How NMR is Used for the Study of Bio-macromolecules

1. 02/04/09 How NMR is Used for theStudy of Bio-macromolecules • Analytical biochemistry • Comparative analysis • Interactions between biomolecules • Structure determination • Biomolecular dynamics from NMR “Dynamic Personalities of Proteins” K. Henzler-Wildman & D. Kern Nature 450 (Dec. 13), 964-972 (2007)

2. Analytical Protein Biochemistry • Purity (can detect >99%)- heterogeneity, degradation, contamination

3. Analytical Protein Biochemistry • Purity (can detect >99%)- heterogeneity, degradation, contamination • Is a protein structured?- fast and easy assay, detects aggregation and folding • Check using sequence (fingerprint regions)

4. NMR Assay of StructureDon’t Need Resonance Assignments or Labeling • 1D requires only 10-50 mM protein concentration

5. 2D Provides A More Detailed Assay 1H COSY 15N-1H HSQC 13C HSQC also! • Analyze tertiary structure, check on sequence

6. Comparative Analysis • Different preparations, changes in conditions • Binding of ligands • Chemical/conformational heterogeneity (discrete signals for different states) • Assaying structural independence of domains (fragments versus intact protein) • Mutants, homologous proteins, engineered proteins

7. B A B A Folding and Domain StructureAre domains packed together or independent? • Chemical shift is extremely sensitive • If peaks are the same, structure is the same • If peaks are different, the structure is different but we don’t know how much RPA70 15N 15N 15N 2 2 3 1H 3 1 1 1H 1H Arunkumar et al., JBC (2003)

8. Effect of MutationsNMR assays for proper folding/stability Wild-type Partially destabilized Structural heterogeneity Unfolded Ohi et al., NSB (2003)

9. Structural Basis for TS PhenotypeWhat is the cause of defective RNA splicing by Prp19-1? Initial interpretation was defect in some binding interface  NMR showed U-box folding defect Ohi et al., NSB (2003)

10. NMR to Study Interactions • Detect the binding of molecules • Determine binding constants (discrete off rates, on rates) • Sequence and 3D structural mapping of binding interfaces

11. The Thousand Dollar Pull-down! After adding binding partner Before Yes, binding did occur - more sensitive than all other methods!

12. NMR- The Master Spectroscopy Titration monitored by 15N-1H HSQC NMR Provides • Site-specific • Multiple probes • In-depth information • Perturbations can be mapped on structure • Structural models of complexes

13. Binding Constants FromChemical Shift Changes Stronger Weaker Molar ratio of d-CTTCA • Fit change in chemical shift to binding equation Arunkumar et al., JBC (2003)

14. Characterize Binding Interactions15N-RPA32C + Unlabeled XPA1-98 15N-1H HSQC • Only 19 residues affected • Discrete binding site • Signal broadening  exchange between the bound and un-bound state • Kd ~ 1 mM RPA32C RPA32C + XPA 1-98 Mer et al., Cell (2000)

15. C N Map XPA Binding Site on RPA32C Using NMR • Map chemical shift perturbations on the structure of RPA32C • Can even map directly on to sequence with no structure Mer et al., Cell (2000)

16. Generate Models of Complexes From Chemical Shift Mapping RPA32C SV40 Tag OBD Arunkumar et al., NSMB (2005)

17. NMR Structure Determination

18. NMR Experimental Observables Providing Structural Information • Distances from dipolar couplings (NOEs) • Backbone and side chain dihedral angles from measurement of scalar couplings • Backbone conformation from chemical shifts (Chemical Shift Index- CSI): , • Hydrogen bonds- NH exchange + NOES • Orientations of inter-nuclear vectors from measurement of residual dipolar coupling

19. NMR Structure Calculations • Programs initially search with restraints but with imperfect chemistry (bond lengths, etc.) • Molecular force fields are then used to improve molecular properties and refine • Data are not perfect (noise, incomplete)  multiple solutions (ensemble) • Final output is an ensemble of conformers, which together represent the conformational space consistent with the experimental data

20. Characteristics of Structures Determined in Solution by NMR • Secondary structures well defined, loops variable • Interiors well defined, surfaces more variable • RMSD provides measure of variability/precision (but not accuracy!) Kordel et al., JMB (1993)

21. Restraints and Uncertainty • Large # of restraints = low values of RMSD • The most important restraints are long-range Kordel et al., JMB (1993)

22. Assessing the Accuracy and Precisionof NMR Structures • Number of experimental restraints (A/P) • Violation of constraints- number, magnitude (A) • Comparison of model and exptl. parameters (A) • Comparison to known structures: PROCHECK (A) • Molecular energies (?A?, subjective) • RMSD of structural ensemble (P, biased)

23. Biomolecular Dynamics from NMR Why? Function requires motion/kinetic energy • Characterize protein motions/flexibility and correlate to function • Folded vs. unfolded states • Entropic contributions to binding events • Basis for uncertainty in NMR/crystal structures • Calibration of computational methods that predict protein properties (predict motions)

24. Characterizing Protein Dynamics: Parameters/Timescales Residual Dipolar Couplings

25. B A B A 15N 15N 15N 1H 1H 1H Linewidth is Dependent on MW • Linewidth determined by size of particle • Fragments have narrower linewidths Arunkumar et al., JBC (2003)

26. Independent Domains in Large Proteins 2H,15N-RPA (116 kDa) TROSY-HSQC Brosey et al., (2009)

27. Correlating Structure and Dynamics      Weak correlation • Measurements show if high RMSD is due to high flexibility (low S2)   Strong correlation  