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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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The effect of the addition of lipids on LCP crystallization of RCs. (a) The number of conditions producing crystals out of a total of 48 from the ammonium sulfate and Jeffamine grid screen, as a function of increasing amounts of extracted R. sphaeroides lipids (squares, dotted line), polar E. coli lipids (triangles, unbroken line) and polar brain lipid (circles, dashed line). (b) The quality of the RC crystals produced in LCP trials as a function of increasing amounts of lipid additives. Data for extracted R. sphaeroides, polar E. coli and polar brain lipids were averaged. Values plotted represent either the total number of crystals observed (diamonds, unbroken line) or a high-quality (triangles, dotted line), medium-quality (circles, dotted line) or low-quality (squares, dotted line) visual score based on the size, degree of symmetry and edge definition of the crystals obtained.
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fig6: The effect of the addition of lipids on LCP crystallization of RCs. (a) The number of conditions producing crystals out of a total of 48 from the ammonium sulfate and Jeffamine grid screen, as a function of increasing amounts of extracted R. sphaeroides lipids (squares, dotted line), polar E. coli lipids (triangles, unbroken line) and polar brain lipid (circles, dashed line). (b) The quality of the RC crystals produced in LCP trials as a function of increasing amounts of lipid additives. Data for extracted R. sphaeroides, polar E. coli and polar brain lipids were averaged. Values plotted represent either the total number of crystals observed (diamonds, unbroken line) or a high-quality (triangles, dotted line), medium-quality (circles, dotted line) or low-quality (squares, dotted line) visual score based on the size, degree of symmetry and edge definition of the crystals obtained.

Mentions: Since the formation of RC crystals in LCP-based crystallization trials was robust when challenged with non-RC protein, we tested whether the same holds for lipids. In this regard, the data show that the amount and source (polar brain, polar E. coli or extracted R. sphaeroides lipids) of the lipids added to the LCP-based crystallization experiments had no negative effect on crystallization success (Fig. 6 ▶ a). The number of crystal-producing conditions was similar for all three lipid sources and slight increases were observed in the number of crystal-producing conditions containing more than 5% lipid, with an average of two additional crystal conditions relative to those experiments containing less than 5% lipid (Fig. 6 ▶ a).


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)

The effect of the addition of lipids on LCP crystallization of RCs. (a) The number of conditions producing crystals out of a total of 48 from the ammonium sulfate and Jeffamine grid screen, as a function of increasing amounts of extracted R. sphaeroides lipids (squares, dotted line), polar E. coli lipids (triangles, unbroken line) and polar brain lipid (circles, dashed line). (b) The quality of the RC crystals produced in LCP trials as a function of increasing amounts of lipid additives. Data for extracted R. sphaeroides, polar E. coli and polar brain lipids were averaged. Values plotted represent either the total number of crystals observed (diamonds, unbroken line) or a high-quality (triangles, dotted line), medium-quality (circles, dotted line) or low-quality (squares, dotted line) visual score based on the size, degree of symmetry and edge definition of the crystals obtained.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2748966&req=5

fig6: The effect of the addition of lipids on LCP crystallization of RCs. (a) The number of conditions producing crystals out of a total of 48 from the ammonium sulfate and Jeffamine grid screen, as a function of increasing amounts of extracted R. sphaeroides lipids (squares, dotted line), polar E. coli lipids (triangles, unbroken line) and polar brain lipid (circles, dashed line). (b) The quality of the RC crystals produced in LCP trials as a function of increasing amounts of lipid additives. Data for extracted R. sphaeroides, polar E. coli and polar brain lipids were averaged. Values plotted represent either the total number of crystals observed (diamonds, unbroken line) or a high-quality (triangles, dotted line), medium-quality (circles, dotted line) or low-quality (squares, dotted line) visual score based on the size, degree of symmetry and edge definition of the crystals obtained.
Mentions: Since the formation of RC crystals in LCP-based crystallization trials was robust when challenged with non-RC protein, we tested whether the same holds for lipids. In this regard, the data show that the amount and source (polar brain, polar E. coli or extracted R. sphaeroides lipids) of the lipids added to the LCP-based crystallization experiments had no negative effect on crystallization success (Fig. 6 ▶ a). The number of crystal-producing conditions was similar for all three lipid sources and slight increases were observed in the number of crystal-producing conditions containing more than 5% lipid, with an average of two additional crystal conditions relative to those experiments containing less than 5% lipid (Fig. 6 ▶ a).

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