Applications of NMR spectroscopy in structural biology

- protein structure determination- ligand screening for drug discovery- analysis of mobility in proteins and protein-ligand interactions- macromolecular complexes- protein folding- imaging

Protein mobility

Proteins are not always rigid.In this example, the protein structure changes in theleft part upon phosphorylation (=attachment of a phosphate group to the side chain of a specific amino acid).

The picture shows the superposition of the backbonestructure of the proteins with and without phosphate bound. The structures were determined by NMR.

Science 291, 2429 (2001)

Protein structure determination

This example shows that two proteinscan have a ‘random coil’ conformation,yet bind specifically to each other and,when they bind, assume a definedthree-dimensional structure.Spectra a and b are 15N-HSQC spectraof the individual proteins (black) and thesame proteins in the complex.In a, protein A had been labelled with15N, but not protein B. (so only peaks ofProtein A are visible). In b, protein B hadBeen labelled with 15N, but not protein A(so only peaks of protein B are visible).

In both spectra, the narrow distribution of1H chemical shifts in the free proteins (blackpeaks) indicates ‘random coil’ conformation.The much wider distribution of 1H chemicalshifts in the complex (red peaks) ischaracteristic of a folded protein.

How to label a protein with 15N and 13C:

Express the protein in E. coli, using a medium containing 15NH4Cl as the only nitrogen source and13C-glucose as the only carbon source. It’s straightforward and not too expensive.

Why label?

The most abundant isotopes of nitrogen and carbon are 14N (>99%) and 12C (>98%), but 14N has a spin 3/2which means that its nuclear magnetization relaxes too quickly to be useful for NMR, and 12C has spin 0, i.e.no magnetic moment at all. 15N and 13C are non-radioactive, naturally occurring isotopes (0.3% of nitrogenand 1% of carbon). They have a spin ½ (like 1H) and are great for NMR. Compounds enriched to >99% with these rare isotopes are commercially available.

Labelling with 15N and 13C allows to record 3D and 4D NMR spectra efficiently which yield better resolution than 2D NMR spectra. Hence, larger proteins can be studied by NMR, if the proteins are labelled.

Ligand screening for drug development

Recipe: -label protein with 15N-record 15N-HSQC spectra with and without ligand-the shifted peaks are from those amino acids for which the chemical environment is changed by the presence of the ligand = great method for the identification of ligand binding sites

Amino-acid selective labelling of proteinsWhy:The assignment of cross-peaks in 15N-HSQC spectrum to individual amino-acid residues in the protein takes time,If the protein is uniformly labelled with 15N. With selectivelabelling, individual cross-peaks can be assigned veryquickly.

How:Express protein with a mixture of amino acids, only one ortwo of them isotope labelled.

Example:PpiB is an enzyme in E. coli which isomerizes peptide bondsinvolving proline. The amino acid sequence is shown below,the 15N-HSQC spectrum of the uniformly labelled proteinat the left.Labelling with 15N-arginine (commercial compound) resultsin a 15N-HSQC spectrum with only 5 cross-peaks.

15N-HSQC spectrum of 15N-arginine labelled PpiB

It’s a much simpler spectrum, but westill need to know which peak belongsto which arginine in the amino acidsequence.

Assignment of the 15N-HSQC cross-peak of Arg87 by double-selective labelling

Recipe:-protein made with 15N-arginine (R) and 13C-alanine (A)-only Arg87 is preceded by an alanine, i.e. only the 15N of Arg87 couples to 13C-an HNCO spectrum transfers magnetization selectively from HN to 15N and 13C, and contains only one cross-peak

HN-15N cross peaks in 15N-arginine labelled PpiB

HN-15N cross peak of Arg87 in 15N-arginine/13C-alaninedouble-labelled PpiB

Addition of a ligand (signals from the ligand are circled).Only the cross-peak of Arg87 shifts, showing that theligand binds near Arg87.

HSQC

HNCO

HSQC+ ligand

Arg87

Screening for protein-binding ligands, if the protein is very large

Transverse magnetization relaxes much faster for systems of high molecular weight than for small molecules. Fast relaxationmeans broad NMR signals that disappear during longer pulse sequences. In these NMR spectra, the protein signal has been suppressed intentionally (by relaxation), so that only the signals from the ligand cocktail are left.

Quart. Rev. Biophys. 32, 211 (1999)

Mixture of 9 small molecules

Mixture of 9 small molecules + protein. The signals of the molecule binding to the protein disappeared, because the magnetization from small molecules relaxes like the protein, once the molecules are bound.

