Biomolecular Nuclear Magnetic Resonance Spectroscopy BIOCHEMISTRY BEYOND STRUCTURE Protein dynamics...
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Transcript of Biomolecular Nuclear Magnetic Resonance Spectroscopy BIOCHEMISTRY BEYOND STRUCTURE Protein dynamics...
Biomolecular Nuclear Magnetic Resonance Spectroscopy
BIOCHEMISTRY BEYOND STRUCTURE
• Protein dynamics from NMR
• Analytical Biochemistry
• Comparative Analysis
01/22/03
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
• Number of signals per atom: multiple signals
for slow exchange between conformational states
A B
Populations ~ relative stability
Rex < (A) - (B)
Rate
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 B
1H
1H
15N
15N
1H
15N
Same shifts, same structure
Linewidth determined by size of particle
Fragments have narrower linewidths
Detecting Functionally Independent Domains in Multi-Domain Proteins
Why?
Flexibility facilitates interactions with protein targets
RPA32
RPA14
173
P
40
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, s-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 OriginOf Structural Uncertainty
Measurements show if high RMSD is due to high flexibility (low S2)
Strong correlation
Weak correlation
Analytical Protein Biochemistry
•Purity (1-2%)- heterogeneity, degradation, buffer
•Check on sequence (fingerprint regions)
•Binding constants, off rates, on rates
Protein Fingerprints
Assay structure from residue counts in each fingerprint
1515N-N-11H HSQCH HSQC11H COSYH COSY
13C HSQC also!
Monitoring Binding Events
NMR Provides
Site-specific
Multiple probes
In-depth information
Spatial distribution of responses can be mapped on structure
Titration followed by 15N-1H HSQC
Binding Constants From NMR
Fit change in chemical shift to binding equation
Molar ratio of d-CTTCA
Stronger Weaker
Comparative Analysis
•Different preparations, chemical modifications
•Conformational heterogeneity (e.g. cis-trans isomerization)
•Homologous proteins, mutants, engineered proteins
Comparative Analysis of StructureIs the protein still the same when we cut it in half?
1H
1H
15N
15N
1H
15N
A B
RPA70
AB
3
1 1
2 23
If the peaks are in the same place, the structure is the same
Same idea for comparing mutants or homologs
Biochemical Assay of MutationsMutations can effect folding and stability
Wild-type
Partially destabilized
& hetero-geneous
Partially destabilized
Unfolded
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
Probing Binding of Protein TargetsStructure is the Starting Point!
C
N
Winged Helix-Loop-Helix
Mer et al., Cell (2000)
• Only 19 residues affected Discrete binding site
• Signal broadening exchange between the bound and un-bound state Kd > 1 M
RPA32CRPA32C + XPA 1-98
Probe Binding Events by NMR15N-RPA32C + Unlabeled XPA1-98
1515N-N-11H HSQCH HSQC
NMR Identification of theXPA Binding Site on RPA32C
C
N
Map of chemical Map of chemical
shift perturbations shift perturbations
on the structure of on the structure of
RPA32CRPA32C
XPA1-98
domainXPA29-46
peptide
• Same residues bind to peptide and protein
Same binding site
• Slower exchange for peptide
Kd < 1 M
Localization of Binding Site
Manual Database Search Predicts Binding Sites in Other
DNA Repair Proteins
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
XPA29-46
UDG79-88
RAD257-274