Biomolecular Nuclear Magnetic Resonance Spectroscopy

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Biomolecular Nuclear Magnetic Resonance
Spectroscopy
01/28/04

• BIOCHEMISTRY BEYOND STRUCTURE
• Protein dynamics from NMR
• Analytical biochemistry
• Comparative analysis
• Interactions between biomolecules

Tutorial on resonance assignments (see the
website)
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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)

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Characterizing Protein Dynamics
Parameters/Timescales
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Dynamics From NMR Parameters

• Number of signals per atom multiple signals for
  slow exchange between conformational states

Two resonances (A,B) for one atom Populations
relative stability
Rex lt w (A) - w (B)
Rate Estimates
A
B

• Multiple states are hard to detect by Xray
  crystallography

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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

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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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Detecting Functionally Independent Domains in
Multi-Domain Proteins
RPA32
RPA14
gt 300 residues / 80 signals

• Why?
• Flexibility facilitates interactions with
  protein targets

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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

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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, ms-ms)
• R1 (1/T1) spin-lattice relaxation rate (z-axis)
• R2 (1/T2) spin-spin relaxation rate (xy-plane)
• Heteronuclear NOE (e.g. 15N- 1H)

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Dynamics To Probe The OriginOf Structural
Uncertainty
?

• Measurements show if high RMSD is due to high
  flexibility (low S2)

Weak correlation
?
?
Strong correlation
?
?
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Analytical Protein Biochemistry

• Purity (can detect gt99)- heterogeneity,
  degradation, buffer
• Check on sequence (fingerprint regions)

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

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

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Comparative Analysis of StructureIs the protein
still the same when we cut it in half?
RPA70

• Chemical shift is extremely sensitive
• If peaks are the same, structure is the same
• But, if peaks are different, differences not
  directly interpretable

15N
15N
15N
2
2
3
1H
3
1
1
1H
1H
Same idea for comparing mutants or homologs
Arunkumar et al., JBC (2003)
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Biochemical Assay of MutationsMutations can
effect folding and stability
Wild-type
Partially destabilized
Partially destabilized hetero-geneous
Unfolded
Ohi et al., NSB (2003)
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Biochemical Assay of MutationsWhat is the cause
of the Prp19-1 defect?
Not perturbation at binding interface
? Destabilized U-box leads to drop in activity
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)
• Identify binding interfaces

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Monitoring Binding Events
Titration monitored by 15N-1H HSQC

• NMR Provides
• Site-specific
• Multiple probes
• In-depth information
• Spatial distribution of responses can be mapped
  on structure

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Binding Constants From NMR
Stronger
Weaker
Molar ratio of d-CTTCA
Fit change in chemical shift to binding equation
Arunkumar et al., JBC (2003)
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Probing Protein InteractionsStructure is the
Starting Point!
Mer et al., Cell (2000)
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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 gt 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 of chemical shift perturbations on the
structure of RPA32C
Mer et al., Cell (2000)
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Map Site for RPA32C on XPA

• Same residues bind to peptide and protein
• Same binding site
• Slower exchange for peptide
• Kd lt 1 mM

XPA1-98 domain
XPA29-46 peptide
Mer et al., Cell (2000)
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Manual Database Search Predicts Binding Sites in
Other DNA Repair Proteins
XPA29-46 UDG79-88 RAD257-274
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
Mer et al., Cell (2000)
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All Three Proteins Bind to RPA32CBinding Sites
are Identical
UDG79-88
RAD257-274
XPA29-46
Mer et al., Cell (2000)