Difference between (a) and (b)

NMR spectrum of pure compound

Structures of protein-ligand complexes

A is a proteinB is a ligand

If we know where B binds to A (e.g. from chemicalshift changes observed between A with and withoutligand), we still don’t know the orientation of B with respect to A.

Attachment of a paramagnetic ion to A causes enhancedrelaxation (and therefore signal broadening) of the protonsnearby.

Example: protein-DNA complexJ. Am. Chem. Soc. 125, 6634 (2003)

in which orientation does the protein bind to DNA?

recipe: synthesize DNA with a chemical groupwhich binds metal ions (here: EDTA). Twodifferent DNA molecules were synthesized, withthe EDTA group at opposite ends (green and red).

Ca2+ is not paramagnetic.Mn2+ is paramagnetic.15N-HSQC spectra of the 15N-labelled protein showthat some cross-peaks broaden in the presence of Mn2+.

Hydration dynamics

J. Am. Chem. Soc. 111, 1871 (1989)andScience 254, 974 (1991)

NOESY experiments have shown that the residence times of hydration water molecules on protein surfacesis much shorter than 500 ps. Therefore, protein solvation presents no kinetic hindrance to protein function.

Here is the proof:Part of a NOESY spectrum recorded with a special type of water suppression, so that NOEs betweenthe water protons and the protein protons can be observed:

position of thewater resonance

Surprisingly few cross-peaks are observed between the water and the protein, because only those NOEs show up which are to water molecules buried deeply inside the protein structure.

NOEs with surface hydration water can be observed in a peptide which is too small to have internal water molecules.The NOESY cross-peaks with water are weak and negative even under conditions, where all intra-peptide cross-peaksare positive. NOE theory shows that negative cross-peaks occur only within small molecules or for very short-livedintermolecular interactions.

Cross-section through NOESY cross-peaks with water

Conventional 1H NMR spectrum

Protein folding

Protein folding can be studied by NMR spectroscopy in different ways (Acc. Chem. Res. 31, 773 (1998)). Here is one:- freeze-dry protein- redissolve in 100% D2O- measure NMR spectra of amide protons as a function of timeThe signal intensity of the amide protons will go down as they exchange with deuterium fromthe solvent. Even the amide protons most deeply buried in the interior of the protein structureexchange with time, because protein structures unfold every now and again, exposing theamides to the solvent. This amide proton exchange experiment thus yields information aboutthe most stable and least stable parts of the structure. Under suitable conditions, the hydrogen exchanges each time the protein unfolds. In this way the frequency of unfolding events can be measured.

HN exchange experiment, monitored by a series of 2D COSY spectra

J. Mol. Biol. 160, 343 (1982)

Imaging (MRI)

Anatomical images

Usually, only the 1H NMR spectrum of water is recorded; the contrast in the images is based ondifferent water properties: concentration,T1 relaxation time, T2 relaxation time, flow rate.

recipe:take the difference between two images which arerecorded while the subject does and does not perform a certain task (e.g. finger tapping)

the way it works:Blood vessels widen in brain areas of increased activity.With the in-flow of blood, concentrations of oxygenatedhemoglobin rise. The iron in hemoglobin is paramagnetic.The 1H NMR signal of water relaxes more quickly in thePresence of a paramagnetic ion.

Functional imaging by MRI

Angiography by MRI

recipe:inject a paramagnetic compound into the blood vesselto enhance the relaxation rate of water in the blood

Principle of MRI

Record 1H NMR spectrum of water in the presence of a magnetic field gradient. This means that the magneticfield B0 (and, hence, the Larmor frequency of the protons) varies as a function of coordinates -> the frequencyspectrum shows the water distribution as a function of coordinates.

Simple pulse sequence to image a 2D plane:

- a frequency-selective pulse applied during a B0 gradient in the z-direction excites only spins with a certain z-coordinate- recording the FID in the presence of a B0 gradient in the y-direction yields the water signal intensity as a function of the y-coordinate- the experiment is performed as a 2D experiment, where the intensity of a B0 field gradient in the x-direction is systematically increased from FID to FID. (Increasing the gradient strength is equivalent to increasing its duration, but not its strength.)- 2D FT yields a picture of the water distribution in the xy-plane.

A mathematical description can be found at http://dutnsic.tn.tudelft.nl:8080/c59_to_html/node45.html