The use of styrene-maleic acid (SMA) copolymers to extract and purify transmembrane proteins, while retaining their native bilayer environment, overcomes many of the disadvantages associated with conventional detergent-based procedures. This approach has huge potential for the future of membrane protein structural and functional studies. In this investigation, we have systematically tested a range of commercially available SMA polymers, varying in both the ratio of styrene and maleic acid and in total size, for the ability to extract, purify and stabilise transmembrane proteins. Three different membrane proteins (BmrA, LeuT and ZipA), which vary in size and shape, were used. Our results show that several polymers, can be used to extract membrane proteins, comparably to conventional detergents. A styrene:maleic acid ratio of either 2:1 or 3:1, combined with a relatively small average molecular mass (7.5-10 kDa), is optimal for membrane extraction, and this appears to be independent of the protein size, shape or expression system. A subset of polymers were taken forward for purification, functional and stability tests. Following a one-step affinity purification, SMA 2000 was found to be the best choice for yield, purity and function. However, the other polymers offer subtle differences in size and sensitivity to divalent cations that may be useful for a variety of downstream applications.

Amphipathic co-polymers such as styrene-maleic acid (SMA) have gained popularity over the last few years due to their ability and ease of use in solubilising and purifying membrane proteins in comparison to conventional methods of extraction such as detergents. SMA2000 is widely used for membrane protein studies and is considered as the optimal polymer for this technique. In this study a side-by-side comparison of SMA2000 with the polymer SZ30010 was carried out as both these polymers have similar styrene:maleic acid ratios and average molecular weights. Ability to solubilise, purify and stabilise membrane proteins was tested using three structurally different membrane proteins. Our results show that both polymers can be used to extract membrane proteins at a comparable efficiency to conventional detergent dodecylmaltoside (DDM). SZ30010 was found to give a similar protein yield and, SMALP disc size as SMA2000, and both polymers offered an increased purity and increased thermostability compared to DDM. Further investigation was conducted to investigate SMALP sensitivity to divalent cations. It was found that the sensitivity is polymer specific and not dependent on the protein encapsulated. Neither is it affected by the concentration of SMALPs. Larger divalent cations such as Co 2+ and Zn 2+ resulted in an increased sensitivity. Highlights: SMA polymers SZ300110 and SMA2000 are comparable for protein solubilisation, yield, purity and thermostability.  Sensitivity of SMALPs to Mg 2+ is similar for different membrane proteins.  The sensitivity to Mg 2+ is independent of the concentration of SMALPs  SMALPs are even more sensitive to larger divalent cations

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.

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