Crystallisation of wild-type and variant forms of a recombinant plant enzyme β-D-glucan glucohydrolase from barley (Hordeum vulgare L.) and preliminary X-ray analysis.
Wild-type and variant crystals of a recombinant enzyme beta-d-glucan glucohydrolase from barley (Hordeum vulgare L.) were obtained by macroseeding and cross-seeding with microcrystals obtained from native plant protein. Crystals grew to dimensions of up to 500 x 250 x 375 mum at 277 K in the hanging-drops by vapour-diffusion. Further, the conditions are described that yielded the wild-type crystals with dimensions of 80 x 40 x 60 mum by self-nucleation vapour-diffusion in sitting-drops at 281 K. The wild-type and recombinant crystals prepared by seeding techniques achived full size within 5-14 days, while the wild-type crystals grown by self-nucleation appeared after 30 days and reached their maximum size after another two months. Both the wild-type and recombinant variant crystals, the latter altered in the key catalytic and substrate-binding residues Glu220, Trp434 and Arg158/Glu161 belonged to the P4(3)2(1)2 tetragonal space group, i.e., the space group of the native microcrystals was retained in the newly grown recombinant crystals. The crystals diffracted beyond 1.57-1.95 A and the cell dimensions were between a = b = 99.2-100.8 A and c = 183.2-183.6 A. With one molecule in the asymmetric unit, the calculated Matthews coefficients were between 3.4-3.5 A(3).Da(-1) and the solvent contents varied between 63.4% and 64.5%. The macroseeding and cross-seeding techniques are advantageous, where a limited amount of variant proteins precludes screening of crystallisation conditions, or where variant proteins could not be crystallized.
Luang
Sukanya
S
School of Biochemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand; E-Mail: jeab_bc@hotmail.com (S.L.); cairns@sut.ac.th (J.R.K.C.).
Ketudat Cairns
James R
JR
Streltsov
Victor A
VA
Hrmova
Maria
M
D003460:Crystallization; D018360:Crystallography, X-Ray; D005959:Glucosidases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D001467:Hordeum / Q000201:enzymology*; D009154:Mutation; D011994:Recombinant Proteins; D014961:X-Ray Diffraction
D011994:Recombinant Proteins; D005959:Glucosidases; C446280:beta-D-glucan glucohydrolase