Biomolecular Nuclear Magnetic Resonance Spectroscopy

1. 01/28/04 Biomolecular Nuclear Magnetic Resonance Spectroscopy BIOCHEMISTRY BEYOND STRUCTURE • Protein dynamics from NMR • Analytical biochemistry • Comparative analysis • Interactions between biomolecules Tutorial on resonance assignments (see the website)

2. Why The Interest In Dynamics? • Function requires motion/kinetic energy • Entropic contributions to binding events • Protein Folding/Unfolding • Uncertainty in NMR and crystal structures • Effect on NMR experiments-spin relaxation is dependent on rate of motions  know dynamics to predict outcomes and design new experiments • Quantum mechanics/prediction (masochism)

3. Characterizing Protein Dynamics: Parameters/Timescales

4. Dynamics From NMR Parameters • Number of signals per atom: multiple signals for slow exchange between conformational states Two resonances (A,B) for one atom Populations ~ relative stability Rex < w (A) - w (B) Rate Estimates A B • Multiple states are hard to detect by Xray crystallography

5. Dynamics From NMR Parameters • Number of signals per atom: multiple signals for slow exchange between conformational states • Linewidths: narrow = faster motion, wide = slower; dependent on MW and structure

6. 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)

7. 40 173 P Detecting Functionally Independent Domains in Multi-Domain Proteins RPA32 RPA14 > 300 residues / ~80 signals Why? • Flexibility facilitates interactions with protein targets

8. Dynamics From NMR Parameters • Number of signals per atom: multiple signals for slow exchange between conformational states • Linewidths: narrow = faster motion, wide = slower; dependent on MW and conformational states • Exchange of NH with solvent:slow timescales (milliseconds to years!) • Requires local and/or global unfolding events • NH involved in H-bond exchanges slowly • Surface or flexible region: NH exchanges rapidly

9. Dynamics From NMR Parameters • Number of signals per atom: multiple signals for slow exchange between conformational states • Linewidths: narrow = faster motion, wide = slower; dependent on MW and conformational states • Exchange of NH with solvent:slow timescales • NMR relaxation measurements (ps-ns, ms-ms) • R1 (1/T1) spin-lattice relaxation rate (z-axis) • R2 (1/T2) spin-spin relaxation rate (xy-plane) • Heteronuclear NOE (e.g. 15N- 1H)

10. Dynamics To Probe The OriginOf Structural Uncertainty  Weak correlation • Measurements show if high RMSD is due to high flexibility (low S2)   Strong correlation  

11. Analytical Protein Biochemistry • Purity (can detect >99%)- heterogeneity, degradation, buffer • Check on sequence (fingerprint regions)

12. Protein Fingerprints 1H COSY 15N-1H HSQC 13C HSQC also! Assay structure from residue counts in each fingerprint

13. Comparative Analysis • Different preparations, chemical modifications • Conformational heterogeneity (e.g. cis-trans isomerization) • Homologous proteins, mutants, engineered proteins

14. B A B A Comparative Analysis of StructureIs the protein still the same when we cut it in half? RPA70 • Chemical shift is extremely sensitive • If peaks are the same, structure is the same • But, if peaks are different, differences not directly interpretable 15N 15N 15N 2 2 3 1H 3 1 1 1H 1H Same idea for comparing mutants or homologs Arunkumar et al., JBC (2003)

15. Biochemical Assay of MutationsMutations can effect folding and stability Wild-type Partially destabilized Partially destabilized & hetero-geneous Unfolded Ohi et al., NSB (2003)

16. Biochemical Assay of MutationsWhat is the cause of the Prp19-1 defect? Not perturbation at binding interface  Destabilized U-box leads to drop in activity Ohi et al., NSB (2003)

17. NMR to Study Interactions • Monitor the binding of molecules • Determine binding constants (discrete off rates, on rates) • Identify binding interfaces

18. Monitoring Binding Events Titration monitored by 15N-1H HSQC NMR Provides • Site-specific • Multiple probes • In-depth information • Spatial distribution of responses can be mapped on structure

19. Binding Constants From NMR Stronger Weaker Molar ratio of d-CTTCA Fit change in chemical shift to binding equation Arunkumar et al., JBC (2003)

20. C N Winged Helix-Loop-Helix Probing Protein InteractionsStructure is the Starting Point! Mer et al., Cell (2000)

21. Probe Binding Events by NMR15N-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)

22. C N Map XPA Binding Site on RPA32C Using NMR Map of chemical shift perturbations on the structure of RPA32C Mer et al., Cell (2000)

23. Map Site for RPA32C on XPA • Same residues bind to peptide and protein • Same binding site • Slower exchange for peptide • Kd < 1 mM XPA1-98 domain XPA29-46 peptide Mer et al., Cell (2000)

24. Manual Database Search Predicts Binding Sites in Other DNA Repair Proteins XPA29-46 UDG79-88 RAD257-274 E R K RQR A L ML R QA R L A A R R I Q RNK A A AL L RL A A R R K L RQK Q L Q Q Q F R E R M E K Mer et al., Cell (2000)

25. All Three Proteins Bind to RPA32CBinding Sites are Identical UDG79-88 RAD257-274 XPA29-46 Mer et al., Cell (2000)