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Effects of impurities on membrane-protein crystallization in different systems.

Kors CA, Wallace E, Davies DR, Li L, Laible PD, Nollert P - Acta Crystallogr. D Biol. Crystallogr. (2009)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biosciences Division, Argonne National Laboratory, 9700 South Cass Avenue, Argonne, IL 60439, USA.

ABSTRACT
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.

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Related in: MedlinePlus

Characterization of protein and lipid impurities found within partially purified RC samples used for crystallization experiments. Protein analyses (a, c) are depicted as digitized intensities from Coomassie-stained SDS–PAGE gels. Lipid analyses (b, d) comprise iodine-stained TLC plates. For the partially purified samples (a) and (b), the RC content was kept constant using 12 and 30 µg in each gel and TLC lane, respectively. The numbers above the lanes indicate the A                  280/A                  800 ratio of the sample. Assignments of spots on the TLC plate in (b) signify the detergent and lipid components present in the samples that are resolved by this solvent system: Pigments, a mixture comprised of bacteriochlorophylls, bacteriopheophytins, carotenoids and quinones; LDAO, N,N-dimethyl­dodecylamine-N-oxide; MGDG, monogalactosyldiacylglycerol; CL, cardiolipin; PE, phosphatidyl­ethanolamine; PC, phosphatidylcholine; PG, phosphatidylglycerol. Increases in LDAO intensities observed on the TLC plate in (b) can be attributed to an overall increase in total protein in samples with decreasing purity (increasing levels of impurities), resulting in a higher overall level of PDCs (daCosta & Baenziger, 2003 ▶). For samples with lipids or membranes added (c, d), the exact contents of the LCP-based trials (0.5 mg of each) in the absence of MO were loaded for analysis. The samples contained (1) RCs only, (2) RCs plus 12% polar brain lipids, (3) RCs plus 18% polar E. coli lipids, (4) RCs plus 1.2% extracted R. sphaeroides lipids, (5) RCs plus 12% R. sphaeroides whole membranes. For both sets of samples, bands corresponding to the three protein subunits (L, M and H; ∼25–30 kDa) of the R. sphaeroides RC complex are marked with a bracket and the lanes containing molecular-weight standards [ProSieve Protein Markers from Lonza in (a) and Full-Range Rainbow Marker from GE Healthcare in (c)] are indicated (lane L).
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fig1: Characterization of protein and lipid impurities found within partially purified RC samples used for crystallization experiments. Protein analyses (a, c) are depicted as digitized intensities from Coomassie-stained SDS–PAGE gels. Lipid analyses (b, d) comprise iodine-stained TLC plates. For the partially purified samples (a) and (b), the RC content was kept constant using 12 and 30 µg in each gel and TLC lane, respectively. The numbers above the lanes indicate the A 280/A 800 ratio of the sample. Assignments of spots on the TLC plate in (b) signify the detergent and lipid components present in the samples that are resolved by this solvent system: Pigments, a mixture comprised of bacteriochlorophylls, bacteriopheophytins, carotenoids and quinones; LDAO, N,N-dimethyl­dodecylamine-N-oxide; MGDG, monogalactosyldiacylglycerol; CL, cardiolipin; PE, phosphatidyl­ethanolamine; PC, phosphatidylcholine; PG, phosphatidylglycerol. Increases in LDAO intensities observed on the TLC plate in (b) can be attributed to an overall increase in total protein in samples with decreasing purity (increasing levels of impurities), resulting in a higher overall level of PDCs (daCosta & Baenziger, 2003 ▶). For samples with lipids or membranes added (c, d), the exact contents of the LCP-based trials (0.5 mg of each) in the absence of MO were loaded for analysis. The samples contained (1) RCs only, (2) RCs plus 12% polar brain lipids, (3) RCs plus 18% polar E. coli lipids, (4) RCs plus 1.2% extracted R. sphaeroides lipids, (5) RCs plus 12% R. sphaeroides whole membranes. For both sets of samples, bands corresponding to the three protein subunits (L, M and H; ∼25–30 kDa) of the R. sphaeroides RC complex are marked with a bracket and the lanes containing molecular-weight standards [ProSieve Protein Markers from Lonza in (a) and Full-Range Rainbow Marker from GE Healthcare in (c)] are indicated (lane L).

Mentions: The purest preparations of R. sphaeroides RCs that retained all aspects of their light-driven charge-separation function were characterized by A 280/A 800 ratios (total protein/bound monomeric bacteriochlorophyll) of 1.2. SDS–PAGE gels of these ultrapure RCs (stained with Coomassie Blue or Silver) revealed few if any impurities and such a sample was defined in this study as being 100% pure. Purified RC samples having absorption ratios of ≤0.4 (∼85% pure) are known to be highly crystallizable (Pokkuluri et al., 2002 ▶). To explore the limits of such samples with various crystallization approaches, two types of RC samples were purified from membranes of R. sphaeroides (A 280/A 800 = 1.4 and 2.4). Samples of intermediate purity (A 280/A 800 = 1.5, 1.6, 2.0 and 2.2) resulted by simple linear mixing. Analysis by UV–Vis–near-IR absorption spectroscopy, complemented by SDS–PAGE (Fig. 1 ▶ a), where band intensities were quantified by ImageJ, suggested that these RC samples ranged in purity from 86 to 50%, respectively. This implies that 14–50% of the samples were contaminating proteins. As expected, a gradual increase in background staining was observed on gels for samples with increasing A 280/A 800 ratios and was the direct result of rising contamination levels of proteins of various sizes.


