An important complementary method for determining
the three-dimensional structures of macromolecules is
nuclear magnetic resonance (NMR). Modern NMR
techniques are being used to determine the structures
of ever-larger macromolecules, including carbohydrates,
nucleic acids, and small to average-sized proteins.
An advantage of NMR studies is that they are carried out on macromolecules in solution, whereas xray
crystallography is limited to molecules that can be
crystallized. NMR can also illuminate the dynamic side
of protein structure, including conformational changes,
protein folding, and interactions with other molecules.
NMR is a manifestation of nuclear spin angular
momentum, a quantum mechanical property of atomic
nuclei. Only certain atoms, including 1H, 13C, 15N, 19F,
and 31P, possess the kind of nuclear spin that gives
rise to an NMR signal. Nuclear spin generates a magnetic
dipole. When a strong, static magnetic field is
applied to a solution containing a single type of macromolecules,
the magnetic dipoles are aligned in the field
in one of two orientations, parallel (low energy) or anti-parallel(high energy). A short (~10 s) pulse of
electromagnetic energy of suitable frequency (the resonant
frequency, which is in the radio frequency
range) is applied at right angles to the nuclei aligned
in the magnetic field. Some energy is absorbed as nuclei
switch to the high-energy state, and the absorption
spectrum that results contains information about
the identity of the nuclei and their immediate chemical
environment. The data from many such experiments
performed on a sample are averaged, increasing
the signal-to-noise ratio, and an NMR spectrum is generated.
1H is particularly important in NMR experiments
because of its high sensitivity and natural abundance.
For macromolecules, 1H NMR spectra can become
quite complicated. Even a small protein has hundreds
of 1H atoms, typically resulting in a one-dimensional
NMR spectrum too complex for analysis. Structural
analysis of proteins became possible with the advent
of two-dimensional NMR techniques. These
methods allow measurement of distance-dependent
coupling of nuclear spins in nearby atoms through
space (the nuclear Overhauser effect (NOE), in a
method dubbed NOESY) or the coupling of nuclear
spins in atoms connected by covalent bonds (total correlation
spectroscopy, or TOCSY).
Translating a two-dimensional NMR spectrum into
a complete three-dimensional structure can be a laborious
process. The NOE signals provide some information
about the distances between individual atoms,
the three-dimensional structures of macromolecules is
nuclear magnetic resonance (NMR). Modern NMR
techniques are being used to determine the structures
of ever-larger macromolecules, including carbohydrates,
nucleic acids, and small to average-sized proteins.
An advantage of NMR studies is that they are carried out on macromolecules in solution, whereas xray
crystallography is limited to molecules that can be
crystallized. NMR can also illuminate the dynamic side
of protein structure, including conformational changes,
protein folding, and interactions with other molecules.
NMR is a manifestation of nuclear spin angular
momentum, a quantum mechanical property of atomic
nuclei. Only certain atoms, including 1H, 13C, 15N, 19F,
and 31P, possess the kind of nuclear spin that gives
rise to an NMR signal. Nuclear spin generates a magnetic
dipole. When a strong, static magnetic field is
applied to a solution containing a single type of macromolecules,
the magnetic dipoles are aligned in the field
in one of two orientations, parallel (low energy) or anti-parallel(high energy). A short (~10 s) pulse of
electromagnetic energy of suitable frequency (the resonant
frequency, which is in the radio frequency
range) is applied at right angles to the nuclei aligned
in the magnetic field. Some energy is absorbed as nuclei
switch to the high-energy state, and the absorption
spectrum that results contains information about
the identity of the nuclei and their immediate chemical
environment. The data from many such experiments
performed on a sample are averaged, increasing
the signal-to-noise ratio, and an NMR spectrum is generated.
1H is particularly important in NMR experiments
because of its high sensitivity and natural abundance.
For macromolecules, 1H NMR spectra can become
quite complicated. Even a small protein has hundreds
of 1H atoms, typically resulting in a one-dimensional
NMR spectrum too complex for analysis. Structural
analysis of proteins became possible with the advent
of two-dimensional NMR techniques. These
methods allow measurement of distance-dependent
coupling of nuclear spins in nearby atoms through
space (the nuclear Overhauser effect (NOE), in a
method dubbed NOESY) or the coupling of nuclear
spins in atoms connected by covalent bonds (total correlation
spectroscopy, or TOCSY).
Translating a two-dimensional NMR spectrum into
a complete three-dimensional structure can be a laborious
process. The NOE signals provide some information
about the distances between individual atoms,
for these distance constraints to be useful, the atoms
giving rise to each signal must be identified. Complementary
TOCSY experiments can help identify which
NOE signals reflect atoms that are linked by covalent
bonds. Certain patterns of NOE signals have been associated
with secondary structures such as helices.
Modern genetic engineering can be used
to prepare proteins that contain the rare isotopes 13C
or 15N. The new NMR signals produced by these atoms,
and the coupling with 1H signals resulting from these
substitutions, help in the assignment of individual 1H
NOE signals. The process is also aided by a knowledge
of the amino acid sequence of the polypeptide.
To generate a three-dimensional structure, researchers
feed the distance constraints into a computer
along with known geometric constraints such as
chirality, van der Waals radii, and bond lengths and
angles. The computer generates a family of closely related
structures that represent the range of conformations
consistent with the NOE distance constraints. The uncertainty in structures generated by
NMR is in part a reflection of the molecular vibrations
(breathing) within a protein structure in solution. Normal experimental
uncertainty can also play a role.
When a protein structure has been determined by
both x-ray crystallography and NMR, the structures
generally agree well. In some cases, the precise locationsgiving rise to each signal must be identified. Complementary
TOCSY experiments can help identify which
NOE signals reflect atoms that are linked by covalent
bonds. Certain patterns of NOE signals have been associated
with secondary structures such as helices.
Modern genetic engineering can be used
to prepare proteins that contain the rare isotopes 13C
or 15N. The new NMR signals produced by these atoms,
and the coupling with 1H signals resulting from these
substitutions, help in the assignment of individual 1H
NOE signals. The process is also aided by a knowledge
of the amino acid sequence of the polypeptide.
To generate a three-dimensional structure, researchers
feed the distance constraints into a computer
along with known geometric constraints such as
chirality, van der Waals radii, and bond lengths and
angles. The computer generates a family of closely related
structures that represent the range of conformations
consistent with the NOE distance constraints. The uncertainty in structures generated by
NMR is in part a reflection of the molecular vibrations
(breathing) within a protein structure in solution. Normal experimental
uncertainty can also play a role.
When a protein structure has been determined by
both x-ray crystallography and NMR, the structures
of particular amino acid side chains on the protein
exterior are different, often because of effects related
to the packing of adjacent protein molecules in
a crystal. The two techniques together are at the heart
of the rapid increase in the availability of structural
information about the macromolecules of living cells.
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