J + D) b 1/4(J + D) -1/4(J + D) aa J + D RDCs are Easily Measured as Contributions to Multiplet Splittings

  6. Some Other Experiments for 15N-1H RDC Measurement • Tolman JR, Prestegard JH: Measurement of one-bond amide N-15-H-1 couplings. J. Magn. Reson., 1996, 112:245-252 • Ottiger M, Delaglio F, Bax A, Measurement of couplings using IPAP. JMR1998,131: 373-378.    • Kontaxis G, Clore GM, Bax A, TROSY-HSQC offsets. J. Magn. Reson., 2000 143:184-190.

  7. Use of 15N-1H RDCs for Structure Validation and Refinement: TM112 rmsd = 1.693 (NMR vs Crystal structure)

  8. REsidual Dipolar Coupling Analysis Tool(REDCAT)Valafar, H., & J.H. Prestegard (2004), J. Mag. Res.167: 228-241 • Given a proposed structure and RDCs, calculates order tensor solutions. • Finds best order tensor solution. • Gives principal elements and Euler angles. • Back-calculates RDCs. • Estimates errors and helps identify problematic data. Another program: Dosset, Hus, Marion & Blackledge (2001), JBNMR, 20: 223-231

  9. Correlation of Experimental 1H-15N RDCs with Calculated RDCs from the NMR Structure of TM112

  10. Correlation of Experimental 1H-15N RDCs with Calculated RDCs from Crystal Structure of TM1112

  11. Structure Refinement Using RDCsSchwieters CD et al. XPLOR-NIH, J. Magn. Res. 160 (1): 65-73 JAN 2003 Write RDCs in principal alignment frame: D = (Da/r3){(3cos2θ – 1)/r3 + (3/2)Rsin2θcos(2)} Write error function in terms of Dmeas and Dcalc ERDC = (Dmeas – Dcalc)2 Seek minimum in ERDC to refine structure – Need to float alignment axes during search

  12. Example of Validation and Refinement – MTH1743 RMSD Before refinement : 0.711, After refinement: 0.672

  13. Using RDCs directly in structure determination:PF1455 – A Protein with a Novel Fold? • 10 kDa protein from Pyrococcus furiosus • No significant sequence identity to PDB entries • No significant threading hits with Genthreader or Prospect • RDC-Prospect finds a structural homolog • Can RDCs and backbone NOEs lead from the homolog to a structure?

  14. Data Collected: • H-N RDCs – in phage - 63 • HaCa RDCs in phage - 61 • H-N RDCs – in C12E5 - 54 • Primary NOEs (45 sequential, 9 long range) • Secondary NOEs (18 sequential, 19 long range) • Ca shifts – 74

  15. Refinement • Started with 1cc8 as template • Constraints: NOE, RDC, torsion, radius of gyration • Three rounds of simulated annealing (400K) in vacuum • One round of simulated annealing in water • 20 independent runs – 1.4Å cluster • Structure moves 2.5Å rmsd from template • Ramachandran statistics: 56%, 30%, 13%, 1%

  16. Comparison of RDC (phage HN) before and after refinement pink symbols excluded – validation set

  17. Structure of PF1455

  18. 3 Ln3+ 1 2 RDCs can be Collected Without Alignment Media: Lanthanide Tagged Proteins: RDC = -(hB2)/(1203r3kT) [½Δ(3cos2θ-1) + ¾sin2θcos] Note: B2 dependence Ikegami, T., et al. (2004) J. Biomol. NMR29:339-349. Wohnert, J., et al. (2003) J. Am. Chem. Soc.125:13338-13339.

  19. Construct for Lanthanide Tagged EB1 MGHHHHHHG*ENLYFQG**YIDTNNDGWYEGDELLA*SAVVYSTSVTSDNLSRHDMLAWINESLQLNLTKIEQLCSGAAYCQFMDMLFPGSIALKKVKFQAKLEHEYIQNFKILQAGFKRMGVDKIIPVDKLVKGKFQDNFEFVQWFKKFFDANYDGKDYDPVAARQGQETAVAPSLVAPALNKPKKPLTSSSAAPQRPISTQRTAAAPKAGPGVVRKNPGVGNGDDEAAELMQQVNVLKLTVEDLEKERDFYFGKLRNIELICQENEGENDPVLQRIVDILYATDEGFVIPDEGGPQEEQEEY Wohnert, J., et al. (2003) J. Am. Chem. Soc.125:13338-13339.

  20. TROSY-HSQC correlations give RDC data.900 MHz Field-Induced Alignment

  21. Paramagnetic Systems Give Other Complementary Information Bertini, I., et al. (2002). Concepts in Magnetic Resonance14: 259-286.

  22. Comparison of Lu3+ and Dy3+ Complexes of Tagged Q15691 gives Pseudo-Contact Shifts Characteristic Diagonal shifts

  23. 20-25 Å 15-20 Å Ln3+ Paramagnetic Enhancement of Spin Relaxation: Distance Mapping Over 30Å Provides Validation of Assignments Lanthanide -Tagged Hum-Q-15691

  24. Test Case:Assignment of Glycine Subset of Amino Acids in Q15691

  25. NIGMS Acknowledgements T. Weldeghiorghis Silvia Mari John Glushka Homay Valafar Greg Benison Fang Tian Nitin Jain Kristen Mayer Sonal Bansal Peter Leblond http://secnmr.org

  26. Example of Multiple Coupling Experiment: Soft HNCA – E.COSY Weisemann, Ruterhans, Schwalbe, Schleucher Bermel, Griesinger, J. Biomol. NMR, 4, 231-240, 1994

  27. Soft HNCA E-COSY Spectra of 15N-Labeled 13C Natural Abundance Rubredoxin Ca chemical shift, Cai to Cai-1 connectivity, 3J-HNHa coupling, Ca-Ha, HNHa and Hai-1HN dipolar coupling

  28. = + f J+D too small J+D correct Inadvertent Mixing of α and β States of Hα can give Systematic Errors in J+D

  29. Inclusion of RDCs Improves Accuracy of Structures

  30. Order Matrix Analysis z rz rx fz q y x

  31. Finding a Principal Order Frame Sx’x’ Sy’y’ Sz’z’ Sxx Sxy .. Syx Syy .. .. .. .. A-1 = A

  32. Simple Modification of Soft HNCA – E.COSYAllows scaled addition of sum and difference E.COSY to compensate for relaxation effects +/- 90 Weisemann, Ruterhans, Schwalbe, Schleucher Bermel, Griesinger, J. Biomol. NMR, 4, 231-240, 1994

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