Biomolecular Nuclear Magnetic Resonance Spectroscopy

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)

Characterizing Protein Dynamics: Parameters/Timescales

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

XPAXPA2929-46UDGUDG79-8879-88 RADRAD257-274257-274

All Three Proteins Bind to RPA32CBinding Sites are Identical