Date Published: September 01, 2017
Publisher: International Union of Crystallography
Author(s): Kazunori D. Yamada, Naoki Kunishima, Yoshinori Matsuura, Koshiro Nakai, Hisashi Naitow, Yoshinori Fukasawa, Kentaro Tomii.
In order to improve the efficiency of protein crystallization, an alternative approach using the mutation of surface residues was devised based on the results of a statistical analysis of the crystal-packing propensity of amino acids. A systematic crystallization experiment validated the results of the statistical analysis.
X-ray crystallographic analysis is currently an important method for the determination of protein structures. Along with advances in structural genomics and other efforts, the methodology of structure determination has been very well established. However, obtaining good protein crystals that are suitable for accurate structural determination persists as a severe bottleneck hindering X-ray crystallographic analysis, especially with respect to biologically important target proteins. To overcome the difficulties associated with crystallization, various approaches for improving the quality of protein crystals have been devised and implemented. Aside from high-throughput screening approaches, which have been widely adopted (Luft et al., 2014 ▸), approaches involving the introduction of a crystallization nucleant (Sugahara et al., 2008 ▸), pH optimization (Meged et al., 2008 ▸), the adjustment of precipitant concentration (Bode & Huber, 1978 ▸), the addition of carrier proteins (Smyth et al., 2003 ▸), homologous DNA shuffling (Keenan et al., 2005 ▸), synthetically symmetrizing proteins (Banatao et al., 2006 ▸), chemical modification (Kobayashi et al., 1999 ▸; Kurinov et al., 2000 ▸; Rypniewski et al., 1993 ▸), proteolytic digestion of proteins (Wernimont & Edwards, 2009 ▸) and crystal contact engineering (Mizutani et al., 2008 ▸; Wine et al., 2009 ▸) have been proposed.
To improve the crystallization success rate and the crystal quality of a protein, methods in which the surface residues of the protein are replaced with other residues have been commonly used in X-ray diffraction studies. Among surface-engineering approaches, the SER method has been widely used, with the aim of reducing the ‘entropic shield’ on the protein surface. In contrast, we have proposed an alternative rational approach to improve protein crystals by using single-site mutation of surface residues based on the results of a statistical analysis using a compiled data set of 918 independent crystal structures, thereby reflecting not only the entropic effect but also other effects upon protein crystallization. Our approach includes the use of a ‘sticky’ or ‘enthalpically favoured’ single-site mutation of surface residue(s) based on the results of statistical analysis, i.e. the distinctive crystal-packing propensity of amino acids depending on three secondary-structure classes.