Protein Structure and Dynamics by NMR in Solution
93
2.2
Choice of the Protein
Two very important criteria for the decision whether a protein can be studied by
NMR are the size of the protein and its solubility.
Concerning the solubility, the protein should be soluble under the above mentioned conditions at a concentration of at least 0.5 mM. For a sample volume
between 300 and 500 Ill, this means that the quantity of protein required for one
sample is at least 15 micromoles.
The maximal size of a protein to be studied by NMR is limited by the characteristics of the technique itself. With increasing molecular size, spectra become
more and more crowded and due to increased relaxation effects the sensitivity of
the experiment significantly decreases. This has led to the development of heteronuclear techniques (3D, 4D ... ), where the frequency of the heteronucleus
ctSN, l3C or both) to which the proton is linked is added as a supplementary
parameter. However, the use of these techniques necessitates isotopic labelling of
the protein (see below). Concerning the relaxation effects, partial deuteration of
the protein (LeMaster 1990, Sattler et al. 1996) or the constructive use of relaxation interference (TROSY, Pervushin et al. 1997) helps to push the size limit to
higher values.
Generally, the following rules apply: a protein which cannot be labelled isotopically should not have more than lOO residues. For proteins up to 150 amino
acids, uniform JSN-Iabelling is sufficient whereas larger proteins should be
double- eSN and l3C) or triple- ctSN, l3C and 2H) labelled. The actual size limit
using the most sophisticated NMR techniques depends on the characteristics of
the protein studied. So far, NMR structure determinations have been reported for
proteins with molecular weights of up to 40 kD.
2.3
Preparation of the Sample
Obviously, the protein should be of high purity. During the sample preparation,
care should be taken not to contaminate the protein with any microorganisms
such as bacteria or fungi, which might result in a digestion of the protein. The
final solution can be filter sterilized or small quantities of antibiotics or sodium
azide might be added to the sample. Sealing the sample under inert athmosphere
also helps to avoid damage during storage. Even with the most sophisticated
purification procedures, proteases are rarely completely eliminated. Therefore,
protease inhibitors are widely used to protect from proteolytic degradation and
can also be added to the final sample. Note however, that the aquisition of the
experimental data takes several days or even weeks during which the protein
should be stable under the experimental conditions given above.
93
2.2
Choice of the Protein
Two very important criteria for the decision whether a protein can be studied by
NMR are the size of the protein and its solubility.
Concerning the solubility, the protein should be soluble under the above mentioned conditions at a concentration of at least 0.5 mM. For a sample volume
between 300 and 500 Ill, this means that the quantity of protein required for one
sample is at least 15 micromoles.
The maximal size of a protein to be studied by NMR is limited by the characteristics of the technique itself. With increasing molecular size, spectra become
more and more crowded and due to increased relaxation effects the sensitivity of
the experiment significantly decreases. This has led to the development of heteronuclear techniques (3D, 4D ... ), where the frequency of the heteronucleus
ctSN, l3C or both) to which the proton is linked is added as a supplementary
parameter. However, the use of these techniques necessitates isotopic labelling of
the protein (see below). Concerning the relaxation effects, partial deuteration of
the protein (LeMaster 1990, Sattler et al. 1996) or the constructive use of relaxation interference (TROSY, Pervushin et al. 1997) helps to push the size limit to
higher values.
Generally, the following rules apply: a protein which cannot be labelled isotopically should not have more than lOO residues. For proteins up to 150 amino
acids, uniform JSN-Iabelling is sufficient whereas larger proteins should be
double- eSN and l3C) or triple- ctSN, l3C and 2H) labelled. The actual size limit
using the most sophisticated NMR techniques depends on the characteristics of
the protein studied. So far, NMR structure determinations have been reported for
proteins with molecular weights of up to 40 kD.
2.3
Preparation of the Sample
Obviously, the protein should be of high purity. During the sample preparation,
care should be taken not to contaminate the protein with any microorganisms
such as bacteria or fungi, which might result in a digestion of the protein. The
final solution can be filter sterilized or small quantities of antibiotics or sodium
azide might be added to the sample. Sealing the sample under inert athmosphere
also helps to avoid damage during storage. Even with the most sophisticated
purification procedures, proteases are rarely completely eliminated. Therefore,
protease inhibitors are widely used to protect from proteolytic degradation and
can also be added to the final sample. Note however, that the aquisition of the
experimental data takes several days or even weeks during which the protein
should be stable under the experimental conditions given above.