lezione 2. different isotopes absorb at different frequencies low frequencyhigh frequency 15 n 2h2h...
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LEZIONE 2
Different Isotopes Absorb at Different Frequencies
low frequency high frequency
15N 2H 13C 19F 1H
50 MHz 77 MHz 125 MHz 200 MHz 470 MHz 500 MHz
31P
Resonance Frequencies Depends on Magnetic Field
low field high field
1H
200 MHz 400 MHz 600 MHz 700 MHz 800 MHz 950 MHz
1H 1H 1H 1H 1H
Rapporto giromagneticoE= 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 = - B0 /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 1H è 500 MHz.
La costante di schermo
Dipende dall’intorno elettronico
Campi magnetici elevati determinano un aumento della risoluzione e della sensibilità
Chemical shift
refrefppm
Es: 1= 500.131 MHz 0=500.13 MHz1-0=1000 Hz
= 1000/500.13x106
(ppm)= 2.0
TMS (Tetramethylsilane)
chemical shift
Spettro 1H NMR di Vanillina
750 MHz 1H NMR Spettro di Tyrosine Kinase
1H NMR Spettro di vari solventi
13C NMR del Fullerene (C60)
1H Chemical Shift Table
tot= local + magn+ rc + el + solv
Pople, 1960
Fattori che influenzano il chemical shift
Caratteristiche funzionaliEffetti induttiviEffetti mesomeri
Effetti attraverso lo spazioEffetti paramagnetici
Effetti induttivi
Effetto della Sostituzione sul Chemical Shift
CHCl3 CH2Cl2 CH3Cl 7.26 5.32 3.05 ppm
-CH2-Br -CH2-CH2Br -CH2-CH2CH2Br 3.30 1.69 1.25 ppm
Shoolery Equation
Il chemical shift dipende dalla sommatoria degli effetti di tutti I sostituenti
Effetti Mesomeri
Competizione traeffetto mesomero ed effetto induttivo
3.74
3.93
Fattori che influenzano il chemical shift
Caratteristiche funzionaliEffetti attraverso lo spazioCorrenti d’anello Anisotropia magneticaEffetti sterici
Effetti paramagnetici= el +anis+st
Correnti d’anello
Correnti d’anello
Modelling 1H NMR Spectra of Organic Compounds: Theory, Applications and NMR prediction Software Di Raymond Abraham,Mehdi Mobli
Pople -Dipole model
0.42
Anisotropia di schermo indotta dai legami chimici
Anisotropia
Equazione di Mc Connell
C-H 90
C-C 140
C≡C -340
x 1036 m-3mol-1
=
= cosR
(Heq-Hax)= ca. 0.50 ppm
NMR in macromolecole NMR in macromolecole biologichebiologiche
Legami a idrogeno
CH3OH
5.34 ppm
CH3OHDiluito in CDCl3
1.1 ppm
C6H5OH C6H5OHDiluito in CDCl3
7.45 ppm 4.60 ppm
12.1 ppm
Water
Benzene(d6) 0.5
CCl4 1.1
CDCl3 1.5
THF 2.5
Ac(d6) 2.8
DMSO 3.3
H2O 4.7
EtOD 5.3
Pyr(d5) 5.0
Solvent Shift (ppm)
pH dipendenza
Catena polipeptidica
CH3
HH
H(helices)H(sheets)
H2O
aromatic
NH sidechains
NH backbone
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
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 methylgroup
methyl regionof protein spectrum
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
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
“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.
Regions of the 1H NMR Spectrumare Further Dispersed by the 3D Fold
What would the unfolded protein look like?
Regions of the 1H NMR Spectrum are Further Dispersed by the 3D Fold
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
“Average” or “random coil” chemical shifts in proteins
poorlydispersed amides
poorlydispersed aromatics
poorlydispersed alphas
poorlydispersed methyls
very shielded methyl
unfoldedubiquitin
foldedubiquitin
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.
13C NMR
The rules discussed for 1H spins, (shielding and deshielding effects) hold also for 13C spins. Some general features of 13C should be pointed out:
Unlike 1H atoms, 13C 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 13C spins spans more than 200 ppm
Range of observed shifts for 13C
A protein 13C NMR spectrum (low resolution)
Backbone CO and side chain COO- signals
Aromatic signals
Aliphatic
13C NMR
The rules discussed for 1H spins, (shielding and deshielding effects) hold also for 13C spins. Some general features of 13C should be pointed out:
The amino acid dependence of chemical shift values is stronger for 13C atoms than in 1H atoms. Therefore, each amino acid has an almost unique
pattern of 13C chemical shifts
13C chemical shifts are residue-specific
13C NMR and Secondary Structure
The chemical shift from secondary structure can be used to get the secondary structure arrangement directly from 13C shifts of Ca, Cb and C’ spins
Fig. 1. Simulated 13C chemical-shift distribution of
(a) Ala and (b) Met. (•) Strand; ( ) coil; (
) helix.
13C
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
http://www.bmrb.wisc.edu/
BMRB – Biological Magnetic Resonance Bank
A Repository for Data from NMR Spectroscopy on Proteins, Peptides,
Nucleic Acids, and other Biomolecules
http://www.bmrb.wisc.edu/