Crystallization chaperone strategies for membrane proteins
School of Chemistry and Biochemistry, Institute for Bioscience and Bioengineering, Georgia Institute of Technology, 901 Atlantic Drive NW, Atlanta, GA 30332, USA. Methods
(Impact Factor: 3.65).
08/2011; 55(4):293-302. DOI: 10.1016/j.ymeth.2011.08.004
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.
Available from: journals.iucr.org
- "Example uses of crystallization chaperones include the application of Fab antibody fragments to study the neurotransmitter sodium symporter LeuT in various conformational states (Krishnamurthy & Gouaux, 2012) and the fusion of T4 lysozyme to G-protein-coupled receptor (Zou et al., 2012). These approaches are proving to be useful for the stabilization of membrane proteins and as aids in their structure determination (Lieberman et al., 2011). "
[Show abstract] [Hide abstract]
ABSTRACT: Protein stability is a topic of major interest for the biotechnology, pharmaceutical and food industries, in addition to being a daily consideration for academic researchers studying proteins. An understanding of protein stability is essential for optimizing the expression, purification, formulation, storage and structural studies of proteins. In this review, discussion will focus on factors affecting protein stability, on a somewhat practical level, particularly from the view of a protein crystallographer. The differences between protein conformational stability and protein compositional stability will be discussed, along with a brief introduction to key methods useful for analyzing protein stability. Finally, tactics for addressing protein-stability issues during protein expression, purification and crystallization will be discussed.
Available from: Isabelle Broutin
- "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.
Available from: Yin Hoe Yau
- "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.
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.