Effects of impurities on membrane-protein crystallization in different systems.

Kors CA, Wallace E, Davies DR, Li L, Laible PD, Nollert P - Acta Crystallogr. D Biol. Crystallogr. (2009)

Characterization of protein and lipid impurities found within partially purified RC samples used for crystallization experiments. Protein analyses (a, c) are depicted as digitized intensities from Coomassie-stained SDS–PAGE gels. Lipid analyses (b, d) comprise iodine-stained TLC plates. For the partially purified samples (a) and (b), the RC content was kept constant using 12 and 30 µg in each gel and TLC lane, respectively. The numbers above the lanes indicate the A                  280/A                  800 ratio of the sample. Assignments of spots on the TLC plate in (b) signify the detergent and lipid components present in the samples that are resolved by this solvent system: Pigments, a mixture comprised of bacteriochlorophylls, bacteriopheophytins, carotenoids and quinones; LDAO, N,N-dimethyl­dodecylamine-N-oxide; MGDG, monogalactosyldiacylglycerol; CL, cardiolipin; PE, phosphatidyl­ethanolamine; PC, phosphatidylcholine; PG, phosphatidylglycerol. Increases in LDAO intensities observed on the TLC plate in (b) can be attributed to an overall increase in total protein in samples with decreasing purity (increasing levels of impurities), resulting in a higher overall level of PDCs (daCosta & Baenziger, 2003 ▶). For samples with lipids or membranes added (c, d), the exact contents of the LCP-based trials (0.5 mg of each) in the absence of MO were loaded for analysis. The samples contained (1) RCs only, (2) RCs plus 12% polar brain lipids, (3) RCs plus 18% polar E. coli lipids, (4) RCs plus 1.2% extracted R. sphaeroides lipids, (5) RCs plus 12% R. sphaeroides whole membranes. For both sets of samples, bands corresponding to the three protein subunits (L, M and H; ∼25–30 kDa) of the R. sphaeroides RC complex are marked with a bracket and the lanes containing molecular-weight standards [ProSieve Protein Markers from Lonza in (a) and Full-Range Rainbow Marker from GE Healthcare in (c)] are indicated (lane L).
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Related In: Results  -  Collection

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Show All Figures
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fig1: Characterization of protein and lipid impurities found within partially purified RC samples used for crystallization experiments. Protein analyses (a, c) are depicted as digitized intensities from Coomassie-stained SDS–PAGE gels. Lipid analyses (b, d) comprise iodine-stained TLC plates. For the partially purified samples (a) and (b), the RC content was kept constant using 12 and 30 µg in each gel and TLC lane, respectively. The numbers above the lanes indicate the A 280/A 800 ratio of the sample. Assignments of spots on the TLC plate in (b) signify the detergent and lipid components present in the samples that are resolved by this solvent system: Pigments, a mixture comprised of bacteriochlorophylls, bacteriopheophytins, carotenoids and quinones; LDAO, N,N-dimethyl­dodecylamine-N-oxide; MGDG, monogalactosyldiacylglycerol; CL, cardiolipin; PE, phosphatidyl­ethanolamine; PC, phosphatidylcholine; PG, phosphatidylglycerol. Increases in LDAO intensities observed on the TLC plate in (b) can be attributed to an overall increase in total protein in samples with decreasing purity (increasing levels of impurities), resulting in a higher overall level of PDCs (daCosta & Baenziger, 2003 ▶). For samples with lipids or membranes added (c, d), the exact contents of the LCP-based trials (0.5 mg of each) in the absence of MO were loaded for analysis. The samples contained (1) RCs only, (2) RCs plus 12% polar brain lipids, (3) RCs plus 18% polar E. coli lipids, (4) RCs plus 1.2% extracted R. sphaeroides lipids, (5) RCs plus 12% R. sphaeroides whole membranes. For both sets of samples, bands corresponding to the three protein subunits (L, M and H; ∼25–30 kDa) of the R. sphaeroides RC complex are marked with a bracket and the lanes containing molecular-weight standards [ProSieve Protein Markers from Lonza in (a) and Full-Range Rainbow Marker from GE Healthcare in (c)] are indicated (lane L).
Mentions: The purest preparations of R. sphaeroides RCs that retained all aspects of their light-driven charge-separation function were characterized by A 280/A 800 ratios (total protein/bound monomeric bacteriochlorophyll) of 1.2. SDS–PAGE gels of these ultrapure RCs (stained with Coomassie Blue or Silver) revealed few if any impurities and such a sample was defined in this study as being 100% pure. Purified RC samples having absorption ratios of ≤0.4 (∼85% pure) are known to be highly crystallizable (Pokkuluri et al., 2002 ▶). To explore the limits of such samples with various crystallization approaches, two types of RC samples were purified from membranes of R. sphaeroides (A 280/A 800 = 1.4 and 2.4). Samples of intermediate purity (A 280/A 800 = 1.5, 1.6, 2.0 and 2.2) resulted by simple linear mixing. Analysis by UV–Vis–near-IR absorption spectroscopy, complemented by SDS–PAGE (Fig. 1 ▶ a), where band intensities were quantified by ImageJ, suggested that these RC samples ranged in purity from 86 to 50%, respectively. This implies that 14–50% of the samples were contaminating proteins. As expected, a gradual increase in background staining was observed on gels for samples with increasing A 280/A 800 ratios and was the direct result of rising contamination levels of proteins of various sizes.

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biosciences Division, Argonne National Laboratory, 9700 South Cass Avenue, Argonne, IL 60439, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus