From G protein-coupled receptors to ion channels, membrane proteins represent over half of known drug targets. Yet, structure-based drug discovery is hampered by the dearth of available three-dimensional models for this large category of proteins. Other than efforts to improve membrane protein expression and stability, current strategies to improve the ability of membrane proteins to crystallize involve examining many orthologs and DNA constructs, testing the effects of different detergents for purification and crystallization, creating a lipidic environment during crystallization, and cocrystallizing with covalent or non-covalent soluble protein chaperones with an intrinsic high propensity to crystallize. In this review, we focus on this last category, highlighting successes of crystallization chaperones in membrane protein structure determination and recent developments in crystal chaperone engineering, including molecular display to enhance chaperone crystallizability, and end with a novel generic approach in development to target any membrane protein of interest.
"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. "
[Show abstract][Hide abstract] ABSTRACT: 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.
"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. "
[Show abstract][Hide abstract] ABSTRACT: 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.
PLoS ONE 10/2013; 8(10):e77984. DOI:10.1371/journal.pone.0077984 · 3.23 Impact Factor
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