Presentation on theme: "LEZIONE 2. Different Isotopes Absorb at Different Frequencies low frequencyhigh frequency 15 N 2H2H 13 C 19 F 1H1H 50 MHz 77 MHz 125 MHz 200 MHz 470 MHz."— Presentation transcript:
Different Isotopes Absorb at Different Frequencies low frequencyhigh frequency 15 N 2H2H 13 C 19 F 1H1H 50 MHz 77 MHz 125 MHz 200 MHz 470 MHz 500 MHz 31 P
Resonance Frequencies Depends on Magnetic Field low field high field 1H1H 200 MHz 400 MHz 600 MHz 700 MHz 800 MHz 950 MHz 1H1H 1H1H 1H1H 1H1H 1H1H
Rapporto giromagnetico E= ħ m B E= ħ B La separazione in energia dipende dal valore del rapporto giromagnetico
La frequenza di precessione di un determinato nucleo ad un determinato campo magnetico è detta FREQUENZA DI PRECESSIONE DI LARMOR Frequenza di precessione 0 = - B 0 /2π Se cosi fosse, ogni nucleo attivo entrerebbe in risonanza con il campo esterno alla sua frequenza e tutti gli isotopi uguali si comporterebbero allo stesso modo (un unico segnale). Es: al campo magnetico di 11.7 T, La FREQUENZA DI PRECESSIONE DI LARMOR del nuclide 1 H è 500 MHz.
La costante di schermo Dipende dallintorno elettronico
Campi magnetici elevati determinano un aumento della risoluzione e della sensibilità
CH 3 H H H (helices) H (sheets) H2OH2O aromatic NH sidechains NH backbone
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 Questo lo hai già visto nella descrizione delle molecole organiche
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
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
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.
Regions of the 1 H NMR Spectrum are Further Dispersed by the 3D Fold
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 Average or random coil chemical shifts in proteins
poorly dispersed amides poorly dispersed aromatics poorly dispersed alphas poorly dispersed methyls very shielded methyl unfolded ubiquitin folded ubiquitin 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. Well 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. Well 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:
13 C NMR The rules discussed for 1 H spins, (shielding and deshielding effects) hold also for 13 C spins. Some general features of 13 C should be pointed out: Unlike 1 H atoms, 13 C atoms may form a different number and type of chemical bonds. Therefore, the so called paramagnetic contributions (see later) are much more effective for deshielding. The chemical shift range of 13 C spins spans more than 200 ppm
Range of observed shifts for 13 C
A protein 13 C NMR spectrum (low resolution) Backbone CO and side chain COO- signals Aromatic signals Aliphatic
13 C NMR The rules discussed for 1 H spins, (shielding and deshielding effects) hold also for 13 C spins. Some general features of 13 C should be pointed out: The amino acid dependence of chemical shift values is stronger for 13 C atoms than in 1 H atoms. Therefore, each amino acid has an almost unique pattern of 13 C chemical shifts
13 C chemical shifts are residue-specific
13 C NMR and Secondary Structure The chemical shift from secondary structure can be used to get the secondary structure arrangement directly from 13 C shifts of Ca, Cb and C spins
Fig. 1. Simulated 13 C chemical-shift distribution of ( a) Ala and (b) Met. () Strand; ( ) coil; ( ) helix.
Use of chemical shifts as source of structural information CSI Molecular fragement replacement (3 to 9 aa) BMRB – Biological Magnetic Resonance Bank A Repository for Data from NMR Spectroscopy on Proteins, Peptides, Nucleic Acids, and other Biomolecules BMRB – Biological Magnetic Resonance Bank A Repository for Data from NMR Spectroscopy on Proteins, Peptides, Nucleic Acids, and other Biomolecules