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Solubilization of membrane proteins by poly(styrene-co-maleic acid) salts (pSMA-S) has significant potential for membrane protein studies. This approach provides an opportunity to overcome many disadvantages associated with traditional detergent-based technique including protein denaturation and displacement of boundary lipids which may offer both...
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... spectroscopy was also used to verify the completion of hydrolysis of the maleic anhydride moiety. In Fig. 5, the parent SMA copolymers have distinct anhydride bands around 1770-1780 cm -1 . For the hydrolyzed SMA copolymers, these bands are almost completely displaced by the stretching vibrations of carboxylate ion at 1400 and 1560 cm -1 along with the appearance of the OH band near 3200 cm -1 . Presence of a band centered at 1770-1780 cm -1 ...
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... completely displaced by the stretching vibrations of carboxylate ion at 1400 and 1560 cm -1 along with the appearance of the OH band near 3200 cm -1 . Presence of a band centered at 1770-1780 cm -1 in spectra of hydrolyzed polymers suggests that the hydrolyzed form of SMA copolymer contains a small amount (less than 5 %) of anhydride groups (Fig. 5, red arrows). The degree of anhydride hydrolyses was quantified by a decrease in the integrated area of the anhydride carbonyl band from absorbance spectra after its baseline correction and normalization using internal reference bands (699 cm -1 for ...
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... 35−37 Recent high-throughput evaluation of SMA polymers has identified approaches that allow efficient solubilization of the thylakoid. 38,39 To attempt to isolate LHCII clusters with SMA (Figure 2A), we utilized lincomycin-treated Arabidopsis minor antennaknockout mutants (NoM) that constitutively overexpress the qE locus and downregulate PSII and photosystem I (PSI) reaction centers while remaining green. 3 This system has been well characterized in Arabidopsis, 3,40,41 with similar methods also employed in the green alga Chlamydomonas. ...
The ability to harvest light effectively in a changing environment is necessary to ensure efficient photosynthesis and crop growth. One mechanism, known as qE, protects photosystem II (PSII) and regulates electron transfer through the harmless dissipation of excess absorbed photons as heat. This process involves reversible clustering of the major light-harvesting complexes of PSII (LHCII) in the thylakoid membrane and relies upon the ΔpH gradient and the allosteric modulator protein PsbS. To date, the exact role of PsbS in the qE mechanism has remained elusive. Here, we show that PsbS induces hydrophobic mismatch in the thylakoid membrane through dynamic rearrangement of lipids around LHCII leading to observed membrane thinning. We found that upon illumination, the thylakoid membrane reversibly shrinks from around 4.3 to 3.2 nm, without PsbS, this response is eliminated. Furthermore, we show that the lipid digalactosyldiacylglycerol (DGDG) is repelled from the LHCII-PsbS complex due to an increase in both the pKa of lumenal residues and in the dipole moment of LHCII, which allows for further conformational change and clustering in the membrane. Our results suggest a mechanistic role for PsbS as a facilitator of a hydrophobic mismatch-mediated phase transition between LHCII-PsbS and its environment. This could act as the driving force to sort LHCII into photoprotective nanodomains in the thylakoid membrane. This work shows an example of the key role of the hydrophobic mismatch process in regulating membrane protein function in plants.
... Interestingly, we observed the increased specific enzyme activity in the samples of photo-PSMA and 1000P in comparison to the 2000P sample. This is quite different from most of the previous published studies [40][41][42]. It is unclear what causes the worsening of 2000P. ...
... The decreased Z-average diameters for the 1:1 PSMA vesicles (photo-PSMA and 1000P) as compared to that of 2000P (Fig. 12) together with the enhanced specific enzyme activity by the 1:1 PSMA extractions (Fig. 11), added support for the need of amphipathic PSMA type (50:50 mol%) in recovery of soybean lipolytic enzymes. This was different from the previous studies in which the 2:1 PSMA was more preferable [40][41][42]. As such, our method may be suitable for certain protein types, such as those amphipathic soybean esterases and lipases, but not for a wider range of membrane protein systems. ...
