International
Tables for
Crystallography
Volume F
Crystallography of biological macromolecules
Edited by M. G. Rossmann and E. Arnold

International Tables for Crystallography (2006). Vol. F, ch. 4.3, p. 101   | 1 | 2 |

Section 4.3.4. Mutations to accelerate crystallization

D. R. Daviesa* and A. Burgess Hickmana

aLaboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0560, USA
Correspondence e-mail:  david.davies@nih.gov

4.3.4. Mutations to accelerate crystallization

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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)[link] 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[link]). 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 [\hbox{Asn68}\rightarrow \hbox{Cys}] and [\hbox{Ala93}\rightarrow \hbox{Cys}]. 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[link]) 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 [\hbox{Ala90}\rightarrow \hbox{Tyr}] and [\hbox{Ala86}\rightarrow \hbox{His}]. 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.

References

Bell, J. A., Wilson, K. P., Zhang, X.-J., Faber, H. R., Nicholson, H. & Matthews, B. W. (1991). Comparison of the crystal structure of bacteriophage T4 lysozyme at low, medium, and high ionic strengths. Proteins, 10, 10–21.
Heinz, D. W. & Matthews, B. W. (1994). Rapid crystallization of T4 lysozyme by intermolecular disulfide cross-linking. Protein Eng. 7, 301–307.
Mittl, P. R. E., Berry, A., Scrutton, N. S., Perham, R. N. & Schulz, G. E. (1994). A designed mutant of the enzyme glutathione reductase shortens the crystallization time by a factor of forty. Acta Cryst. D50, 228–231.








































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