around a given nucleus in a molecule tends to slightly disturb the
local magnetic field exerted on that nucleus and to affect its transition energy. This dependence of the transition energy on the position of a particular atom in a molecule makes NMR extremely
useful for determining the structure of molecules.
The sample placed in an intense magnetic field will be disturbed
by radiofrequency pulses. The recording of the return to the equilibrium of the spins makes it possible to have access to the chemical
environment of the atoms. This information offers the possibility of
structural and dynamic analyzes as well as the study of interactions
involving biological macromolecules.
NMR can be used to characterize the degree of folding of a
protein by observing the dispersion of the resonance peaks. Indeed,
in the one-dimensional (1D)
1 H spectrum or the two-dimensional
(2D)
1 H15 N correlation spectra if the protein is
15
N-labeled, the
peaks for a well-folded protein are narrow and sharp and distributed
over a large range of chemical shifts (good signal dispersion)
(Fig. 6).
1 H resonances can be especially found at values
<0.5 ppm (corresponding to high field-shifted methyl group protons) or > 8.5 ppm (corresponding to down field-shifted amide
protons). In contrast, the peaks can be broader and not as widely
dispersed in the spectrum of an unfolded or partially folded protein.
Moreover, observed linewidths of peaks are related to the molecular
weight of the protein, and then may be indicative of autoassociation
or aggregation. In addition to the evaluation of the folding and the
stability of a protein, a 1D
1 H spectrum provides information about
purity. Indeed, impurities with low molecular weight and observable nuclei give rise to sharp signals amongst the broader envelope
of the protein resonances.
The most common 2D spectrum to obtain structural information about a protein is the
1 H15 N heteronuclear single quantum
coherence (HSQC) spectrum. It correlates the nitrogen atom of an
amide group with the directly attached amide proton. Since there is
only one backbone H
N per amino acid, except for Pro, each HSQC
signal represents one single amino acid. The HSQC also contains
signals from the NH 2 groups of the side chains of asparagine and
glutamine (also lysine and arginine depending on the pH values)
and of the aromatic H
N protons of Trp and Histidine. The signals
may cover a spectral range from 6.0 to 12 ppm. If a protein is
folded, its signals are distributed over the complete spectral range;
if not, signals are located between 7.5 and 8.5 ppm in the proton
dimension. Signals outside these regions indicate that the protein is
folded and in a defined three-dimensional (3D) state. The chemical
shift is very sensitive to the overall structure so that even slight
conformational changes will affect the signals in a
1 H15 N HSQC
spectrum, making it a very efficient tool to check conformational
stability and folding states.
PDZ Sample Quality Assessment
101
local magnetic field exerted on that nucleus and to affect its transition energy. This dependence of the transition energy on the position of a particular atom in a molecule makes NMR extremely
useful for determining the structure of molecules.
The sample placed in an intense magnetic field will be disturbed
by radiofrequency pulses. The recording of the return to the equilibrium of the spins makes it possible to have access to the chemical
environment of the atoms. This information offers the possibility of
structural and dynamic analyzes as well as the study of interactions
involving biological macromolecules.
NMR can be used to characterize the degree of folding of a
protein by observing the dispersion of the resonance peaks. Indeed,
in the one-dimensional (1D)
1 H spectrum or the two-dimensional
(2D)
1 H15 N correlation spectra if the protein is
15
N-labeled, the
peaks for a well-folded protein are narrow and sharp and distributed
over a large range of chemical shifts (good signal dispersion)
(Fig. 6).
1 H resonances can be especially found at values
<0.5 ppm (corresponding to high field-shifted methyl group protons) or > 8.5 ppm (corresponding to down field-shifted amide
protons). In contrast, the peaks can be broader and not as widely
dispersed in the spectrum of an unfolded or partially folded protein.
Moreover, observed linewidths of peaks are related to the molecular
weight of the protein, and then may be indicative of autoassociation
or aggregation. In addition to the evaluation of the folding and the
stability of a protein, a 1D
1 H spectrum provides information about
purity. Indeed, impurities with low molecular weight and observable nuclei give rise to sharp signals amongst the broader envelope
of the protein resonances.
The most common 2D spectrum to obtain structural information about a protein is the
1 H15 N heteronuclear single quantum
coherence (HSQC) spectrum. It correlates the nitrogen atom of an
amide group with the directly attached amide proton. Since there is
only one backbone H
N per amino acid, except for Pro, each HSQC
signal represents one single amino acid. The HSQC also contains
signals from the NH 2 groups of the side chains of asparagine and
glutamine (also lysine and arginine depending on the pH values)
and of the aromatic H
N protons of Trp and Histidine. The signals
may cover a spectral range from 6.0 to 12 ppm. If a protein is
folded, its signals are distributed over the complete spectral range;
if not, signals are located between 7.5 and 8.5 ppm in the proton
dimension. Signals outside these regions indicate that the protein is
folded and in a defined three-dimensional (3D) state. The chemical
shift is very sensitive to the overall structure so that even slight
conformational changes will affect the signals in a
1 H15 N HSQC
spectrum, making it a very efficient tool to check conformational
stability and folding states.
PDZ Sample Quality Assessment
101