Effects of impurities on membrane-protein crystallization in different systems
- Biosciences Division, Argonne National Laboratory, 9700 South Cass Avenue, Argonne, IL 60439 (United States)
- deCODE biostructures, 7869 NE Day Road West, Bainbridge Island, WA 98110 (United States)
- Department of Chemistry and Institute for Biophysical Dynamics, University of Chicago, 929 East 57th Street, Chicago, IL 60637 (United States)
The effects of commonly encountered impurities on various membrane-protein crystallization regimes are investigated and it is found that the lipidic cubic phase crystallization methodology is the most robust, tolerating protein contamination levels of up to 50%, with little effect on crystal quality. If generally applicable, this tolerance may be exploited (i) in initial crystallization trials to determine the ‘crystallizability’ of a given membrane-protein and (ii) to subject partially pure membrane-protein samples to crystallization trials. When starting a protein-crystallization project, scientists are faced with several unknowns. Amongst them are these questions: (i) is the purity of the starting material sufficient? and (ii) which type of crystallization experiment is the most promising to conduct? The difficulty in purifying active membrane-protein samples for crystallization trials and the high costs associated with producing such samples require an extremely pragmatic approach. Additionally, practical guidelines are needed to increase the efficiency of membrane-protein crystallization. In order to address these conundrums, the effects of commonly encountered impurities on various membrane-protein crystallization regimes have been investigated and it was found that the lipidic cubic phase (LCP) based crystallization methodology is more robust than crystallization in detergent environments using vapor diffusion or microbatch approaches in its ability to tolerate contamination in the forms of protein, lipid or other general membrane components. LCP-based crystallizations produced crystals of the photosynthetic reaction center (RC) of Rhodobacter sphaeroides from samples with substantial levels of residual impurities. Crystals were obtained with protein contamination levels of up to 50% and the addition of lipid material and membrane fragments to pure samples of RC had little effect on the number or on the quality of crystals obtained in LCP-based crystallization screens. If generally applicable, this tolerance for impurities may avoid the need for samples of ultrahigh purity when undertaking initial crystallization screening trials to determine preliminary crystallization conditions that can be optimized for a given target protein.
- OSTI ID:
- 22347922
- Journal Information:
- Acta Crystallographica. Section D: Biological Crystallography, Vol. 65, Issue Pt 10; Other Information: PMCID: PMC2748966; PMID: 19770503; PUBLISHER-ID: bw5288; OAI: oai:pubmedcentral.nih.gov:2748966; Copyright (c) Kors et al. 2009; This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.; Country of input: International Atomic Energy Agency (IAEA); ISSN 0907-4449
- Country of Publication:
- Denmark
- Language:
- English
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