Section 12.1.7. Analogues of amino acids

Attempts to replace amino acids by heavy-atom substituted synthetic analogues with a similar charge and shape have not proved successful in large proteins, although a selenocystine was used successfully in the analysis of oxytocin (Wood et al., 1986). However, the production of proteins labelled by selenium using biological substitution of selenomethionine (SeMet) for methionine (Hendrickson, 1985) has been stimulated by multiple-wavelength anomalous dispersion (MAD) (Hendrickson et al., 1990). Methionine biosynthesis is blocked in the cells in which the protein is produced and SeMet is substituted for Met in the growth medium. The generality of the labelling scheme for proteins is the root of its success, as discussed by Doublié (1997).

SeMet has been incorporated into proteins expressed in Escherichia coli strains that are auxotrophic for Met [strain DL421 (Hendrickson et al., 1990); strain B834 (Leahy et al., 1994); strain LE392 (Ceska et al., 1996)]. Nearly complete incorporation has also been reported in non-auxotrophic bacterial strains, E. coli strain XA90 (Labahn et al., 1996), in a mammalian cell line (Lustbader et al., 1995) and in baculovirus-infected insect cells (Chen & Bahl, 1991). Usually, somewhat higher than normal concentrations of disulfide reducing agents, such as dithiothreitol or mercaptoethanol, are sufficient to protect SeMet from air oxidation to the selenoxide, although crystallization in an inert atmosphere may be necessary. Proteins usually have SeMet substituted for Met at levels approaching 100%. The cells are viable and the proteins are functional.

Site-directed mutagenesis offers an alternative approach for the introduction of specific heavy-atom binding sites. A common procedure is to replace residue(s) in the variable part of the primary structure with cysteine. The selection of the residue to mutate in a protein of unknown structure remains a challenge.

Although selenocysteine is toxic to cells, cysteine auxotrophic strains, in which proteins can be synthesized with the seleno derivative, have been developed (Miller, 1972; Muller et al., 1994). The bacteria are grown under limiting amounts of cysteine with no other sulfur source. They are induced for 10 min and then resuspended in selenocysteine for a 3 h incubation. The protein is purified with a reducing agent. In general, the substitution at the selenocysteine seems to be less satisfactory than selenomethionine, with occupancy often as low as 20%.

Budisa et al. (1997) have experimented on incorporating a range of novel amino-acid analogues using in vitro suppression. This is achieved by suppressing the stop colons and engineering tRNA synthases to incorporate the analogue. Possible candidates are telluromethionine, 5-bromotryptophan, 5-iodotryptophan, selenotryptophan and tellurotryptophan. The bioincorporation of TeMet into derivatized crystals did not greatly affect their stability in buffer solutions and to X-radiation. Isomorphism was maintained despite the C—Te bond being longer than C—Se or C—S. TeMet crystals are not as suitable for MAD analysis as SeMet crystals due to the 0.3 Å absorption edge of tellurium. The method is restricted to methionine residues located in the hydrophobic regions, since solvent accessibility may cause undefined chemical reactions with the highly reactive C—Te side chain. Thus the protein must be expressed in the folded form.