NMR in macromolecole biologiche. The amount of shielding the nucleus experiences will vary with the density of the surrounding electron cloud If a.

NMR in macromolecole NMR in macromolecole biologichebiologiche

The amount of shielding the nucleus experiences will vary with the density of the surrounding electron cloud If a 1H nucleus is bound to a more electronegative atome.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 electronwithdrawing--less shielded

less electronwithdrawing--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 mostHC 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 ina 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 aminoacids 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 Hshifts range from ~4-4.8, but Hshifts 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 theprevious page because they are measured on the free aminoacids, not on amino acids within peptides

Tabella 1H chemical shift in

peptidi e proteine

chemical shifts in proteins. Secondary structure

Note that the Ha shifts range from ~4-4.8, but Ha

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 1H 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 nearrandom coilvalue

Regions of the 1H NMR Spectrumare Further Dispersed by the 3D Fold

What would the unfolded protein look like?

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 methylgroup

methyl regionof protein spectrum

poorlydispersed amides

poorlydispersed aromatics

poorlydispersed alphas

poorlydispersed methyls

very shielded methyl

unfoldedubiquitin

foldedubiquitin

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

noticearomatic/amideprotons withshifts above 9and below 7

notice alpha protonswith shifts above 5

notice all these methyl peaks withchemical shifts around zero or evennegative

Linewidths in 1D spectra: aggregation andconformational 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 poordispersion and broad lines, despite being very stably foldedand not aggregated (circular dichroism and analytical ultra- centrifugation measurements). These mutants are folded but flexible.

Hill & DeGrado (2000) Structure 8: 471-9.

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 H2O è ruotata. Tanto piu’ lungo è l’impulso applicato tanto maggiore sarà la rotazione

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

The NMR Experiment The NMR Experiment After the pulse is After the pulse is switched off, the switched off, the magnetization magnetization

precesses in the xy precesses in the xy plane and relaxes plane and relaxes to equilibriumto equilibrium

The current induced in a coil The current induced in a coil by the magnetization by the magnetization precessing in the xy plane is precessing in the xy plane is recorded. It is called FID.recorded. It is called FID.

zz

yy

xx

zz

yy

xx

zz

yy

xx

MM

BB11

90°90° tt

II

II

tt

2T

t

e

)(

2

1I

To have a spin transition, a To have a spin transition, a magnetic field Bmagnetic field B11 , oscillating , oscillating

in the range of in the range of radiofrequencies and radiofrequencies and

perpendicular to z, is applied perpendicular to z, is applied ((perturbing pulseperturbing pulse) )

The BThe B11 field creates field creates

coherence among the coherence among the spins (they all have spins (they all have

the same phase) and the same phase) and net net magnetizationmagnetization in in

the x,y plane is the x,y plane is createdcreated

FTrelax.

x90

Preparation Detection

x

y

z

x90 t2

0

dte)t(f)(F ti

FOURIER TRANSFORMATIONS

F()=(0)

F()=A(sin)/ centered at 0

F()=T2/1+(2T2)2 -i 2(T2)2/1+(2T2)20

F()=T2/1+(2T2)2 -i 2(T2)2/1+(2T2)20

F(t)=exp(-t/T2)

F(t)=exp(-t/T2)exp(i2A)

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

-y -y

-y -y

-y-y

-y

y

The rotation of magnetization under the effect of 90° pulses according to the convention

of Ernst et al..

Signal to noise

Signal to noiseScans S/N1 1.00 80 8.94 8 2.83 800 28.28 16 4.00