TB3 - NMR Flashcards
How does NMR collect data?
Data collection relies on placing the sample inside a powerful magnet, sending radio frequency signals through the sample, and measuring the absorption of those signals. Depending on the environment of atoms within the protein, the nuclei of individual atoms will absorb different frequencies of radio signals. Furthermore, the absorption signals of different nuclei may be perturbed by adjacent nuclei. This information can be used to determine distances between nuclei and thus give the overall protein structure.
What happens when a nucleus with a magnetic moment enters a strong magnetic field?
it will begin to precess like a spinning top, whereby the magnetic moment wants to align with the magnetic field. If the sample placed in this magnetic field is irradiated with radio waves at the same frequency, an NMR spectrum can be obtained.
What is Larmor precession?
When a nucleus with a nuclear magnetic moment is placed in an external magnetic field, B0, the magnetic field of the nuclei will not simply be oriented opposite to the orientation of the magnetic field. Because the nucleus is rotating, the nuclear magnetic field will instead precess around the axis of the external field vector.
Equation for ω
ω = γ B0
Equation for ΔE
ΔE = hv = ℏ γ B0
How do we observe the magnetic moment?
In order to observe the nuclear magnetization, we want to bring it perpendicular to the applied field. Applying a radio frequency pulse, which is perpendicular to the external magnetic field, can do this. If the pulse has the same frequency as the Larmor frequency of the nuclei, the magnetization can be directed from the direction of the magnetic field to a direction perpendicular to this.
Explain bulk magnetisation.
In general, a typical NMR sample contains nearly Avogadro’s number of spins. In the presence of an external field, each of these dipoles will have random orientations along the x-and y-axes, but one of two orientations along the z-axis. The collective behavior of these spins is given by the vector sum of the individual magnetic vectors, known as the bulk magnetization, M0.
What is the minimum sample required for NMR?
300μM
Explain free induction decay
The nuclear magnetization perpendicular to the magnetic field will decrease with time, partly because the nuclear magnetization gets out of phase and because the magnetization returns to the direction of the external field. This can be measured as a frequency and a decay rate as a function of time, referred to as the FID.
How is sensitivity increased in NMR?
using stronger magnets
How do you go from the FID to an NMR spectrum?
FID is a function of time, so applying a fourier transform converts this to a function of frequency (NMR spectrum).
What is chemical shift?
When a molecule is placed in an external magnetic field, this will induce the molecular electrons to produce local currents. These currents will produce an alternative field, which opposes the external magnetic field. The total effective magnetic field that acts on the nuclear magnetic moment will therefore be reduced depending on the strength of the locally induced magnetic field. This is known as chemical shift.
How does lower shielding impact ppm?
It causes a higher ppm as it’s more exposed to the external magnetic field
Why are additional pulses with differing times introduced in NMR?
These create extra dimensions (time dimensions) to the spectrum.
Define J-coupling
Two nuclei in a molecule, which are connected by one, two, and three bonds, can be seen to be coupling in the NMR spectrum. The coupling is observed by a splitting of the NMR signal.
What is the coupling constant?
The separation between the two components of the split J-coupled signal. Measured in Hz and helps define dihedral angles.
What can coupling constants be used for in protein structure determination?
The three-bond coupling constant depends on the dihedral angle defined by rotation around the middle bond in the coupling system. The J-coupling may also be used to distinguish between trans and gauche conformations.
One particularly important application of the coupling constant is as a measure of the coupling between the Hα and the HN in the peptide backbone. This coupling depends on the φ-angle in the peptide bond. The coupling may also be measured by the coupling constant between the HN and Cβ.
What are COSY spectra?
- ONLY SHOW THROUGH-BOND COUPLINGS
The two-dimensional COSY spectrum is recorded so that the spectrum contains two types of signals: diagonal peaks and off-diagonal peaks, often called cross peaks. The diagonal peaks represent the signals from each of the 1H types in an amino acid. Cross peaks report the couplings between pairs of nuclei.
What are TOCSY spectra?
By recording the correlation NMR spectrum in a way so that all the spins in a spin system of an amino acid are all correlated, you get a more complicated spectrum. However, this type of spectrum becomes very useful in particular in the heteronuclear NMR experiments.
Why do we use 13C and 15N?
By introducing 13C and 15N, the spins in a protein are almost all being connected by one-bond couplings, facilitating the study tremendously.
What is 1H-15N HSQC?
The 1H-15N coupling in the peptide bond is the starting point for the heteronuclear NMR analysis of proteins. This bond is present in every amino acid residue in a protein, except proline residues. The correlation spectroscopy method used to record this coupling is called a 1H-15N HSQC. Each ‘spot’ is an NMR signal representing the 1H-15N coupling from one of the residues in the protein, generating a ‘protein fingerprint’.
What are sequential assignments?
This is a process by which a particular amino acid spin system identified in the spectrum is assigned to a particular residue in the amino acid sequence.
Why can’t dipole-dipole interactions always been seen?
In solution, where there’s molecular motion, these interactions are averaged out so aren’t always observed.
How can we get dipole-dipole interactions back?
In very strong magnetic fields, it’s possible to orient homogenously charged particles. Protein molecules placed in such environments will no longer have free molecular motion and the dipole-dipole interactions will be partially re-established. The degree to which the residual dipolar coupling is re-established can be controlled by the concentration of colloidal solution.
