Crystallization chaperone strategies for membrane proteins

Lieberman, Raquel L.; Culver, Jeffrey A.; Entzminger, Kevin C.; Pai, Jennifer C.; Maynard, Jennifer A.

doi:10.1016/j.ymeth.2011.08.004

Cited by 34 publications

(21 citation statements)

References 100 publications

(120 reference statements)

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“…The co-crystallization of binding proteins with IMPs is an emerging technique, which has led to structure determination of a diverse set of membrane proteins [11]. The formation of an IMP/binding protein complex can not only enhance the solubility of IMPs and stabilize certain conformations, but also increases the hydrophilic protein surface for crystal contact formation.…”

Section: Discussionmentioning

confidence: 99%

“…Extraction of these proteins from the natural membrane environment in a detergent solution might enhance conformational dynamics contributing to increased sample heterogeneity and lower success rates in crystallization. A promising approach to increase the likelihood of crystal formation and to improve diffraction quality is the use of crystallization chaperones [8-11]. These chaperones typically represent macromolecules that have been selected to bind specifically to a given target protein.…”

Section: Introductionmentioning

confidence: 99%

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Nanobody Mediated Crystallization of an Archeal Mechanosensitive Channel

et al. 2013

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Mechanosensitive channels (MS) are integral membrane proteins and allow bacteria to survive sudden changes in external osmolarity due to transient opening of their pores. The efflux of cytoplasmic osmolytes reduces the membrane tension and prevents membrane rupture. Therefore these channels serve as emergency valves when experiencing significant environmental stress. The preparation of high quality crystals of integral membrane proteins is a major bottleneck for structure determination by X-ray crystallography. Crystallization chaperones based on various protein scaffolds have emerged as promising tool to increase the crystallization probability of a selected target protein. So far archeal mechanosensitive channels of small conductance have resisted crystallization in our hands. To structurally analyse these channels, we selected nanobodies against an archeal MS channel after immunization of a llama with recombinant expressed, detergent solubilized and purified protein. Here we present the characterization of 23 different binders regarding their interaction with the channel protein using analytical gel filtration, western blotting and surface plasmon resonance. Selected nanobodies bound the target with affinities in the pico- to nanomolar range and some binders had a profound effect on the crystallization of the MS channel. Together with previous data we show that nanobodies are a versatile and valuable tool in structural biology by widening the crystallization space for highly challenging proteins, protein complexes and integral membrane proteins.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Nanobody Mediated Crystallization of an Archeal Mechanosensitive Channel

et al. 2013

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“…In contrast, proteoliposome-targeted strategies have been used in anti-GPCR antibody screening via traditional hybridoma technology to successfully identify monoclonal antibodies against the human b 2 adrenergic receptor (b 2 AR), (21,22) human orphan receptor RAI3 (also known as GPRC5A and RAIG-1), (23) and human adenosine A 2A receptor (A 2A R). (24) Fab fragments to the b 2 AR and A 2A R were used as ''crystallization chaperones'' (25)(26)(27)(28) to facilitate structural determination.…”

Section: Epitope Mappingmentioning

confidence: 99%

Proteoliposome-based Selection of a Recombinant Antibody Fragment Against the Human M2 Muscarinic Acetylcholine Receptor

Suharni

Nomura

Arakawa

et al. 2014

Monoclonal Antibodies in Immunodiagnosis and Immunotherapy

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The development of antibodies against human G-protein-coupled receptors (GPCRs) has achieved limited success, which has mainly been attributed to their low stability in a detergent-solubilized state. We herein describe a method that can generally be applied to the selection of phage display libraries with human GPCRs reconstituted in liposomes. A key feature of this approach is the production of biotinylated proteoliposomes that can be immobilized on the surface of streptavidin-coupled microplates or paramagnetic beads and used as a binding target for antibodies. As an example, we isolated a single chain Fv fragment from an immune phage library that specifically binds to the human M2 muscarinic acetylcholine receptor with nanomolar affinity. The selected antibody fragment recognized the GPCR in both detergent-solubilized and membrane-embedded forms, which suggests that it may be a potentially valuable tool for structural and functional studies of the GPCR. The use of proteoliposomes as immunogens and screening bait will facilitate the application of phage display to this difficult class of membrane proteins.

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“…Using MPs in complex with a specific and tight-binding protein partner can considerably facilitate the challenging process of MP crystallization. This strategy is often referred to as ''crystallization chaperones'' (Koide 2009;Lieberman et al 2011), even though the term ''chaperone'' is used here in a somewhat relaxed sense. Indeed, a chaperone is not supposed to play a role in the final association states of its transiently protected partner, whereas the ''crystallization chaperone'' is still present in the final crystal.…”

Section: Introductionmentioning

confidence: 99%

Amphipol-Mediated Screening of Molecular Orthoses Specific for Membrane Protein Targets

et al. 2014

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Specific, tight-binding protein partners are valuable helpers to facilitate membrane protein (MP) crystallization, because they can i) stabilize the protein, ii) reduce its conformational heterogeneity, and iii) increase the polar surface from which well-ordered crystals can grow. The design and production of a new family of synthetic scaffolds (dubbed αReps, for "artificial alpha repeat protein") have been recently described. The stabilization and immobilization of MPs in a functional state are an absolute prerequisite for the screening of binders that recognize specifically their native conformation. We present here a general procedure for the selection of αReps specific of any MP. It relies on the use of biotinylated amphipols, which act as a universal "Velcro" to stabilize, and immobilize MP targets onto streptavidin-coated solid supports, thus doing away with the need to tag the protein itself.

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Crystallization chaperone strategies for membrane proteins

Cited by 34 publications

References 100 publications

Nanobody Mediated Crystallization of an Archeal Mechanosensitive Channel

Nanobody Mediated Crystallization of an Archeal Mechanosensitive Channel

Proteoliposome-based Selection of a Recombinant Antibody Fragment Against the Human M2 Muscarinic Acetylcholine Receptor

Amphipol-Mediated Screening of Molecular Orthoses Specific for Membrane Protein Targets

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