title: Crystallizing Membrane Proteins in the Lipidic Mesophase. Experience with Human Prostaglandin E2 Synthase 1 and an Evolving Strategy.
abstract: The lipidic mesophase or in meso method for crystallizing membrane proteins has several high profile targets to its credit and is growing in popularity. Despite its success, the method is in its infancy as far as rational crystallogenesis is concerned. Consequently, significant time, effort, and resources are still required to generate structure-grade crystals, especially with a new target type. Therefore, a need exists for crystallogenesis protocols that are effective with a broad range of membrane protein types. Recently, a strategy for crystallizing a prokaryotic α-helical membrane protein, diacylglycerol kinase (DgkA), by the in meso method was reported (Cryst. Growth. Des.2013, 14, 2846-2857). Here, we describe its application to the human α-helical microsomal prostaglandin E2 synthase 1 (mPGES1). While the DgkA strategy proved useful, significant modifications were needed to generate structure-quality crystals of this important therapeutic target. These included protein engineering, using an additive phospholipid in the hosting mesophase, performing multiple rounds of salt screening, and carrying out trials at 4 °C in the presence of a tight binding ligand. The crystallization strategy detailed here should prove useful for generating structures of other integral membrane proteins by the in meso method.
authors.affiliation: Membrane Structural and Functional Biology Group, School of Medicine and School of Biochemistry and Immunology, Trinity College Dublin, Dublin 2, Ireland.
authors.forename: Dianfan
authors.initials: D
authors.lastname: Li
authors.affiliation: Membrane Structural and Functional Biology Group, School of Medicine and School of Biochemistry and Immunology, Trinity College Dublin, Dublin 2, Ireland.
authors.forename: Nicole
authors.initials: N
authors.lastname: Howe
authors.affiliation: Membrane Structural and Functional Biology Group, School of Medicine and School of Biochemistry and Immunology, Trinity College Dublin, Dublin 2, Ireland.
authors.forename: Abhiram
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authors.lastname: Dukkipati
authors.affiliation: Membrane Structural and Functional Biology Group, School of Medicine and School of Biochemistry and Immunology, Trinity College Dublin, Dublin 2, Ireland.
authors.forename: Syed T A
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authors.affiliation: Molecular Discovery Research, GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage, SG1 2PA, U.K.
authors.forename: Benjamin D
authors.initials: BD
authors.lastname: Bax
authors.affiliation: Molecular Discovery Research, GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage, SG1 2PA, U.K.
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authors.affiliation: Molecular Discovery Research, GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage, SG1 2PA, U.K.
authors.forename: Angela
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authors.affiliation: Molecular Discovery Research, GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage, SG1 2PA, U.K.
authors.forename: Phil
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authors.lastname: Hardwicke
authors.affiliation: Molecular Discovery Research, GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage, SG1 2PA, U.K.
authors.forename: Onkar M P
authors.initials: OM
authors.lastname: Singh
authors.affiliation: Molecular Discovery Research, GlaxoSmithKline, Medicines Research Centre, Gunnels Wood Road, Stevenage, SG1 2PA, U.K.
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authors.lastname: Giblin
authors.affiliation: Departments of Lead Identification and Optimization Support, Boehringer Ingelheim Pharma GmbH & Co. KG, 88400 Biberach an der Riss, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Pautsch
authors.affiliation: Departments of Lead Identification and Optimization Support, Boehringer Ingelheim Pharma GmbH & Co. KG, 88400 Biberach an der Riss, Germany.
authors.forename: Roland
authors.initials: R
authors.lastname: Pfau
authors.affiliation: Departments of Lead Identification and Optimization Support, Boehringer Ingelheim Pharma GmbH & Co. KG, 88400 Biberach an der Riss, Germany.
authors.forename: Gisela
authors.initials: G
authors.lastname: Schnapp
authors.affiliation: Swiss Light Source, Paul Scherrer Institute , Villigen, Switzerland.
authors.forename: Meitian
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authors.lastname: Wang
authors.affiliation: Swiss Light Source, Paul Scherrer Institute , Villigen, Switzerland.
authors.forename: Vincent
authors.initials: V
authors.lastname: Olieric
authors.affiliation: Membrane Structural and Functional Biology Group, School of Medicine and School of Biochemistry and Immunology, Trinity College Dublin, Dublin 2, Ireland.
authors.forename: Martin
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authors.lastname: Caffrey