Structure and function of membrane proteins encapsulated in a polymer-bound lipid bilayer

Abstract

New technologies for the purification of stable membrane proteins have emerged in recent years, in particular methods that allow the preparation of membrane proteins with their native lipid environment. Here, we look at the progress achieved with the use of styrene-maleic acid copolymers (SMA) which are able to insert into biological membranes forming nanoparticles containing membrane proteins and lipids. This technology can be applied to membrane proteins from any host source, and, uniquely, allows purification without the protein ever being removed from a lipid bilayer. Not only do these SMA lipid particles (SMALPs) stabilise membrane proteins, allowing structural and functional studies, but they also offer opportunities to understand the local lipid environment of the host membrane. With any new or different method, questions inevitably arise about the integrity of the protein purified: does it retain its activity; its native structure; and ability to perform its function? How do membrane proteins within SMALPS perform in existing assays and lend themselves to analysis by established methods? We outline here recent work on the structure and function of membrane proteins that have been encapsulated like this in a polymer-bound lipid bilayer, and the potential for the future with this approach. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain.

Examining the stability of membrane proteins within SMALPs

“…The sensitivity to Ni 2+ was particularly interesting given that we routinely use Ni-NTA affinity chromatography to purify SMALPencapsulated proteins. Binding of SMALP-encapsulated proteins to Ni-NTA resin has previously been reported to be problematic at times [5,14,18,29], but this has generally been considered to result from interactions between the resin and excess free SMA [18,29], and despite these issues there have been many reported success stories using Ni-NTA [6-11, 14, 16, 17, 29]. This suggests that the effective concentration of Ni 2+ in the bead slurry must be below 2 mM or perhaps the spatial arrangement of Ni 2+ attached to the beads doesn't allow multiple nickel ions to bind to the same SMALP in the manner that leads to precipitation.…”

“…In 2009 this issue was partially resolved by the use of styrene-maleic acid (SMA) copolymers for extracting small discs of membrane, termed SMA lipid particles (SMALPs) from native membranes [3]. This method has greatly simplified how membrane proteins can be purified and studied, whilst maintaining the lipid bilayer environment of the protein [3][4][5].…”

Mechanism of gating and partial agonist action in the glycine receptor

“…Here we isolated the full length GlyR with native lipids and determined high resolution structures of the receptor bound to glycine, taurine or GABA. The receptors were imaged by cryo-electron microscopy (cryo-EM) in MSP nanodiscs or after solubilization by styrene maleic acid (SMA) copolymers 26 , showing by MD simulations that the SMA complex adopts a physiologically relevant open state. Importantly, cryo-EM structures of GlyR bound to taurine or GABA reveal, for the first time, agonist-bound closed states as well as open and desensitized states.…”

Structural Mass Spectrometry of Membrane Proteins within Their Native Lipid Environments

“…Here, we consider the most basic native system to be a membrane protein retained within a lipid bilayer consisting of its native cellular lipid composition, where it has never been delipidated from its surrounding cellular lipid milieu. This can be achieved using the rapidly developing native nanodisc technologies, which have become increasingly used in biochemical and structural biology investigations of membrane proteins . Native nanodiscs utilize an expanding repertoire of amphipathic polymers, styrene‐maleic acid (SMA) being the most popular, to solubilize a membrane protein within its surrounding lipid bilayer directly from a cell membrane forming a “native nanodisc” (Figure a).…”

“…This can be achieved using the rapidly developing native nanodisc technologies, [11] which have become increasingly used in biochemical and structural biology investigations of membrane proteins. [12] Native nanodiscs utilize an expanding repertoire of amphipathic polymers,s tyrene-maleic acid (SMA) being the most popular,t os olubilize am embrane protein within its surrounding lipid bilayer directly from ac ell membrane forming a "native nanodisc" (Figure 2a). Importantly,t he membrane protein can be contained within its native lipid bilayer,h as never been in contact with potentially destabilizing detergents and/ or has not been reconstituted into al ipid bilayer of non-native composition.…”