Transcript Center for Structural Biology

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?
• 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)
Characterizing Protein Dynamics:
Parameters/Timescales
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 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
A
B
A
15N
B
Linewidth
determined by
size of particle
15N
15N
Fragments
have narrower
linewidths
1H
1H
1H
Arunkumar et al., JBC (2003)
Detecting Functionally Independent
Domains in Multi-Domain Proteins
40
RPA32
173
P
RPA14
> 300 residues / ~80 signals
Why?
 Flexibility facilitates
interactions with protein
targets
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 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

Weak correlation
Strong correlation 


Measurements
show if high RMSD
is due to high
flexibility (low S2)
Analytical Protein Biochemistry
•Purity (can detect >99%)- heterogeneity,
degradation, buffer
•Check on sequence (fingerprint regions)
Protein Fingerprints
15N-1H
HSQC
13C
1H
COSY
HSQC also!
Assay structure from residue counts in each fingerprint
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?
A
B
A
B
RPA70
15N
Chemical shift
is extremely
sensitive
15N
15N
2
3
2
1H
3
1
1
1H
1H
If peaks are the
same, structure
is the same
But, if peaks are
different,
differences not
directly
interpretable
Same idea for comparing mutants or homologs
Arunkumar et al., JBC (2003)
Biochemical Assay of Mutations
Mutations can effect folding and stability
Wild-type
Partially
destabilized
& heterogeneous
Partially
destabilized
Unfolded
Ohi et al., NSB (2003)
Biochemical Assay of Mutations
What 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
• Monitor the binding of molecules
• Determine binding constants (discrete
off rates, on rates)
• Identify binding interfaces
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
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
Mer et al., Cell (2000)
Probe Binding Events by NMR
15N-RPA32C
15N-1H
+ Unlabeled XPA1-98
HSQC
• Only 19 residues affected
 Discrete binding site
• Signal broadening 
exchange between the
bound and un-bound state
RPA32C
RPA32C + XPA 1-98
 Kd > 1
mM
Mer et al., Cell (2000)
Map XPA Binding Site on
RPA32C Using NMR
Map of chemical
shift perturbations
C
on the structure of
RPA32C
N
Mer et al., Cell (2000)
Map Site for RPA32C on XPA
• Same residues bind
to peptide and protein
 Same binding site
• Slower exchange for
peptide
XPA1-98
domain
XPA29-46
peptide
 Kd < 1 mM
Mer et al., Cell (2000)
Manual Database Search
Predicts Binding Sites in Other
DNA Repair Proteins
XPA29-46
E R K R Q R A L M L R Q A R L A A R
UDG79-88
R I Q R N K A A A L L R L A A R
RAD257-274
R K L R Q K 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
XPA29-46
UDG79-88
RAD257-274
Mer et al., Cell (2000)