How NMR is Used for the Study of Bio-macromolecules

1. 02/05/10 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” Henzler-Wildman & Kern Nature 450, 964-972 (2007) “Probing ribosome nascent chain complexes produced in vivo by NMR spectroscopy” Cabrita, Hsu, Launay, Dobson, Christodoulou PNAS 106, 22239-22234 (2009)

2. Analytical Protein Biochemistry • Purity (can detect >99%)- heterogeneity, degradation, contamination, 1D • Is a protein structured?- fast and easy assay, detects aggregation and folding, even 1D is effective • Checks using knowledge of sequence (fingerprint regions), 2D

3. NMR Assay of Purity and FoldingDon’t Need Resonance Assignments or Labeling • 1D requires only 10-50 mM protein concentration

4. 2D Provides A More Detailed Assay 1H COSY 15N-1H HSQC 13C HSQC also! • Analyze tertiary structure, check sequence

5. Comparative Analysis • Different preparations, changes in conditions • Chemical/conformational heterogeneity (discrete signals for different states) • Mutants, homologous proteins, engineered proteins • Binding of ligands, molecular interactions

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

7. 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)

8. NMR to Study Ligand Bindingand Molecular Interactions • Detect the binding of metals, molecules • Sequence and 3D structural mapping of binding sites and molecular interfaces • Determine binding constants (discrete off rates, on rates)

9. B A B A NMR Chemical Shift PerturbationAre 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)

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

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

12. Characterize Binding Events15N-RPA32C + Unlabeled XPA1-98 15N-1H HSQC Key Observations • 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)

13. C N NMR to Map Binding SitesXPA binding site on RPA32C • Map chemical shift perturbations on the structure of RPA32C • Can even map directly on to sequence with no structure!! Mer et al., Cell (2000)

14. Generate Models of Complexes from Chemical Shift Perturbations RPA32C SV40 Tag OBD Arunkumar et al., NSMB (2005)

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

16. NMR Structure Determination

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

18. NMR Structure Calculations • Initial search to get a general idea • Molecular force fields to improve molecular properties and optimize conformations • 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

19. 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)

20. Restraints and Uncertainty • Large # of restraints = low values of RMSD Kordel et al., JMB (1993)

21. 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)

22. Biomolecular Dynamics from NMR Why? Function requires motion/kinetic energy • Characterize protein motions/flexibility and correlate to function - Direct coupling to enzyme kinetics - Action of multi-protein machinery - Folded vs. unfolded states - Entropic contributions to binding events - Uncertainty in NMR/crystal structures - Calibration of computational methods

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

24. 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)

25. NMR to Monitor Architectural Remodeling 2H,15N-RPA (116 kDa) TROSY-HSQC Brosey et al., (2009)

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