Please use this identifier to cite or link to this item:
https://hdl.handle.net/2440/60790
Citations | ||
Scopus | Web of Science® | Altmetric |
---|---|---|
?
|
?
|
Type: | Journal article |
Title: | 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 |
Other Titles: | Crystallisation of wild-type and variant forms of a recombinant plant enzyme beta-D-glucan glucohydrolase from barley (Hordeum vulgare L.) and preliminary X-ray analysis |
Author: | Luang, S. Cairns, J. Streltsov, V. Hrmova, M. |
Citation: | International Journal of Molecular Sciences, 2010; 11(7):2759-2769 |
Publisher: | Molecular Diversity Preservation International (MDPI) AG. |
Issue Date: | 2010 |
ISSN: | 1422-0067 1422-0067 |
Statement of Responsibility: | Sukanya Luang, James R. Ketudat Cairns, Victor A. Streltsov and Maria Hrmova |
Abstract: | Wild-type and variant crystals of a recombinant enzyme β-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 µm 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 µm by self-nucleation vapour-diffusion in sitting-drops at 281 K. The wild-type and recombinant crystals prepared by seeding techniques achieved 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 P4₃2₁2 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 Å and the cell dimensions were between a = b = 99.2-100.8 Å and c = 183.2-183.6 Å. With one molecule in the asymmetric unit, the calculated Matthews coefficients were between 3.4-3.5 ų.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. |
Keywords: | macro- and cross-seeding wild-type and mutant protein X-ray diffraction |
Rights: | © 2010 by the authors; licensee MDPI, Basel, Switzerland. This article is an Open Access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/). |
DOI: | 10.3390/ijms11072759 |
Grant ID: | ARC |
Published version: | http://dx.doi.org/10.3390/ijms11072759 |
Appears in Collections: | Agriculture, Food and Wine publications Aurora harvest |
Files in This Item:
File | Description | Size | Format | |
---|---|---|---|---|
hdl_60790.pdf | Published version | 4.21 MB | Adobe PDF | View/Open |
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.