1. Biomolecular Nuclear Magnetic Resonance Spectroscopy BIOCHEMISTRY BEYOND STRUCTURE Protein dynamics from NMR Analytical biochemistry Comparative analysis Interactions between biomolecules 01/28/04 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 AB Two resonances (A,B) for one atom Populations ~ relative stability R ex <  (A) -  (B) Rate Estimates  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. Linewidth is Dependent on MW A B A B 1H1H 1H1H 15 N 1H1H  Linewidth determined by size of particle  Fragments have narrower linewidths Arunkumar et al., JBC (2003)

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

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,  s-ms )  R 1 (1/T 1 ) spin-lattice relaxation rate (z-axis)  R 2 (1/T 2 ) spin-spin relaxation rate (xy-plane)  Heteronuclear NOE (e.g. 15 N- 1 H)

10. Dynamics To Probe The Origin Of Structural Uncertainty  Measurements show if high RMSD is due to high flexibility (low S 2 ) Strong correlation Weak correlation     

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

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

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

14. Comparative Analysis of Structure Is the protein still the same when we cut it in half? 1H1H 1H1H 15 N 1H1H A B RPA70 A B 3 1 1 2 2 3 Chemical shift is extremely sensitive  If peaks are the same, structure is the same  But, if peaks are different, differences not directly interpretable Same idea for comparing mutants or homologs Arunkumar et al., JBC (2003)

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

16. Biochemical Assay of Mutations What 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 NMR Provides  Site-specific  Multiple probes  In-depth information  Spatial distribution of responses can be mapped on structure Titration monitored by 15 N- 1 H HSQC

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

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

21. Only 19 residues affected  Discrete binding site Signal broadening  exchange between the bound and un-bound state  Kd > 1  M RPA32C RPA32C + XPA 1-98 Probe Binding Events by NMR 15 N-RPA32C + Unlabeled XPA 1-98 15 N- 1 H HSQC Mer et al., Cell (2000)

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

23. XPA 1-98 domain XPA 29-46 peptide Same residues bind to peptide and protein  Same binding site Slower exchange for peptide  Kd < 1  M Map Site for RPA32C on XPA Mer et al., Cell (2000)

24. Manual Database Search Predicts Binding Sites in Other DNA Repair Proteins E R K R Q R A L M L R Q A R L A A R R I Q R N K A A A L L R L A A R R K L R Q K Q L Q Q Q F R E R M E K XPA 29-46 UDG 79-88 RAD 257-274 Mer et al., Cell (2000)

25. XPA 29 XPA 29-46 UDG 79-88 RAD 257-274 All Three Proteins Bind to RPA32C Binding Sites are Identical Mer et al., Cell (2000)