The Novel Application of Geometric Morphometrics with Principal Component Analysis to Existing G Protein-Coupled Receptor (GPCR) Structures.

The G protein-coupled receptor (GPCR) superfamily is a large group of membrane proteins which, because of their vast involvement in cell signalling pathways, are implicated in a plethora of disease states and are therefore considered to be key drug targets. Despite advances in techniques to study these receptors, current prophylaxis is often limited due to the challenging nature of their dynamic, complex structures. Greater knowledge and understanding of their intricate structural rearrangements will therefore undoubtedly aid structure-based drug design against GPCRs. Disciplines such as anthropology and palaeontology often use geometric morphometrics to measure variation between shapes and we have therefore applied this technique to analyse GPCR structures in a three-dimensional manner, using principal component analysis. Our aim was to create a novel system able to discriminate between GPCR structures and discover variation between them, correlated with a variety of receptor characteristics. This was conducted by assessing shape changes at the extra- and intracellular faces of the transmembrane helix bundle, analysing the XYZ coordinates of the amino acids at those positions. We have demonstrated that GPCR structures can be classified based on characteristics such as activation state, bound ligands and fusion proteins, with the most significant results focussed at the intracellular face. Conversely, our analyses provide evidence that thermostabilising mutations do not cause significant differences when compared to non-mutated GPCRs. We believe that this is the first time geometric morphometrics has been applied to membrane proteins on this scale, and believe it can be used as a future tool in sense-checking newly resolved structures and planning experimental design.

Expression and purification of recombinant G protein-coupled receptors: A review.

Given their extensive role in cell signalling, GPCRs are significant drug targets; despite this, many of these receptors have limited or no available prophylaxis. Novel drug design and discovery significantly rely on structure determination, of which GPCRs are typically elusive. Progress has been made thus far to produce sufficient quantity and quality of protein for downstream analysis. As such, this review highlights the systems available for recombinant GPCR expression, with consideration of their advantages and disadvantages, as well as examples of receptors successfully expressed in these systems. Additionally, an overview is given on the use of detergents and the styrene maleic acid (SMA) co-polymer for membrane solubilisation, as well as purification techniques.

Single molecule fluorescence for membrane proteins.

The cell membrane is a complex milieu of lipids and proteins. In order to understand the behaviour of individual molecules is it often desirable to examine them as purified components in in vitro systems. Here, we detail the creation and use of droplet interface bilayers (DIBs) which, when coupled to TIRF microscopy, can reveal spatiotemporal and kinetic information for individual membrane proteins. A number of steps are required including modification of the protein sequence to enable the incorporation of appropriate fluorescent labels, expression and purification of the membrane protein and subsequent labelling. Following creation of DIBs, proteins are spontaneously incorporated into the membrane where they can be imaged via conventional single molecule TIRF approaches. Using this strategy, in conjunction with step-wise photobleaching, FRET and/or single particle tracking, a host of parameters can be determined such as oligomerisation state and dynamic information. We discuss advantages and limitations of this system and offer guidance for successful implementation of these approaches.