How NMR is Used for the Study of Biomacromolecules Analytical biochemistry Comparative analysis Interactions between biomolecules Structure determination Biomolecular dynamics from NMR 01/30/08 Arunkumar et al., JBC 278, (2003) Mer et al. Cell 103, (2000) Ohi et al. NSB 11, (2003)

Analytical Protein Biochemistry Purity (can detect >99%)- heterogeneity, degradation, contamination Is a protein structured?- fast and easy assay, detects aggregation and folding Check on sequence (fingerprint regions)  Don’t need the sequence-specific assignments! Start with 1D NMR (50  M)

NMR Assay of Structural Integrity Tertiary structure, check on sequence 15 N- 1 H HSQC 1 H COSY 13 C HSQC also!

Comparative Analysis Different preparations, changes in conditions Binding of ligands Chemical/conformational heterogeneity Assaying structural/functional independence of domains Homologous proteins, mutants, engineered proteins

Folding and Domain Structure Are 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 1H1H 1H1H 15 N 1H1H A B RPA70 A B Arunkumar et al., JBC (2003)

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

Structural Basis for TS Phenotype What 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)

NMR to Study Interactions Detect the binding of molecules Determine binding constants (discrete off rates, on rates) Sequence and 3D structural mapping of binding interfaces

The Thousand Dollar Pull-down! Before After adding binding partner Yes, binding did occur!

NMR- The Master Spectroscopy NMR Provides  Site-specific  Multiple probes  In-depth information  Perturbations can be mapped on structure Titration monitored by 15 N- 1 H HSQC

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

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 Characterize Binding Events 15 N-RPA32C + Unlabeled XPA N- 1 H HSQC Mer et al., Cell (2000)

Map XPA Binding Site on RPA32C Using NMR C N  Map chemical shift perturbations on the structure of RPA32C  Can even map directly on to sequence with no structure Mer et al., Cell (2000)

Determine 3D Structure of Complexes by NMR

NMR Structure Determination

NMR Experimental Observables Providing Structural Information Distances from dipolar couplings (NOEs) Backbone and side chain dihedral angles from scalar couplings Backbone conformation from chemical shifts (Chemical Shift Index- CSI): ,  Hydrogen bonds- NH exchange or J Relative inter-nuclear orientations from residual dipolar couplings (RDCs)

NMR Structure Calculations Programs initially search with restraints disregarding chemistry (bond lengths, etc.) Molecular force fields are then used to improve molecular properties and refine Data are not perfect (noise, incomplete)  multiple solutions (conformational ensemble) Final output is all conformations consistent with the experimental data

Secondary structures well defined, loops variable Interiors well defined, surfaces more variable RMSD provides measure of variability/precision (but not accuracy!) Characteristics of Structures Determined in Solution by NMR

Restraints and Uncertainty  Large # of restraints = low values of RMSD  The most important restraints are long- range

Assessing the Accuracy and Precision of 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)

Biomolecular Dynamics from NMR Why? Function requires motion/kinetic energy Entropic contributions to binding events Differences in folded vs. unfolded states Basis for uncertainty in NMR/crystal structures Effect on NMR experiments  dynamics to predict outcomes and design new experiments Calibration of computational methods that predict protein properties (predict motions)

Biomolecular Dynamics from NMR “Dynamic Personalities of Proteins” K. Henzler-Wildman & D. Kern Nature 450 (Dec. 13), (2007) Lecture by Dorothee Kern: April 7, 2008

Characterizing Protein Dynamics: Parameters/Timescales Residual Dipolar Couplings

Linewidth is Dependent on MW A B 1H1H 1H1H 15 N A B 1H1H  Linewidth determined by size of particle  Fragments have narrower linewidths Arunkumar et al., JBC (2003)

Independent Domains in Large Proteins Why? A structurally-independent functional domain RPA32 RPA P 40 > 400 residues / ~80 signals Mer et al., Cell (2000)

Correlating Structure and Dynamics  Measurements show if high RMSD is due to high flexibility (low S 2 ) Strong correlation Weak correlation       