Transcript NMR Powerpoint 2 - Center for Structural Biology

How NMR is Used for the
Study of Bio-macromolecules
02/05/10
• Analytical biochemistry
• Comparative analysis
• Interactions between biomolecules
• Structure determination
• Biomolecular dynamics from NMR
“Dynamic personalities of proteins”
Henzler-Wildman & Kern
Nature 450, 964-972 (2007)
“Probing ribosome nascent chain complexes
produced in vivo by NMR spectroscopy”
Cabrita, Hsu, Launay, Dobson, Christodoulou
PNAS 106, 22239-22234 (2009)
Analytical Protein Biochemistry
• Purity (can detect >99%)- heterogeneity,
degradation, contamination, 1D
• Is a protein structured?- fast and easy
assay, detects aggregation and folding,
even 1D is effective
• Checks using knowledge of sequence
(fingerprint regions), 2D
NMR Assay of Purity and Folding
Don’t Need Resonance Assignments or Labeling
 1D requires only 10-50 mM protein concentration
2D Provides A More Detailed Assay
15N-1H
HSQC
13C
1H
COSY
HSQC also!
 Analyze tertiary structure, check sequence
Comparative Analysis
• Different preparations, changes in conditions
• Chemical/conformational heterogeneity
(discrete signals for different states)
• Mutants, homologous proteins, engineered
proteins
• Binding of ligands, molecular interactions
Effect of Mutations
NMR assays for proper folding/stability
Wild-type
Structural
heterogeneity
Partially
destabilized
Unfolded
Ohi et al., NSB (2003)
Structural Basis for TS Phenotype
What 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)
NMR to Study Ligand Binding
and Molecular Interactions
• Detect the binding of metals, molecules
• Sequence and 3D structural mapping
of binding sites and molecular
interfaces
• Determine binding constants (discrete
off rates, on rates)
NMR Chemical Shift Perturbation
Are domains packed together or independent?
A
B
A
B
RPA70
15N
 If peaks are the
same, structure
is the same
15N
15N
2
3
2
1H
3
1
1
1H
Chemical shift
is extremely
sensitive
 If peaks are
different, the
structure is
different but we
don’t know how
much
1H
Arunkumar et al., JBC (2003)
The Thousand Dollar Pull-down!
After adding
Before
binding partner
Yes, binding did occur - more sensitive than all other methods!
NMR- The Master Spectroscopy
Titration monitored by 15N-1H HSQC
NMR Provides
 Site-specific
 Multiple probes
 Atomic information
 Perturbations can be
mapped on structure
 Structural models of
complexes
Characterize Binding Events
15N-RPA32C
15N-1H
HSQC
+ Unlabeled XPA1-98
Key Observations
• 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)
NMR to Map Binding Sites
XPA binding site on RPA32C
C
 Map chemical shift
perturbations on the
structure of RPA32C
 Can even map
directly on to
sequence with no
structure!!
N
Mer et al., Cell (2000)
Generate Models of Complexes from
Chemical Shift Perturbations
RPA32C
SV40 Tag
OBD
Arunkumar et al., NSMB (2005)
Binding Constants From
Chemical Shift Changes
Stronger
Weaker
Molar ratio
 Fit change in chemical shift to binding equation
Arunkumar et al., JBC (2003)
NMR Structure Determination
NMR Experimental Observables
Providing Structural Information
• Distances from dipolar couplings (NOEs)
• Orientations of inter-nuclear vectors from
residual dipolar coupling (RDCs)
• Backbone and side chain dihedral angles
from scalar couplings (J)
• Backbone (f,y) angles from chemical
shifts (Chemical Shift Index- CSI, TALOS)
• Hydrogen bonds: NH exchange + NOES, J
NMR Structure Calculations
• Initial search to get a general idea
• Molecular force fields to improve molecular
properties and optimize conformations
• Data are not perfect (noise, incomplete) 
multiple solutions (ensemble)
Final output is an ensemble of conformers,
which together represent the conformational
space consistent with the experimental data
Characteristics of Structures
Determined in Solution by NMR
• Secondary structures well defined, loops variable
• Interiors well defined, surfaces more variable
• RMSD provides measure of variability/precision
(but not accuracy!)
Kordel et al., JMB (1993)
Restraints and Uncertainty
Large # of restraints
= low values of RMSD
Kordel et al., JMB (1993)
Assessing the Accuracy and Precision
of NMR Structures
• Number of experimental restraints (A/P)
• Violation of constraints- number, magnitude (A)
• Comparison of model and exptl. parameters (A)
• Comparison to known structures: PROCHECK (A)
• Molecular energies (?A?, subjective)
• RMSD of structural ensemble (P, biased)
Biomolecular Dynamics from NMR
Why? Function requires motion/kinetic energy
• Characterize protein motions/flexibility
and correlate to function
- Direct coupling to enzyme kinetics
- Action of multi-protein machinery
- Folded vs. unfolded states
- Entropic contributions to binding events
- Uncertainty in NMR/crystal structures
- Calibration of computational methods
Characterizing Protein Dynamics:
Parameters/Timescales
Residual Dipolar Couplings
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)
NMR to Monitor Architectural Remodeling
2H,15N-RPA
(116 kDa)
TROSY-HSQC
Brosey et al., (2009)
Correlating Structure and Dynamics



Weak correlation
Strong correlation 




Measurements
show if high RMSD
is due to high
flexibility (low S2)