The use of styrene maleic acid co-polymer (SMA) for membrane protein extraction and purification has grown in recent years. SMA inserts in the membrane and assembles into small discs of bilayer encircled by polymer, termed SMA lipid particles (SMALPs). This allows purification of membrane proteins whilst maintaining their lipid bilayer environment. SMALPs offer several improvements over conventional detergent approaches, however there are limitations, most notably a sensitivity to low pH and divalent cations. Recently it was shown that the aliphatic diisobutylene-maleic acid (DIBMA) copolymer, was also able to directly solubilize membranes forming DIBMALPs (DIBMA lipid particles), and that this polymer overcame some of the limitations of SMA. In this study the ability of DIBMA to solubilize and purify functional membrane proteins has been compared to SMA. It was found that DIBMA is able to solubilize several different membrane proteins from different expression systems, however for some proteins it gives a lower yield and lower degree of purity than SMA. DIBMA extracted G protein-coupled receptors retain ligand-and G proteinbinding. DIBMALPS are larger than SMALPs and display a decreased sensitivity to magnesium. However the stability of DIBMALPs appears to be lower than SMALPs. The lower purity and lower stability are likely linked to the larger size of the DIBMALP particle. However, this also offers a potentially less rigid lipid environment which may be more amenable to protein dynamics. Therefore the optimal choice of polymer will depend on which features of a protein are to be investigated.

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Combining the technologies of encapsulation of GPCRs in SMA lipid particles with fluorescence correlation spectroscopy provides a versatile characterisation platform.

Calcitonin gene-related peptide (CGRP) binds to the complex of the calcitonin receptor-like receptor (CLR) with receptor activity-modifying protein 1 (RAMP1). How CGRP interacts with the transmembrane domain (including the extracellular loops) of this family B receptor remains unclear. In this study, a photoaffinity cross-linker, p-azido l-phenylalanine (azF), was incorporated into CLR, chiefly in the second extracellular loop (ECL2) using genetic code expansion and unnatural amino acid mutagenesis. The method was optimized to ensure efficient photolysis of azF residues near the transmembrane bundle of the receptor. A CGRP analogue modified with fluorescein at position 15 was used for detection of ultraviolet-induced cross-linking. The methodology was verified by confirming the known contacts of CGRP to the extracellular domain of CLR. Within ECL2, the chief contacts were I284 on the loop itself and L291, at the top of the fifth transmembrane helix (TM5). Minor contacts were noted along the lip of ECL2 between S286 and L290 and also with M223 in TM3 and F349 in TM6. Full length molecular models of the bound receptor complex suggest that CGRP sits at the top of the TM bundle, with Thr of the peptide making contacts with L291 and H295. I284 is likely to contact Leu and Ala of CGRP, and Leu of CGRP is at the ECL/extracellular domain boundary of CLR. The reduced potency, E, and affinity of [LeuAla]-human α CGRP are consistent with this model. Contacts between Thr of CGRP and H295 may be particularly important for receptor activation.

AbstractThe β2-adrenoceptor (β2AR) is a well-established target in asthma and a prototypical GPCR for biophysical studies. Solubilisation of membrane proteins has classically involved the use of detergents. However, the detergent environment differs from the native membrane environment and often destabilises membrane proteins. Use of amphiphilic copolymers is a promising strategy to solubilise membrane proteins within their native lipid environment in the complete absence of detergents. Here we show the isolation of the β2AR in the polymer Diisobutylene maleic acid (DIBMA). We demonstrate that β2AR remains functional in the DIBMA lipid particle (DIBMALP) and shows improved thermal stability compared to the n-Dodecyl-β-D-Maltopyranoside (DDM) detergent solubilised β2AR. This unique method of extracting β2AR offers significant advantages over previous methods routinely employed such as the introduction of thermostabilising mutations and the use of detergents, particularly for functional biophysical studies.