A mutagenesis and screening strategy to generate optimally thermostabilized membrane proteins for structural studies

Abstract

The thermostability of an integral membrane protein in detergent solution is a key parameter that dictates the likelihood of obtaining well-diffracting crystals suitable for structure determination. However, many mammalian membrane proteins are too unstable for crystallisation. We developed a thermostabilisation strategy based on systematic mutagenesis coupled to a radioligand-binding thermostability assay that can be applied to receptors, ion channels and transporters. It takes approximately 6-12 months to thermostabilise a G protein-coupled receptor (GPCR) containing 300 amino acid residues. The resulting thermostabilised membrane proteins are more easily crystallised and result in high-quality structures. This methodology has facilitated structure-based drug design applied to GPCRs, because it is possible to determine multiple structures of the thermostabilised receptors bound to low affinity ligands. Protocols and advice are given on how to develop thermostability assays for membrane proteins and how to combine mutations to make an optimally stable mutant suitable for structural studies.

Strategic Screening and Characterization of the Visual GPCR-mini-G Protein Signaling Complex for Successful Crystallization

“…However, most membrane proteins do not have such a convenient chromophore to follow functionality during purification. Other options would be to make a ligand detectable by adding a light-detectable chromophore or by using radioligand-binding and thermal shift assays 25 .…”

Membrane protein engineering to the rescue

“…However, advances within the field of protein engineering are providing new tools to overcome these difficulties. Proteins can be edited to yield water soluble domains [23] , fused to solubilising protein tags to produce water soluble variants [29] , or have their stability enhanced to aid in crystallisation or detergent compatibility [46] . The ingenuity of protein engineers has led to many new innovations and it is truly an exciting time to be working in the area of membrane protein research.…”

“…This methodology has the ability to produce a stabilised GPCR with a favoured conformation, making it ideal for crystallisation and structural characterisation [43,45] . This technique relies on a program of single residue substitutions, typically switched to alanine, systematically introduced at each position along the protein sequence [46] [47] . Each variant is then expressed in cells, detergent solubilised, and heated to just above the melting temperature (Tm) of the native protein.…”

“…Preparing, expressing and screening these mutations (potentially hundreds of constructs for a single GPCR) is a large undertaking, but using high throughput techniques and automation where necessary can reduce some of the experimental burden [46] . An alternative strategy is to generate random mutations by processes such as error prone PCR and screen for binding on the pool of variants using fluorescent ligands and fluorescently activated cell sorting (FACS) equipment [49,50] .…”

Crystal structure of a ligand-free stable TSH receptor leucine-rich repeat domain

“…Combined mutations improved the thermostability of the adenosine A 2A receptor (226-fold; Lebon et al 2011) and of the β 1 -adrenergic receptor (900fold; Miller & Tate 2011). Such increased thermostability has enabled the purification and crystallisation of several membrane proteins including GPCRs, transporters and ion channels (Magnani et al 2016).…”