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

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)

Characterizing Protein Dynamics: Parameters/Timescales

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

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

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

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

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

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)

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     

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

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

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

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

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

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

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

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

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

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 N- 1 H HSQC

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

XPA 1-98 domain XPA peptide Same residues bind to peptide and protein  Same binding site Slower exchange for peptide  Kd < 1  M Localization of Binding Site

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 UDG RAD

XPA 29 XPA UDG RAD All Three Proteins Bind to RPA32C Binding Sites are Identical