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Structural Biology > Biological NMR > Flashcards

Biological NMR Flashcards

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1
Q

Applications

A
  • analytical
  • chemical structure determination
  • 3D structure determination
  • map molecular interactions and dynamics
  • metabolomics
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2
Q

Nuclear Spin

A
  • elementary particles possess a intrinsic angular momentum (spin)
  • spin is quantised and has multiples of 1/2
  • nuclei comprising odd proton/neutron numbers have integral spin quantum number / even has I=0
  • the rest have 1/2 integral spin
  • The overall spin, I, is important. Quantum mechanics tells us that a nucleus of spin I will have 2I + 1 possible orientations. A nucleus with spin 1/2 will have 2 possible orientations
  • charged nucleus rotating with angular frequency creates magnetic field B (behaves like a bar magnet)
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3
Q

Spin Energy Levels

A
  • strength of applied magnetic field determines energy gap of given nuclei
  • nuclei placed in external field has 2 states dependent of relative orientation of spin factor with magnetic field
  • In the absence of an external magnetic field, these orientations are of equal energy. If a magnetic field is applied, then the energy levels split. Each level is given a magnetic quantum number, m.
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4
Q

Larmor Precession of Spin

A
  • magnetic moment associated with spinning spherical charge will precess in external magnetic field
  • The nucleus is spinning on its axis. In the presence of a magnetic field, this axis of rotation will precess around the magnetic field
  • If energy is absorbed by the nucleus, then the angle of precession, q, will change. For a nucleus of spin 1/2, absorption of radiation “flips” the magnetic moment so that it opposes the applied field (the higher energy state)
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5
Q

Macroscopic Magnetization

A
  • large numbers of spin 1/2 nuclei at equilibrium in strong external field
  • net magnetisation along z axis
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6
Q

Relaxation

A
  • nuclei in higher energy state return to lower energy state
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7
Q

Pulse Effect

A
  • net magnetisation shifts towards y axis due to spin flips between 1/2 and -1/2 states until spin precessions about z-axis becomes coherent
  • pulse applied at angle with respect to direction of magnetisation exerts torque and causes rotation about the plane
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8
Q

Pulsed Fourier Transform

A
  • decaying radiofrequency signal generated in receiver coil is called the free induction decay
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9
Q

Spectra Parameters

A
  1. chemical shift
  2. integral
  3. scalar coupling
  4. relaxation times
  5. dipolar coupling
  6. nuclear overhauser effect
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10
Q

Chemical Shift

A
  • information about types of chemical groups
  • electrons associated with atoms circulate about the direction of an applied magnetic field causing a local shielding magnetic field at the nucleus
  • electronegative groups withdraw electrons away from nucleus reducing shielding effect
  • The difference between the applied magnetic field and the field at the nucleus is termed the nuclear shielding.
  • less shielding = higher shift
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11
Q

Shift Equivalence

A
  • nuclei exchangeable by a symmetry operation or rapid exchange on NMR timescale (conformational motion change) have the same chemical shifts
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12
Q

Integrals

A
  • area under peak gives relative numbers of nuclei and proton numbers
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13
Q

Scalar Coupling

A
  • two spin systems
  • adjacent non equivalent spin 1/2 nuclei will experience a spin-spin interaction
  • coupling communicated through electrons (spin 1/2) in bonds
  • indirect spin spin interactions
  • split is equal to n+1 of adjacent nuclei
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14
Q

Exchangeable OH/NH

A
  • biological samples in water show no OH resonance due to rapid exchange with high solvent water
  • samples in organic solvents show OH splitting (add D20)
  • if you dissolve sample in organic solvent with no free exchangeable protons the sample will only exchange with itself
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15
Q

Spin Lattice

A
  • longitudinal relaxation
  • enthalpic recovery of Z magnetization
  • Nuclei in an NMR experiment are in a sample. The sample in which the nuclei are held is called the lattice. Nuclei in the lattice are in vibrational and rotational motion, which creates a complex magnetic field. The magnetic field caused by motion of nuclei within the lattice is called the lattice field. This lattice field has many components. Some of these components will be equal in frequency and phase to the Larmor frequency of the nuclei of interest. These components of the lattice field can interact with nuclei in the higher energy state, and cause them to lose energy (returning to the lower state). The energy that a nucleus loses increases the amount of vibration and rotation within the lattice (resulting in a tiny rise in the temperature of the sample).

The relaxation time, T1 (the average lifetime of nuclei in the higher energy state) is dependant on the magnetogyric ratio of the nucleus and the mobility of the lattice. As mobility increases, the vibrational and rotational frequencies increase, making it more likely for a component of the lattice field to be able to interact with excited nuclei. However, at extremely high mobilities, the probability of a component of the lattice field being able to interact with excited nuclei decreases.

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16
Q

Spin-spin relaxation

A
  • transverse relaxation
  • entropic loss of x,y magnetization
  • Spin - spin relaxation describes the interaction between neighbouring nuclei with identical precessional frequencies but differing magnetic quantum states. In this situation, the nuclei can exchange quantum states; a nucleus in the lower energy level will be excited, while the excited nucleus relaxes to the lower energy state. There is no net change in the populations of the energy states, but the average lifetime of a nucleus in the excited state will decrease. This can result in line-broadening.
17
Q

Dipolar Coupling

A
  • Magnetic dipole–dipole interaction, also called dipolar coupling, refers to the direct interaction between two magnetic dipoles.
  • defined by direct coupling
  • fields of 2 spins directly interaction in solution
18
Q

Nuclear Overhauser Effect

A
  • transfer of nuclear spin polarization from one population of spin-active nuclei (e.g. 1H, 13C, 15N etc.) to another via cross-relaxation
  • the change in the intensity of one spin when the spin transition of another nuclei is perturbed from equilibrium population (by saturation or inversion). Nuclear spin that are coupled through space (due to spatial proximity) give rise to nuclear Overhauser effect (NOE).
19
Q

Carbon-13 NMR

A
  • C13 nuclei split by directly attached protons and those attached to adjacent C atoms
  • directly attached protons couple to carbon
  • saturate H1 signals to remove coupling
20
Q

H1 decoupling

A
  1. broadband: saturates signals to give singlets

2. off-resonance: split only by protons attached directly to them (n+1 peaks)

21
Q

2D NMR

A
  • add second frequency dimension
  • allow spins to share frequencies to they can carry 2 frequencies
  • cross peaks reveal correlation between frequencies on two axes (either through bonds or space)
22
Q

COSY

A
  • 2D H-H correlation spectroscopyuseful method for
  • determining which signals arise from neighboring protons (usually up to four bonds). Correlations appear when there is spin-spin coupling between protons, but where there is no coupling, no correlation is expected to appear
  • useful for resolving overlap
23
Q

NOESY

A
  • 2D NMR spectroscopic method used to identify nuclear spins undergoing cross-relaxation and to measure their cross-relaxation rates.
  • Therefore, the cross peaks of a NOESY spectrum indicate which protons are close to each other in space. In this respect, the NOESY experiment differs from the COSY experiment that relies on J-coupling to provide spin-spin correlation, and whose cross peaks indicate which 1H’s are close to which other 1H’s through the chemical bonds of the molecule.
  • measures coupling irrespective of bonds
24
Q

HMBC

A
  • correlates C13 with H1 spectrum
  • both long range and directly bonded couplings give cross peaks
  • coupling to proton of attached carbon
25
Q

HMQC

A
  • correlates the C13 with H1 spectrum
  • only directly bonded couplings give cross peaks
  • correlates proton to directly attached heteronucleus

Structural Biology (10 decks)

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