Download presentation
Presentation is loading. Please wait.
Published byBrandon Flowers Modified over 9 years ago
1
How NMR is Used for the Study of Bio-macromolecules Analytical biochemistry Comparative analysis Interactions between biomolecules Structure determination Biomolecular dynamics from NMR 02/05/10 “Dynamic personalities of proteins” Henzler-Wildman & Kern Nature 450, 964-972 (2007) “Probing ribosome nascent chain complexes produced in vivo by NMR spectroscopy” Cabrita, Hsu, Launay, Dobson, Christodoulou PNAS 106, 22239-22234 (2009)
2
Analytical Protein Biochemistry Purity (can detect >99%)- heterogeneity, degradation, contamination, 1D Is a protein structured?- fast and easy assay, detects aggregation and folding, even 1D is effective Checks using knowledge of sequence (fingerprint regions), 2D
3
NMR Assay of Purity and Folding Don’t Need Resonance Assignments or Labeling 1D requires only 10-50 M protein concentration
4
2D Provides A More Detailed Assay Analyze tertiary structure, check sequence 15 N- 1 H HSQC 1 H COSY 13 C HSQC also!
5
Comparative Analysis Different preparations, changes in conditions Chemical/conformational heterogeneity (discrete signals for different states) Mutants, homologous proteins, engineered proteins Binding of ligands, molecular interactions
6
Effect of Mutations NMR assays for proper folding/stability Wild-type Structural heterogeneity Partially destabilized Unfolded Ohi et al., NSB (2003)
7
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)
8
NMR to Study Ligand Binding and Molecular Interactions Detect the binding of metals, molecules Sequence and 3D structural mapping of binding sites and molecular interfaces Determine binding constants (discrete off rates, on rates)
9
NMR Chemical Shift Perturbation 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 3 1 1 2 2 3 Arunkumar et al., JBC (2003)
10
The Thousand Dollar Pull-down! Before After adding binding partner Yes, binding did occur - more sensitive than all other methods!
11
NMR- The Master Spectroscopy NMR Provides Site-specific Multiple probes Atomic information Perturbations can be mapped on structure Structural models of complexes Titration monitored by 15 N- 1 H HSQC
12
Key Observations 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 1-98 15 N- 1 H HSQC Mer et al., Cell (2000)
13
NMR to Map Binding Sites XPA binding site on RPA32C 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)
14
Generate Models of Complexes from Chemical Shift Perturbations Arunkumar et al., NSMB (2005) RPA32C SV40 Tag OBD
15
Binding Constants From Chemical Shift Changes Fit change in chemical shift to binding equation Molar ratio StrongerWeaker Arunkumar et al., JBC (2003)
16
NMR Structure Determination
17
NMR Experimental Observables Providing Structural Information Distances from dipolar couplings (NOEs) Orientations of inter-nuclear vectors from residual dipolar coupling (RDCs) Backbone and side chain dihedral angles from scalar couplings (J) Backbone ( angles from chemical shifts (Chemical Shift Index- CSI, TALOS) Hydrogen bonds: NH exchange + NOES, J
18
NMR Structure Calculations Initial search to get a general idea Molecular force fields to improve molecular properties and optimize conformations Data are not perfect (noise, incomplete) multiple solutions (ensemble) Final output is an ensemble of conformers, which together represent the conformational space consistent with the experimental data
19
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 Kordel et al., JMB (1993)
20
Restraints and Uncertainty Large # of restraints = low values of RMSD Kordel et al., JMB (1993)
21
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)
22
Biomolecular Dynamics from NMR Why? Function requires motion/kinetic energy Characterize protein motions/flexibility and correlate to function - Direct coupling to enzyme kinetics - Action of multi-protein machinery - Folded vs. unfolded states - Entropic contributions to binding events - Uncertainty in NMR/crystal structures - Calibration of computational methods
23
Characterizing Protein Dynamics: Parameters/Timescales Residual Dipolar Couplings
24
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)
25
NMR to Monitor Architectural Remodeling 2 H, 15 N-RPA (116 kDa) TROSY-HSQC Brosey et al., (2009)
26
Correlating Structure and Dynamics Measurements show if high RMSD is due to high flexibility (low S 2 ) Strong correlation Weak correlation
Similar presentations
© 2024 SlidePlayer.com. Inc.
All rights reserved.