94
B. BERSCH et al.
2.4
Obtention of Isotopically Labelled Proteins.
Whereas any source can be used for the obtention of protein samples for homonuclear NMR experiments, the obtention of isotopically labelled proteins necessitates the overexpression of the protein in a convenient host.
Isotopic labelling is achieved by providing completely labelled nitrogen- and/
or carbon sources to the culture medium (for a review see McIntosh 1990). To
reduce costs, such cultures are usually grown in minimal media supplemented
with 15NH4CI and/or l3C6-glucose. As a consequence, the protein should be overexpressed in an organism capable of growing on minimal media such as E. coli or
P. pastoris, the latter is able to grow on methanol as sole carbon source (in this
case, the medium should contain l3C-methanol). However, in some cases it might
be necessary to increase the expression by the use of rich media which are
derived from partially hydrolysed algae protein. In addition, isotopically labelled
media are available for insect cells (baculovirus expression system) as well as
CHO cells.
Before starting cultures in expensive isotopically labelled media, culture and
purification methods should be optimized to a maximum. The use of tags or
fusion proteins for an optimized purification is possible but it should be born in
mind that the addition of any residues might have consequences for the threedimensional structure of the protein. Protease mediated cleavage of the fusion
protein or the tag is widely used, but does not always give satisfactory results due
to side reactions. Very recently, a ubiquitin fusion has been proposed for the
expression of small, isotopically labelled peptides which should also work for
bigger proteins (Kohno et al. 1998). One advantage of this system is the very specific cleavage reaction by a ubiquitin-specific hydrolase.
If complete deuteration of cells is desired, they must be adapted to deuterated
media in a stepwise manner (Katz and Crespi 1966). Note, however, that in contrast to 15N_ or l3C-Iabelling, which have no effect on cell metabolism, deuteration
drastically reduces growth and consequently protein production (Haon et al.
1993). In addition, a protocol for the obtention of deuterated proteins which are
selectively protonated on the aliphatic side chains has been described (Rosen et
al. 1996).
3
NMR Spectral Parameters
Nuclear magnetic resonance can be used to probe the structure and dynamics of
biomolecules, because the spins act as spies of their intimate environment: each
nuclear spin interacts with its direct surroundings consisting of other nuclei and
electrons. These numerous pair-wise interactions are detected by a smaller number of spectral parameters, such as the chemical shift, the scalar coupling constants or the relaxation rate constants. Structural or dynamical information concerning the protein itself can then be obtained by the conversion of the spectral
parameters into structural parameters, such as distances, angles or frequencies of
motions. However, one should keep in mind that solution state NMR provides an
B. BERSCH et al.
2.4
Obtention of Isotopically Labelled Proteins.
Whereas any source can be used for the obtention of protein samples for homonuclear NMR experiments, the obtention of isotopically labelled proteins necessitates the overexpression of the protein in a convenient host.
Isotopic labelling is achieved by providing completely labelled nitrogen- and/
or carbon sources to the culture medium (for a review see McIntosh 1990). To
reduce costs, such cultures are usually grown in minimal media supplemented
with 15NH4CI and/or l3C6-glucose. As a consequence, the protein should be overexpressed in an organism capable of growing on minimal media such as E. coli or
P. pastoris, the latter is able to grow on methanol as sole carbon source (in this
case, the medium should contain l3C-methanol). However, in some cases it might
be necessary to increase the expression by the use of rich media which are
derived from partially hydrolysed algae protein. In addition, isotopically labelled
media are available for insect cells (baculovirus expression system) as well as
CHO cells.
Before starting cultures in expensive isotopically labelled media, culture and
purification methods should be optimized to a maximum. The use of tags or
fusion proteins for an optimized purification is possible but it should be born in
mind that the addition of any residues might have consequences for the threedimensional structure of the protein. Protease mediated cleavage of the fusion
protein or the tag is widely used, but does not always give satisfactory results due
to side reactions. Very recently, a ubiquitin fusion has been proposed for the
expression of small, isotopically labelled peptides which should also work for
bigger proteins (Kohno et al. 1998). One advantage of this system is the very specific cleavage reaction by a ubiquitin-specific hydrolase.
If complete deuteration of cells is desired, they must be adapted to deuterated
media in a stepwise manner (Katz and Crespi 1966). Note, however, that in contrast to 15N_ or l3C-Iabelling, which have no effect on cell metabolism, deuteration
drastically reduces growth and consequently protein production (Haon et al.
1993). In addition, a protocol for the obtention of deuterated proteins which are
selectively protonated on the aliphatic side chains has been described (Rosen et
al. 1996).
3
NMR Spectral Parameters
Nuclear magnetic resonance can be used to probe the structure and dynamics of
biomolecules, because the spins act as spies of their intimate environment: each
nuclear spin interacts with its direct surroundings consisting of other nuclei and
electrons. These numerous pair-wise interactions are detected by a smaller number of spectral parameters, such as the chemical shift, the scalar coupling constants or the relaxation rate constants. Structural or dynamical information concerning the protein itself can then be obtained by the conversion of the spectral
parameters into structural parameters, such as distances, angles or frequencies of
motions. However, one should keep in mind that solution state NMR provides an