1. Biomolecular Nuclear Magnetic Resonance Spectroscopy BIOCHEMISTRY BEYOND STRUCTURE Protein dynamics from NMR Analytical Biochemistry Comparative Analysis 01/22/03

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 Populations ~ relative stability R ex <  (A) -  (B) Rate

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  Same shifts, same structure  Linewidth determined by size of particle  Fragments have narrower linewidths

7. Detecting Functionally Independent Domains in Multi-Domain Proteins Why?  Flexibility facilitates interactions with protein targets RPA32 RPA14 173 P 40

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 (1-2%)- heterogeneity, degradation, buffer Check on sequence (fingerprint regions) Binding constants, off rates, on rates

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

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

14. Binding Constants From NMR Fit change in chemical shift to binding equation Molar ratio of d-CTTCA StrongerWeaker

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

16. 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 If the peaks are in the same place, the structure is the same Same idea for comparing mutants or homologs

17. Biochemical Assay of Mutations Mutations can effect folding and stability Wild-type Partially destabilized & hetero- geneous Partially destabilized Unfolded

18. 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

19. Probing Binding of Protein Targets Structure is the Starting Point! C N Winged Helix-Loop-Helix Mer et al., Cell (2000)

20. 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

21. NMR Identification of the XPA Binding Site on RPA32C C N Map of chemical shift perturbations on the structure of RPA32C

22. 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 Localization of Binding Site

23. 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

24. XPA 29 XPA 29-46 UDG 79-88 RAD 257-274 All Three Proteins Bind to RPA32C Binding Sites are Identical