Detergent-free purification and reconstitution of functional human serotonin transporter (SERT) using diisobutylene maleic acid (DIBMA) copolymer

Abstract

Structure and function analysis of human membrane proteins in lipid bilayer environments is acutely lacking despite the fundame1ntal cellular importance of these proteins and their dominance of drug targets. An underlying reason is that detailed study usually requires a potentially destabilising detergent purification of the proteins from their host membranes prior to subsequent reconstitution in a membrane mimic; a situation that is exacerbated for human membrane proteins due to the inherent difficulties in overexpressing suitable quantities of the proteins. We advance the promising styrene maleic acid polymer (SMA) extraction approach to introduce a detergent-free method of obtaining stable, functional human membrane transporters in bilayer nanodiscs directly from yeast cells. We purify the human serotonin transporter (hSERT) following overexpression in Pichia pastoris using diisobutylene maleic acid (DIBMA) as a superior method to traditional detergents or the more established styrene maleic acid polymer. hSERT plays a pivotal role in neurotransmitter regulation being responsible for the transport of the neurotransmitter 5-hydroxytryptamine (5-HT or serotonin). It is representative of the neurotransmitter sodium symporter (NSS) family, whose importance is underscored by the numerous diseases attributed to their malfunction. We gain insight into hSERT activity through an in vitro transport assay and find that DIBMA extraction improves the thermostability and activity of hSERT over the conventional detergent method.

Differences in SMA-like polymer architecture dictate the conformational changes exhibited by the membrane protein rhodopsin encapsulated in lipid nano-particles

“…For example, the thermostability of the human serotonin transporter (hSERT) is comparable in a SMALP or a DIBMALP but the A 2A R in a DIBMALP was reported to be less stable to long-term storage (6 days) at 4°C than in a SMALP. 44,46 Rho-LP were characterised further using sedimentation velocity analytical ultracentrifugation (AUC; Fig. 5).…”

Biological insights from SMA-extracted proteins

“…Many studies of SMALP-encapsulated proteins have carried out established functional assays to show that the proteins within the SMALPs were indeed functioning, and that this was comparable to either detergent solubilised protein or within native membranes. Techniques included radioligand binding assays [42][43][44]47,60], spectroscopic binding assays [19,30,32,43,47], characteristic spectra [27,40,61], NMR based assays [10,62], and measurement of channel activity [13,63]. One limitation of SMALP structure, with both sides of the membrane freely accessible is the inability to measure vectorial transport.…”

“…One limitation of SMALP structure, with both sides of the membrane freely accessible is the inability to measure vectorial transport. However, several studies have now demonstrated effective reconstitution of proteins from SMALPs or DIBMALPs into proteoliposomes for measurement of transport [ 60 , 64 , 65 ].…”

“…Furthermore, the rhomboid protease from Vibrio cholerae VcROM, which is proteolytically unstable in detergents, remained intact in DIBMA. Similarly, the transport activity of reconstituted human serotonin transporter, hSERT, showed a 5-fold higher level of activity when purified within DIBMALPs than with detergent [ 60 ].…”

Membrane protein extraction and purification using partially-esterified SMA polymers

“…The less restricted environment within DIBMA particles has been shown to allow full function of proteins which were restricted from completing all conformational changes within SMALPs [39] . However, although DIBMA works well for some proteins such as GPCRs, for others, such as the ABC transporter BmrA, it gives a lower yield of protein, lower purity and decreased stability [40] , [41] .…”