How NMR is Used for the Study of Biomacromolecules

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How NMR is Used forthe Study of Biomacromolecules
01/26/07

• Analytical biochemistry
• Comparative analysis
• Interactions between biomolecules
• Structure determination
• Biomolecular dynamics from NMR

Arunkumar et al., JBC 278, 41077-41082 (2003) Mer
et al. Cell 103, 449-456 (2000) Ohi et al. NSB
11, 250-255 (2003)
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Analytical Protein Biochemistry

• Purity (can detect gt99)- heterogeneity,
  degradation, contamination
• Is a protein structured?- fast and easy assay,
  detects aggregation and folding
• Check on sequence (fingerprint regions)
• Dont need the sequence-specific assignments!

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Protein Folding and Fingerprints
1H COSY
15N-1H HSQC
13C HSQC also!
Assay of tertiary structure check sequence
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Comparative Analysis

• Different preparations, changes in conditions
• Domain structure
• Structural heterogeneity (e.g. Pro cis-trans
  isomerization)
• Homologous proteins, mutants, engineered
  proteins

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Folding and Domain StructureAre 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 dont know how much

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Biochemical Effect of MutationsAssay for proper
folding/stability
Wild-type
Partially destabilized
Structural heterogeneity
Unfolded
Ohi et al., NSB (2003)
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Structural Basis for 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)
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NMR to Study Interactions

• Monitor the binding of molecules
• Determine binding constants (discrete off rates,
  on rates)
• Sequence and 3D structural mapping of binding
  interfaces

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NMR- The Master Spectroscopy
Titration monitored by 15N-1H HSQC

• NMR Provides
• Site-specific
• Multiple probes
• In-depth information
• Perturbations can be mapped on structure

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Binding Constants FromChemical Shift Changes
Stronger
Weaker
Molar ratio of d-CTTCA

• Fit change in chemical shift to binding equation

Arunkumar et al., JBC (2003)
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Probe Binding Events by NMR15N-RPA32C
Unlabeled XPA1-98
15N-1H HSQC

• Only 19 residues affected
• Discrete binding site
• Signal broadening ? exchange between the bound
  and un-bound state
• Kd 1 mM

RPA32C RPA32C XPA 1-98
Mer et al., Cell (2000)
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Map XPA Binding Site on RPA32C Using NMR

• Map chemical shift perturbations on the
  structure of RPA32C
• Can even map directly on to sequence with no
  structure

Mer et al., Cell (2000)
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Structure Determination by NMR
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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)

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NMR Structure Calculations

• Objective is to determine all conformations
  consistent with the experimental data
• Programs initially search geometry only
• More calculations using molecular force fields to
  improve molecular properties
• NMR data are not perfect (noise, incomplete) ?
  multiple solutions (conformational ensemble)

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Characteristics of NMR Structures

• Secondary structures well defined, loops
  variable
• Interiors well defined, surfaces more variable
• RMSD provides measure of variability

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Restraints and Uncertainty

• Large of restraints low values of RMSD
• The most important restraints are long-range

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Assessing the Accuracy and Precisionof NMR
Structures

• Number of experimental restraints (A/P)
• Violation of constraints- number, magnitude (A)
• Compare model and exptl. parameters (A)
• Comparison to known structures PROCHECK (A)
• Molecular energies (?A?, subjective)
• RMSD of structural ensemble (P, biased)

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Biomolecular Dynamics from NMR

• Why? Function requires motion/kinetic energy
• Entropic contributions to binding events
• Protein folding/unfolding
• Uncertainty in NMR and crystal structures
• Effect on NMR experiments? dynamics to predict
  outcomes and design new experiments
• Calibration of computational methods

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Characterizing Protein Dynamics
Parameters/Timescales
Residual Dipolar Couplings
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NMR Observables and Dynamics

• 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, ms-ms)
• Direct measurements of motion of atoms
• Parameters R1 (T1), R2 (T2), Het. NOE (e.g. 15N-
  1H)

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Linewidth is Dependent on MW

• Linewidth determined by size of particle
• Fragments have narrower linewidths

Arunkumar et al., JBC (2003)
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Independent Domains in Large Proteins
RPA32
RPA14
gt 400 residues / 80 signals
Why? A structurally-independent functional domain
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Correlating Structure and Dynamics
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• Measurements show if high RMSD is due to high
  flexibility (low S2)

Weak correlation
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Strong correlation
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