Purification, crystallization and preliminary X-ray diffraction analysis of the phage T4 vertex protein gp24 and its mutant forms.
The study of bacteriophage T4 assembly has revealed regulatory mechanisms pertinent not only to viruses but also to macromolecular complexes. The capsid of bacteriophage T4 is composed of the major capsid protein gp23, and a minor capsid protein gp24, which is arranged as pentamers at the vertices of the capsid. In this study the T4 capsid protein gp24 and its mutant forms were overexpressed and purified to homogeneity. The overexpression from plasmid vectors of all the constructs in Escherichia coli yields biologically active protein in vivo as determined by assembly of active virus following infection with inactivated gene 24 mutant viruses. The gp24 mutant was subjected to surface entropy reduction by mutagenesis and reductive alkylation in order to improve its crystallization properties and diffraction quality. To determine if surface mutagenesis targeting would result in diffractable crystals, two glutamate to alanine mutations (E89A,E90A) were introduced. We report here the biochemical observations and consequent mutagenesis experiment that resulted in improvements in the stability, crystallizability and crystal quality of gp24 without affecting the overall folding. Rational modification of the protein surface to achieve crystallization appears promising for improving crystallization behavior and crystal diffracting qualities. The crystal of gp24(E89A,E90A) diffracted to 2.6A resolution compared to wild-type gp24 at 3.80A resolution under the same experimental conditions. Surface mutation proved to be a better method than reductive methylation for improving diffraction quality of the gp24 crystals.
Boeshans
Karen M
KM
X-ray Crystallography Facility/Office of Science and Technology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892-8024, USA.
Liu
Fang
F
Peng
Guihong
G
Idler
William
W
Jang
Shyh-Ing
SI
Marekov
Lyuben
L
Black
Lindsay
L
Ahvazi
Bijan
B
D019943:Amino Acid Substitution; D036022:Capsid Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D018360:Crystallography, X-Ray / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D020125:Mutation, Missense; D017434:Protein Structure, Tertiary; D019065:Virus Assembly / Q000502:physiology
D036022:Capsid Proteins; C074187:gene 24 protein, Enterobacteria phage T4