NMR – a versatile tool in structural proteomics

NMR basics Protein spectra, multidimensional NMR

Resonance assignments Structure determination

Ligand mappning Folding

Dynamics

NMR fundamentals from a quantum mechanical perspective

Certain atomic nuclei possess a property called ’spin’, characterized by the spin quantum number I.

If we put these nuclei in a magnetic field, they will adopt different states with different energies.

I= ½ gives two states: ψ = α , ψ = β

1H: I=½ 12C: I=0 14N: I=1 16O:I=0 31P: I= ½ 2H: I=1 13C: I= ½ 15N:I= ½ 17O: I=5/2

ε

Increasing magnetic field Bo

Δε = hν Energy difference is small => radio freq

Equipment for NMR

Supraconducting, nitrogein/heliumfilled magnets

Pulse- and wave form generators, gradient unit, general electronics

User terminal, pulse programming unit

Probes

Samples

A spectroskopic method where we can study atoms in various amino acids!

… but how do we know which signals belong to which atom???

SÄTT IN SPEKTRUM

Backbone NH

Aromatic Hs

Hα

Aliphatic Hs

Methyl Hs

Resonance assignment requires evaluation of multidimensional spectra

Folding

Ligand bindning

Structure Dynamics

Nuclear spins affect each other - using 2D experiments we can analyse how this is done!

Magnetisation transfer through space – distance dependent (< 5Å), ~ 1/r6

coherence Spinn relaxation (nOe)

Magnetisation transfer between nuclear neighbors

Magnetic labelling: requires gene technology and bacterial

overexpression 1H: I=½ 12C: I=0 14N: I=1 16O:I=0 31P: I= ½ 2H: I=1 13C: I= ½ 15N:I= ½ 17O: I=5/2

A 2D spectrum of the aromatic part of an organic compound

A 2D NMR NOESY spectrum of a protein depicts intermolecular distances between all protons in the biomolecule.

A protein!

3D-NOESY-HSQC

HSQC-sidan

1HN

1H

15N

1HN

15N

The HSQC experiment can be recorded and analyzed separately.

HSQCs show one peak per amino acid.

HSQC

15N Dispersed 1H-1H TOCSY

3 overlapped NH resonances

Same NH, different 15N

F1

F2 F3

1H 1H 15N

t1 t2 t3

TOCSY HSQC

Basic strategy for NMR assignment of proteins:

1.  Identify NMR-resonances for each amino acid:

2. Put them in order: Sequence-specific assignment

1 2 3 4 5 6 7 R - G - S - T - L - G - S

L T G S S R G

Heteronuclear assignment:

Exempel: HNCA!

Other backbone experiments

Names of experiments relate to magnetization transfer pathways

Multiple redundancies gives plenty of back-check options !

Sidechain assignments are required for structure determination by NOEs

Kurt Wüthrich

Nobel laureate 2002 ETH, Zürich

Secondary structure elements can be identified by unique short distances α-helix

(CαH)i – NHi+3, (i+2, i+4)

(CαH)i - (CβH)i+3

β-sheet

NH – NH across the sheet

CαH – CαH across the sheet

CαHi – NHi+1 along the chain

Ηα-NH

Ηα,NHi+3

Ηα,NHi+2

Ηα,NHi+1 Hα

5 Å

NH

Hα R1

O

NH

Hα R2

O

NH

Hα R3

O

NH

Hα R4

O

NH-Hα

Ηα-NH

Ηα,NHi+3

Ηα,NHi+2

Ηα,NHi+1 Hα

5 Å

NH

Hα R1

O

NH

Hα R2

O

NH

Hα R3

O

NH

Hα R4

O

NH-Hα

Long-range NOEs!

This is the location of the helix in the Grx4- structure!

NMR-structures a family of structures consistent with the NMR-derived experimental constraints set (distances, angles, chemical shifts etc)

First generation structures: ~7 NOEs per residue rmsd 1.5 bb / 2.0 all atoms Second generation structures: ~10 NOEs per residue rmsd 0.9 bb / 1.2 all atoms Third generation structures: ~13 NOEs per residue rmsd 0.7 bb / 0.9 all atoms Fourth generation structures: ~16 or more NOEs per residue rmsd 0.4 bb / 0.9 all atoms rmsd 0.5 for ordered side chains

WR41

C-TmZip

ER14

MMP-1

IL13

FGF-2

WR90

WR64

LC8

ER115

JR19

ZR18

OP3

WR33

ZR31 Z-domain

IR24

Examples of NESG structures

Problem vid högre molekylvikter

•  Långsam rörelse i lösning gör att signalen relaxerar snabbare → dåligt signal-brus och breda signaler

•  Växelverkan mellan relaxerande kärnor ökar relaxationen •  Överlappande toppar även i 2D och 3D spektra

Var går gränsen för NMR på proteiner?

Malate synthase G (MSG) from E. coli - a monomeric 723-residue protein 1531 NOE, 1101 dihedral angles, 415 residual dipolar coupling, and 300

carbonyl shift restraints. Tugarinov, Choy, Orekov and Kay, PNAS 2005

Är subdomänerna rörliga? Kan vi utvärdera det med NMR?

Folded or unfolded protein?

1D-spektra HSQC

Ligand bindning studied in HSQC spectra

Binding of the tumor inhibitor chalcone to the p53 interacting protein MDM2; spectra with different ligand concentration are colored differently

15N-relaxation gives information on dynamics in proteins – active sites are flexible!

DHFR, Dyson,Wright and coworkers

NMR - treats J •  High-resolution structures can be determined

also for non-crystallizable proteins •  Rapid and specific mapping of ligand binding

and protein interactions •  Dynamics studies But: •  Technically advanced •  Specific sample requirements •  Current convenient max size limit around 35

kDa