NMR in macromolecole biologiche

The amount of shielding the nucleus experiences will vary with the density of the surrounding electron cloud If a 1 H nucleus is bound to a more electronegative atom e.g. N or O as opposed to C, the density of the electron cloud will be lower and it will be less shielded or “deshielded”. These considerations extend beyond what is directly bonded to the H atom as well. Simple shielding effects--electronegativity N H C H more electron withdrawing-- less shielded less electron withdrawing-- more shielded

less shielded higher resonance frequency more shielded lower resonance frequency amides (HN) aliphatic/alpha/beta etc.(HC) most HN nuclei come between 6-11 ppm while most HC nuclei come between -1 and 6 ppm. Simple shielding effects--electronegativity

One consequence of these effects is that aromatic protons, which are attached to aromatic rings, are deshielded relative to other HC protons. In fact, aromatic ring protons overlap with the amide (HN) region. aromatic region (6-8 ppm) amide region (7-10 ppm) More complex shielding effects: Aromatic protons

It should now be apparent to you that different types of proton in a protein will resonate at different frequencies based on simple chemical considerations. For instance, H  protons will resonate in a region centered around the relatively high shift of 4.4 ppm, based on the fact that they are adjacent to a carbonyl and an amine group, both of which withdraw electron density. But not all H  protons resonate at 4.4 ppm: They are dispersed as low as ~3 and as high as ~5.5. Why? “H  region”

“Average” or “random coil” chemical shifts in proteins One reason for this dispersion is that the side chains of the 20 amino acids are different, and these differences will have some effect on the H  shift. The table at right shows “typical” values observed for different protons in the 20 amino acids. These were measured in unstructured peptides to mimic the environment experienced by the proton averaged over essentially all possible conformations. These are sometimes called “random coil” shift values. Note that the H  shifts range from ~4-4.8, but H  shifts in proteins range from ~3 to 5.5. So this cannot entirely explain the observed dispersion.

Amino acid structures and chemical shifts note: the shifts are somewhat different from the previous page because they are measured on the free amino acids, not on amino acids within peptides

Tabella 1 H chemical shift in peptidi e proteine

chemical shifts in proteins. Secondary structure Note that the H a shifts range from ~4-4.8, but H a shifts in proteins range from ~3 to 5.5. So this cannot entirely explain the observed dispersion.

A simple reason for the increased shift dispersion is that the environment experienced by 1 H nuclei in a folded protein (B) is not the same as in a unfolded, extended protein or “random coil” (A). shift of particular proton in folded protein influenced by groups nearby in space, conformation of the backbone, etc. Not averaged among many structures because there is only one folded structure. So, some protons in folded proteins will experience very particular environments and will stray far from the average. shift of particular proton in unfolded protein is averaged over many fluctuating structures will be near random coil value

Example: shielding by aromatic side chains in folded proteins Picture shows the side chain packing in the hydrophobic core of a protein--the side chains are packed in a very specific manner, somewhat like a jigsaw puzzle a consequence of this packing is that some protons may be positioned within the shielding cone of an aromatic ring such as Phe 51. Such protons will exhibit unusually low resonance frequencies (see picture at left). Note that such effects depend upon precise positioning of side chains within folded proteins + + shielded methyl group methyl region of protein spectrum

poorly dispersed amides poorly dispersed aromatics poorly dispersed alphas poorly dispersed methyls very shielded methyl unfolded ubiquitin folded ubiquitin so you can tell if your protein is folded or not by looking at the 1D spectrum...

What specifically to look for in a nicely folded protein notice aromatic/amide protons with shifts above 9 and below 7 notice alpha protons with shifts above 5 notice all these methyl peaks with chemical shifts around zero or even negative

Linewidths in 1D spectra: aggregation and conformational flexibility Linewidths get broader with larger particle size, due to faster transverse relaxation rates. We’ll learn the physical basis for the faster relaxation later. Broader than expected linewidths can indicate that the protein is aggregated. It can also indicate that the protein has conformational flexibility, i.e. that its structure is fluctuating between several slightly different forms. We’ll learn why this is when we cover the effect of protein dynamics on NMR spectra. Conformational flexibility also tends to reduce dispersion by averaging the environment experienced by a nucleus.

An example of analyzing linewidths and dispersion: Hill & DeGrado used measurements of chemical shift dispersion and line broadening in the methyl region of 1D spectra to gauge the effect of mutations at position 7 on the conformational flexibility of  2D protein leucine and valine mutants have poor dispersion and broad lines, despite being very stably folded and not aggregated (circular dichroism and analytical ultra- centrifugation measurements). These mutants are folded but flexible. Hill & DeGrado (2000) Structure 8:

Trasformata di Fourier

F(t) F( ) Trasformata di Fourier

Esperimento NMR L’effetto di un IMPULSO è di portare il sistema fuori dall’equilibrio La magnetizzazione di H 2 O è ruotata. Tanto piu’ lungo è l’impulso applicato tanto maggiore sarà la rotazione

Esperimento NMR Il segnale osservato nell’esperimento NMR è il segnale che si trova sul piano xy

The NMR Experiment After the pulse is switched off, the magnetization precesses in the xy plane and relaxes to equilibrium The current induced in a coil by the magnetization precessing in the xy plane is recorded. It is called FID. zy xzy xzy x M B1B1B1B1 90°t IIII I t To have a spin transition, a magnetic field B 1, oscillating in the range of radiofrequencies and perpendicular to z, is applied (perturbing pulse) The B 1 field creates coherence among the spins (they all have the same phase) and net magnetization in the x,y plane is created

FT relax. PreparationDetection x y z t2t2 00

FOURIER TRANSFORMATIONS F( )=  ( 0 ) F( )=A  (sin  )/  centered at 0 F( )=T 2 /1+(2  T 2 ) 2 -i 2  (T 2 ) 2 /1+(2  T 2 ) 2 0 F( )=T 2 /1+(2    T 2 ) 2 -i 2    (T 2 ) 2 /1+(2    T 2 ) 2 0 F(t)=exp(-t/T 2 ) F(t)=exp(-t/T 2 )exp(i2  A )

Why bother with FT? FT allows to decompose a function in a sum of sinusoidal function (deconvolution). In NMR FT allows to switch from the time domain, i.e. the signal emitted by the sample as a consequence of the radiofrequency irradiation and detected by the receiving coil to the frequency domain (NMR spectrum) The FT allows to determine the frequency content of a squared function

A “real” F.I.D.

Pulse! -y y The rotation of magnetization under the effect of 90° pulses according to the convention of Ernst et al..

Signal to noise

ScansS/N