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01/28/04. Biomolecular Nuclear Magnetic Resonance Spectroscopy. BIOCHEMISTRY BEYOND STRUCTURE Protein dynamics from NMR Analytical biochemistry Comparative analysis Interactions between biomolecules. Tutorial on resonance assignments (see the website). Why The Interest In Dynamics? .

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biomolecular nuclear magnetic resonance spectroscopy

01/28/04

Biomolecular Nuclear Magnetic Resonance Spectroscopy

BIOCHEMISTRY BEYOND STRUCTURE

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

Tutorial on resonance assignments (see the website)

why the interest in dynamics
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)
dynamics from nmr parameters
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 < w (A) - w (B)

Rate Estimates

A

B

  • Multiple states are hard to detect by Xray crystallography
dynamics from nmr parameters5
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
linewidth is dependent on mw

B

A

B

A

15N

15N

15N

1H

1H

1H

Linewidth is Dependent on MW
  • Linewidth determined by size of particle
  • Fragments have narrower linewidths

Arunkumar et al., JBC (2003)

detecting functionally independent domains in multi domain proteins

40

173

P

Detecting Functionally Independent Domains in Multi-Domain Proteins

RPA32

RPA14

> 300 residues / ~80 signals

Why?

  • Flexibility facilitates interactions with protein targets
dynamics from nmr parameters8
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
dynamics from nmr parameters9
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)
dynamics to probe the origin of structural uncertainty
Dynamics To Probe The OriginOf Structural Uncertainty

Weak correlation

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

Strong correlation

analytical protein biochemistry
Analytical Protein Biochemistry
  • Purity (can detect >99%)- heterogeneity, degradation, buffer
  • Check on sequence (fingerprint regions)
protein fingerprints
Protein Fingerprints

1H COSY

15N-1H HSQC

13C HSQC also!

Assay structure from residue counts in each fingerprint

comparative analysis
Comparative Analysis
  • Different preparations, chemical modifications
  • Conformational heterogeneity (e.g. cis-trans isomerization)
  • Homologous proteins, mutants, engineered proteins
comparative analysis of structure is the protein still the same when we cut it in half

B

A

B

A

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)

biochemical assay of mutations mutations can effect folding and stability
Biochemical Assay of MutationsMutations can effect folding and stability

Wild-type

Partially destabilized

Partially destabilized & hetero-geneous

Unfolded

Ohi et al., NSB (2003)

biochemical assay of mutations what is the cause of the prp19 1 defect
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)

nmr to study interactions
NMR to Study Interactions
  • Monitor the binding of molecules
  • Determine binding constants (discrete off rates, on rates)
  • Identify binding interfaces
monitoring binding events
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
binding constants from nmr
Binding Constants From NMR

Stronger

Weaker

Molar ratio of d-CTTCA

Fit change in chemical shift to binding equation

Arunkumar et al., JBC (2003)

probing protein interactions structure is the starting point

C

N

Winged Helix-Loop-Helix

Probing Protein InteractionsStructure is the Starting Point!

Mer et al., Cell (2000)

probe binding events by nmr 15 n rpa32c unlabeled xpa 1 98
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)

map xpa binding site on rpa32c using nmr

C

N

Map XPA Binding Site on RPA32C Using NMR

Map of chemical shift perturbations on the structure of RPA32C

Mer et al., Cell (2000)

map site for rpa32c on xpa
Map Site for RPA32C on XPA
  • Same residues bind to peptide and protein
    • Same binding site
  • Slower exchange for peptide
    • Kd < 1 mM

XPA1-98 domain

XPA29-46 peptide

Mer et al., Cell (2000)

manual database search predicts binding sites in other dna repair proteins
Manual Database Search Predicts Binding Sites in Other DNA Repair Proteins

XPA29-46

UDG79-88

RAD257-274

E R K RQR A L ML R QA R L A A R

R I Q RNK A A AL L RL A A R

R K L RQK Q L Q Q Q F R E R M E K

Mer et al., Cell (2000)

all three proteins bind to rpa32c binding sites are identical
All Three Proteins Bind to RPA32CBinding Sites are Identical

UDG79-88

RAD257-274

XPA29-46

Mer et al., Cell (2000)