92
B. BERSCH et al.
concrete examples of recent applications from our laboratory. We hope that this
chapter might be a helpful illustration of the various aspects of this technique
and an encouragement to take advantage of the possibilities it offers.
2
Sample Requirements
High resolution NMR information on biological macromolecules is usually
obtained from solution studies. The most frequently observed nuclei are lH
(100 % natural abundance), Be (l.1 % natural abundance or isotopic enrichment)
and 15N (isotopic enrichment). The obtention of isotopically labelled proteins is
presented in section 2.4.
The quality of the experimental data strongly depends on the experimental
conditions, so a careful preparation of the NMR sample is primordial.
2.1
Experimental Conditions
The most physiological solvent used for the studies of biological macromolecules
is obviously water. However, the use of this solvent has two major inconveniences
which can be circumvented by the appropriate choice of experimental parameters. First, the lH nuclei are generally detected during the experimental aquisition due to their high gyromagnetic ratio (y), resulting in a much better sensitivity. As the concentration of water protons (110 M) is several orders of magnitude
higher than that of the sample (0.5-10 mM), the water signal has to be suppressed, otherwise it would completely mask the signals from the protein and
reduce the available dynamic range. Appropriate techniques have been developed: the presaturation of the water resonance, a jump-and-return or a watergate
pulse sequence are most commonly used (Hore 1989, Piotto et al. 1992). In addition, several protons capable of forming hydrogen bonds with water have a tendency to exchange with the solvent (exchangeable or labile protons). Most information gained on proteins relies on the observation of the amide protons which
do exchange with the solvent (this is the reason why D20 is rarely used as a solvent for protein studies). Proton-solvent exchange increases with pH above pH ~
4.0. Therefore, a pH below 7.0 is preferred. Above this value, exchange with the
solvent leads to excessive line-broadening and disappearance of several amide
resonances.
Buffers used in protein NMR should not contain any non-exchangeable protons. Phosphate is a good choice, but deuterated TRIS and acetic acid are also
available. The ionic strength should be kept to a minimum. The temperature is
generally set between 15° and 40°C. Increasing temperature reduces the linewidth, due to faster molecular tumbling, but also reduces the protein stability.
B. BERSCH et al.
concrete examples of recent applications from our laboratory. We hope that this
chapter might be a helpful illustration of the various aspects of this technique
and an encouragement to take advantage of the possibilities it offers.
2
Sample Requirements
High resolution NMR information on biological macromolecules is usually
obtained from solution studies. The most frequently observed nuclei are lH
(100 % natural abundance), Be (l.1 % natural abundance or isotopic enrichment)
and 15N (isotopic enrichment). The obtention of isotopically labelled proteins is
presented in section 2.4.
The quality of the experimental data strongly depends on the experimental
conditions, so a careful preparation of the NMR sample is primordial.
2.1
Experimental Conditions
The most physiological solvent used for the studies of biological macromolecules
is obviously water. However, the use of this solvent has two major inconveniences
which can be circumvented by the appropriate choice of experimental parameters. First, the lH nuclei are generally detected during the experimental aquisition due to their high gyromagnetic ratio (y), resulting in a much better sensitivity. As the concentration of water protons (110 M) is several orders of magnitude
higher than that of the sample (0.5-10 mM), the water signal has to be suppressed, otherwise it would completely mask the signals from the protein and
reduce the available dynamic range. Appropriate techniques have been developed: the presaturation of the water resonance, a jump-and-return or a watergate
pulse sequence are most commonly used (Hore 1989, Piotto et al. 1992). In addition, several protons capable of forming hydrogen bonds with water have a tendency to exchange with the solvent (exchangeable or labile protons). Most information gained on proteins relies on the observation of the amide protons which
do exchange with the solvent (this is the reason why D20 is rarely used as a solvent for protein studies). Proton-solvent exchange increases with pH above pH ~
4.0. Therefore, a pH below 7.0 is preferred. Above this value, exchange with the
solvent leads to excessive line-broadening and disappearance of several amide
resonances.
Buffers used in protein NMR should not contain any non-exchangeable protons. Phosphate is a good choice, but deuterated TRIS and acetic acid are also
available. The ionic strength should be kept to a minimum. The temperature is
generally set between 15° and 40°C. Increasing temperature reduces the linewidth, due to faster molecular tumbling, but also reduces the protein stability.