Biomolecular Nuclear Magnetic Resonance Spectroscopy

FROM ASSIGNMENT TO STRUCTURE

Sequential resonance assignment strategies

NMR data for structure determination

Structure calculations

Properties of NMR structures

01/26/04

Basic Strategy to Assign Resonances in a Protein

1. Identify resonances for each amino acid

2. Put amino acids in order- Sequential assignment (R-G-S,T-L-G-S)- Sequence-specific assignment

1 2 3 4 5 6 7

R - G - S - T - L - G - S

LT G S S R G

Homonuclear 1H Assignment Strategy

• Scalar coupling to identify resonances, dipolar couplings to place in sequence

• Based on backbone NH (unique region of spectrum, greatest dispersion of resonances, least overlap)

• Concept: build out from the backbone to identify the side chain resonances

• 2nd dimension resolves overlaps, 3D rare

1H 1H 1H

Step 1: Identify Spin System

COSY: One coupling

H

N—C

H

A

B

C

H

N—C

H

H

N—C

H

R-COSY: Add A 2nd Coupling

H

N—C—C

H

A

B

C

H

N—C—C

H

H

N—C—CH3

H

H

H

DR-COSY: Add A 3rd Coupling

H

N—C—C

H

A

B

C

H

N—C—C

H

H

N—C—CH3

H

H

H

H

H

TOCSY: All Coupled Spins

H

N—C—C—C—COOH

H H H

H H

H

N—C—C—C—C—C—NH3

H H H H H

H H H HH

N—C—CH3

H

A

B

C

Step 2: Fit Residues in Sequence

Peaks in NOESY spectra

Same as scalar coupling peaks

Peaks from residue i to i+1

A - B - C

A B (B C)

A Minor Problem With NOESYMany Types of NOEs

A B C D Z• • • • Intraresidue

Sequential

Medium-range(helices)

Long Range

Use only these to make sequential assignments

Extended Homonuclear 1H Strategy

• Same basic idea as 1H strategy: based on backbone NH

• Concept: when backbone 1H overlaps disperse with backbone 15N

• Use Het. 3D to increase signal resolution

1H 1H 15N

15N Dispersed 1H-1H TOCSY

3 overlapped NH resonanceswith different side chains

Add a 3rd dimension separating out HN overlaps by their 15N frequency

15N Dispersed 1H-1H TOCSY

3 overlapped NH resonances

Same NH, different 15N

F1

F2F3

1H 1H 15Nt1 t2 t3

TOCSY HSQC

Heteronuclear (1H,13C,15N) Strategy

• One bond at a time - all atoms (except O)

• Even handles backbone 15N1H overlaps disperse with backbone

C’CHCH…

• Het. 3D/4D increases signal resolution

1H 13C 15N 1H

• Works on bigger proteins because one bond scalar couplings are larger

Heteronuclear Assignments:Backbone Experiments

Names of scalar experiments based on atoms detected

Consecutive residues!!NOESY not needed

Heteronuclear Assignments:Side Chain Experiments

Multiple redundancies increase reliability

Tutorial on the website

Heteronuclear Strategy: Key Points

• Bonus: amino acid identification and sequential assignments all at once

• Most efficient, but expts. more complex

• Enables study of much larger proteins (TROSY/CRINEPT 1 MDa: e.g. Gro EL)

• Requires 15N, 13C, [2H] enrichment High expression in minimal media (E. coli) Extra $ ($150/g 13C-glucose, $20/g 15NH4Cl)

Structure Determination by NMRStructure Determination by NMR

NMR Experimental Observables Providing Structural Information

• Backbone conformation from chemical shifts (Chemical Shift Index- CSI): ,

• Distance restraints from NOEs

• Hydrogen bond restraints

• Backbone and side chain dihedral angle restraints from scalar couplings

• Orientation restraints from residual dipolar couplings

11H-H-11H Distances From NOEsH Distances From NOEs

A B C D Z• • • • Intraresidue

Sequential

Medium-range(helices)

Long-range(tertiary structure)

Challenge is to assign all peaks in NOESY spectra

Approaches to Identifying NOEsApproaches to Identifying NOEs

• 15N- or 13C-dispersed 1H-1H NOESY 3D 1H

13C

1H1H15N

1H

1H15N

1H

13C

1H13C

1H

13C

1H15N

1H

15N

4D

1H 1H2D

1H 1H 1H3D

• 1H-1H NOESY

Special NOESY ExperimentsSpecial NOESY Experiments

•Filtered, edited NOE: based on selection of NOEs from two molecules with unique labeling patterns.

1H 1H

13CUnlabeled

peptide

Labeledprotein

Only NOEs at the interface

Only NOEs from bound state

H

H

HH

kon

koff

•Transferred NOE: based on 1) faster build-up of NOEs in large versus small molecules; 2) Fast exchange 3) NOEs of bound state detected at resonance frequencies of free state

Backbone Hydrogen BondsBackbone Hydrogen Bonds

• NH chemical shift at low field (high ppm)

• Slow rate of NH exchange with solvent

• Characteristic pattern of NOEs

• (Scalar couplings across the H-bond)

When H-bonding atoms are known can impose a series of distance/angle constraints to enforce standard H-bond geometries

C=O H-N

• • • •6 Hz

Dihedral Angles FromScalar Couplings

Must accommodate multiple solutions multiple J valuesBut database shows few occupy higher energy conformations

Orientational Constraints From Residual Dipolar Couplings (RDCs)

Requires medium to partially align molecules Must accommodate multiple solutions multiple orientations

1H

15N

1H

15N

Ho

F1

F2F3

1H13C

1H

1H

Reports angle of inter-nuclear vector relative to magnetic field Ho

NMR Structure Calculations

• Objective is to determine all conformations consistent with the experimental data

• Programs that only do conformational search lead to bad chemistry use molecular force fields improve molecular properties Some programs try to do both at once Need a reasonable starting structure

• NMR data is not perfect: noise, incomplete data multiple solutions (conformational ensemble)

Variable Resolution of Structures

• Secondary structures well defined, loops variable

• Interiors well defined, surfaces more variable

• Trends the same for backbone and side chains More dynamics at loops/surface Constraints in all directions in the interior

Restraints and Uncertainty

Large # of restraints = low values of RMSD

Large # of restraints for key hydrophobic side chains

Assessing the Qualityof NMR Structures

• Number of experimental constraints

• RMSD of structural ensemble (subjective!)

• Violation of constraints- number, magnitude

• Molecular energies

• Comparison to known structures: PROCHECK

• Back-calculation of experimental parameters

Read the book chapter!