International
Tables for Crystallography Volume F Crystallography of biological macromolecules Edited by M. G. Rossmann and E. Arnold © International Union of Crystallography 2006 |
International Tables for Crystallography (2006). Vol. F. ch. 4.3, p. 101
Section 4.3.4. Mutations to accelerate crystallization
aLaboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0560, USA |
A common problem encountered in crystallization is that certain crystals appear late and grow slowly. Sometimes, the slow appearance of crystals is the result of proteolytic processing, but often the reasons are not apparent. There are several examples where protein engineering has resulted in an increase in the rate of crystallization.
Heinz & Matthews (1994) explored the crystallization of T4 phage lysozyme using a strategy based on their understanding of the structure of the enzyme and its crystallization properties. The crystallization of the wild-type protein required the presence of β-mercaptoethanol (BME), an additive which could not be replaced with dithiothreitol. It had also been observed that the oxidized form of BME, hydroxyethyl disulfide, was trapped in the dimer interface between two lysozyme molecules (Bell et al., 1991
). It was hypothesized that dimer formation might be the rate-limiting step in crystallization, so dimerization was enhanced by cross-linking two monomers by disulfide-bridge formation. Applying rules developed for constructing S–S bridges, they selected
and
. In the presence of oxidized BME, the rate of crystallization of these mutant proteins was substantially increased, with crystals reaching full size in two days, in contrast to two weeks for the unmutated protein. Furthermore, they were able to crystallize a previously uncrystallizable mutant. Unexpectedly, however, the dimer formed in this way was lacking in activity, despite the selection of mutation sites on the opposite side of the molecule to the active site.
Mittl et al. (1994) wanted to improve the resolution of their crystals of glutathione reductase. From the 3 Å map, they could see a hole in the crystal packing where two molecules within 6 Å of each other just missed forming a crystal contact; they filled this hole by mutating
and
. This designed double mutant did not improve the resolution, but did increase the rate of crystallization 40-fold, i.e., initial crystals were observed within 1.5 h versus 60 h for the wild-type enzyme.
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