How NMR is Used for the Study of Bio-macromolecules Analytical biochemistry Comparative analysis Interactions between biomolecules Structure determination.

How NMR is Used for theStudy of Bio-macromolecules

• Analytical biochemistry

• Comparative analysis

• Interactions between biomolecules

• Structure determination

• Biomolecular dynamics from NMR

02/05/10

“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 FoldingDon’t Need Resonance Assignments or Labeling

1D requires only 10-50 M protein concentration

2D Provides A More Detailed Assay

Analyze tertiary structure, check sequence

1515N-N-11H HSQCH HSQC11H COSYH COSY

13C HSQC also!

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 MutationsNMR assays for proper folding/stability

Wild-type

Structural heterogeneity

Partially destabilized

Unfolded

Ohi et al., NSB (2003)

Structural Basis for TS PhenotypeWhat 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 Bindingand 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 PerturbationAre domains packed together or independent?

Chemical shift is extremely sensitive If peaks are the same, structure is the same

If peaks are different, the structure is different but we don’t know how much

1H

1H

15N

15N

1H

15N

A B

RPA70

AB

3

1 1

2 23

Arunkumar et al., JBC (2003)

The Thousand Dollar Pull-down!

BeforeAfter addingbinding partner

Yes, binding did occur - more sensitive than all other methods!

NMR- The Master Spectroscopy

NMR Provides

Site-specific

Multiple probes

Atomic information

Perturbations can be mapped on structure

Structural models of complexes

Titration monitored by 15N-1H HSQC

Key Observations

• Only 19 residues affected Discrete binding site

• Signal broadening exchange between the bound and un-bound state Kd ~ 1 M

RPA32CRPA32C + XPA 1-98

Characterize Binding Events15N-RPA32C + Unlabeled XPA1-98

1515N-N-11H HSQCH HSQC

Mer et al., Cell (2000)

NMR to Map Binding SitesXPA binding site on RPA32C

C

N

Map chemical shift Map chemical shift perturbations on the perturbations on the structure of RPA32Cstructure of RPA32C Can even map Can even map directly on to directly on to sequence with no sequence with no structure!!structure!!

Mer et al., Cell (2000)

Generate Models of Complexes from Chemical Shift Perturbations

Arunkumar et al., NSMB (2005)

RPA32C

SV40 TagOBD

Binding Constants FromChemical Shift Changes

Fit change in chemical shift to binding equation

Molar ratio

Stronger Weaker

Arunkumar et al., JBC (2003)

NMR Structure DeterminationNMR 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 ( 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

• Secondary structures well defined, loops variable

• Interiors well defined, surfaces more variable

• RMSD provides measure of variability/precision (but not accuracy!)

Characteristics of Structures Determined in Solution by NMR

Kordel et al., JMB (1993)

Restraints and Uncertainty

Large # of restraints = low values of RMSD

Kordel et al., JMB (1993)

Assessing the Accuracy and Precisionof 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

1H

1H

15N

A B

15N

1H

15N

Linewidth determined by size of particle

Fragments have narrower linewidths

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

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

Strong correlation

Weak correlation