how nmr is used for the study of bio-macromolecules analytical biochemistry comparative analysis...
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How NMR is Used for theStudy 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)
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
NMR Assay of Purity and FoldingDon’t Need Resonance Assignments or Labeling
1D requires only 10-50 M protein concentration
2D Provides A More Detailed Assay
Analyze tertiary structure, check sequence
1515N-N-11H HSQCH HSQC11H COSYH COSY
13C HSQC also!
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
Effect of MutationsNMR assays for proper folding/stability
Wild-type
Structural heterogeneity
Partially destabilized
Unfolded
Ohi et al., NSB (2003)
Structural Basis for TS PhenotypeWhat 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 Ligand Bindingand 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)
NMR Chemical Shift PerturbationAre 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
1H
1H
15N
15N
1H
15N
A B
RPA70
AB
3
1 1
2 23
Arunkumar et al., JBC (2003)
The Thousand Dollar Pull-down!
BeforeAfter addingbinding partner
Yes, binding did occur - more sensitive than all other methods!
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 15N-1H HSQC
Key Observations
• Only 19 residues affected Discrete binding site
• Signal broadening exchange between the bound and un-bound state Kd ~ 1 M
RPA32CRPA32C + XPA 1-98
Characterize Binding Events15N-RPA32C + Unlabeled XPA1-98
1515N-N-11H HSQCH HSQC
Mer et al., Cell (2000)
NMR to Map Binding SitesXPA binding site on RPA32C
C
N
Map chemical shift Map chemical shift perturbations on the perturbations on the structure of RPA32Cstructure of RPA32C Can even map Can even map directly on to directly on to sequence with no sequence with no structure!!structure!!
Mer et al., Cell (2000)
Generate Models of Complexes from Chemical Shift Perturbations
Arunkumar et al., NSMB (2005)
RPA32C
SV40 TagOBD
Binding Constants FromChemical Shift Changes
Fit change in chemical shift to binding equation
Molar ratio
Stronger Weaker
Arunkumar et al., JBC (2003)
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
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
• 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)
Assessing the Accuracy and Precisionof 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
• 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
Linewidth is Dependent on MW
A B
1H
1H
15N
A B
15N
1H
15N
Linewidth determined by size of particle
Fragments have narrower linewidths
Arunkumar et al., JBC (2003)
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