Membrane protein extraction and purification using partially-esterified SMA polymers

Abstract

Styrene maleic acid (SMA) polymers have proven to be very successful for the extraction of membrane proteins, forming SMA lipid particles (SMALPs), which maintain a lipid bilayer around the membrane protein. SMALP-encapsulated membrane proteins can be used for functional and structural studies. The SMALP approach allows retention of important protein-annular lipid interactions, exerts lateral pressure, and offers greater stability than traditional detergent solubilisation. However, SMA polymer does have some limitations, including a sensitivity to divalent cations and low pH, an absorbance spectrum that overlaps with many proteins, and possible restrictions on protein conformational change. Various modified polymers have been developed to try to overcome these challenges, but no clear solution has been found. A series of partially-esterified variants of SMA (SMA 2625, SMA 1440 and SMA 17352) has previously been shown to be highly effective for solubilisation of plant and cyanobacterial thylakoid membranes. It was hypothesised that the partial esterification of maleic acid groups would increase tolerance to divalent cations. Therefore, these partially-esterified polymers were tested for the solubilisation of lipids and membrane proteins, and their tolerance to magnesium ions. It was found that all partially esterified polymers were capable of solubilising and purifying a range of membrane proteins, but the yield of protein was lower with SMA 1440, and the degree of purity was lower for both SMA 1440 and SMA 17352. SMA 2625 performed comparably to SMA 2000. SMA 1440 also showed an increased sensitivity to divalent cations. Thus, it appears the interactions between SMA and divalent cations are more complex than proposed and require further investigation.

Mechanisms of Formation, Structure, and Dynamics of Lipoprotein Discs Stabilized by Amphiphilic Copolymers: A Comprehensive Review

“…Divalent ions, predominantly Ca 2+ and Mg 2+ , are essential for a number of enzymes and other proteins, including ion channels [ 48 , 49 , 50 ] and GPCRs [ 51 ]. At the same time, these ions tend to be chelated by carboxylate groups and at some concentration lead to precipitation of carboxyl-containing polymers/lipodiscs, apparently because Ca 2+ and Mg 2+ ions induce conformational strain or a conformational change in the polymer [ 42 , 52 , 53 ]. The most notable effect is observed for SMA, which does not tolerate the concentration of Ca 2+ over 1 [ 42 ] or 2 mM [ 20 , 21 ], though a bit higher tolerable concentration of Mg 2+ (up to 4 mM) has been shown for SMA 2000 (Cray Valley) in [ 42 ].…”

“…However, there exists at least one counterexample. SMA with maleic acid residues partially esterified with 2-butoxyethanol demonstrated increased sensitivity to Mg 2+ [ 53 , 54 ] as compared to unmodified SMA. A possible explanation for this behavior might be similar to the one proposed for SMA-PA as the resulting pending moiety is rather long, flexible, and hydrophobic (see the previous paragraph).…”

Detergent-Free Isolation of Membrane Proteins and Strategies to Study Them in a Near-Native Membrane Environment

“…Additionally, the SMI polymer has been shown to be compatible with the functional reconstitution of human adenosine A 2A receptor (A 2A R) and the human V 1a vasopressin receptor (V 1a R) expressed in HEK 293T cells [ 192 ]. The partial-esterification of SMA polymers caused lower protein yields and increased sensitivity to divalent ions [ 196 ]. The functionalization of maleic acid units in SMA with alkoxy ethoxylates of increasing alkoxy chain length improved the solubilization efficacy of trimeric photosystem-I from the membranes of cyanobacterium Thermosynechococcus elongates [ 176 ].…”

Effect of Cholesterol on the Structure and Composition of Glyco-DIBMA Lipid Particles

“…Polymer–lipid particles composed of SMA and DIBMA, also known as SMALPs and DIBMALPs, respectively, have been used successfully for the stabilization of membrane proteins in synthetic lipid environments and for direct extraction of membrane proteins from their native cellular membrane. ,− SMA was the first co-polymer introduced for successful extraction of membrane protein from the native membrane. However, the ultraviolet (UV) absorption associated with the styrene group prevents the quantification of the protein concentration by light absorption in the UV spectrum, which is an important aspect during sample preparation .…”