Approaches aiming to recover proteins without denaturation represent attractive strategies. To accomplish this, a membrane lysis agent based on poly(styrene-alt-maleic acid) or PSMA was synthesized by photopolymerization using Irgacure® 2959 and carbon tetrabromide (CBr4) as a radical initiator and a reversible chain transfer agent, respectively. Structural elucidation of our in-house synthesized PSMA, so-called photo-PSMA, was performed by using NMR spectroscopy. The use of this photo-PSMA in soybean enzyme extraction was also demonstrated for the first time in this study. Without a severe cell rupture, energy input or any organic solvent, recovery of lipolytic enzymes directly into nanometric-sized particles was accomplished in one-step process. Due to the improved structural regularity along the photo-PSMA backbone, the most effective protective reservoir for enzyme immobilization was generated through the PSMA aggregation. Formation of such reservoir enabled soybean enzymes to be shielded from the surroundings and resolved in their full functioning state. This was convinced by the increased specific lipolytic activity to 1,950 mU/mg, significantly higher than those of sodium dodecyl sulfate (SDS) and the two commercially-available PSMA sources (1000P and 2000P). Our photo-PSMA had thus demonstrated its great potential for cell lyse application, especially for soybean hydrolase extraction.
... Lipids were quantified using tandem mass spectrometry via comparison to internal standards and the protein mass was determined by bicinchoninic acid protein analysis (BCA). However, our group previously discovered that the solubilization efficiency of plant thylakoids by a number of SMA copolymers when determined by protein mass (by BCA) and pigment content (spectrophotometrically) had a similar trend but were not exact, for purposes not fully understood [38]. For this reason, we decided to confirm the ratio of chl:protein between the DDM and SMALP extracted samples using laser flash photolysis (Fig. 7). ...
The use of styrene-maleic acid copolymers (SMAs) to produce membrane protein-containing nanodiscs without the initial detergent isolation has gained significant interest over the last decade. We have previously shown that a Photosystem I SMALP from the thermophilic cyanobacterium, Thermosynechococcus elongatus (PSI-SMALP), has much more rapid energy transfer and charge separation in vitro than detergent isolated PSI complexes. In this study, we have utilized small-angle neutron scattering (SANS) to better understand the geometry of these SMALPs. These techniques allow us to investigate the size and shape of these particles in their fully solvated state. Further, the particle's proteolipid core and detergent shell or copolymer belt can be interrogated separately using contrast variation, a capability unique to SANS. Here we report the dimensions of the Thermosynechococcus elongatus PSI-SMALP containing a PSI trimer. At ~1.5 MDa, PSI-SMALP is the largest SMALP to be isolated; our lipidomic analysis indicates it contains ~1300 lipids/per trimeric particle, >40-fold more than the PSI-DDM particle and > 100 fold more than identified in the 1JB0 crystal structure. Interestingly, the lipid composition to the PSI trimer in the PSI-SMALP differs significantly from bulk thylakoid composition, being enriched ~50 % in the anionic sulfolipid, SQDG. Finally, utilizing the contrast match point for the SMA 1440 copolymer, we also can observe the ~1 nm SMA copolymer belt surrounding this SMALP for the first time, consistent with most models of SMA organization.
... SMA and related copolymers found many diverse applications in structural biology. First of all, they were efficient in solubilization of a wide range of membrane proteins, including GPCRs [107][108][109][110][111][112], ABC transporters [113][114][115][116], ion channels [117][118][119], photoreaction centers [61,120,121], and electron transport chain complexes [62], expressed in bacteria [42,[122][123][124][125], yeast [126][127][128][129], insect [116,130,131], and mammalian cells [115,132,133], as well as plants [134]. As reviewed by Overduin and Esmaili, SMA is effective for solubilizing both monomeric and oligomeric proteins as well as those that are unstable, low-abundance, or lipid-dependent [13]. ...
Amphiphilic copolymers consisting of alternating hydrophilic and hydrophobic units account for a major recent methodical breakthrough in the investigations of membrane proteins. Styrene–maleic acid (SMA), diisobutylene–maleic acid (DIBMA), and related copolymers have been shown to extract membrane proteins directly from lipid membranes without the need for classical detergents. Within the particular experimental setup, they form disc-shaped nanoparticles with a narrow size distribution, which serve as a suitable platform for diverse kinds of spectroscopy and other biophysical techniques that require relatively small, homogeneous, water-soluble particles of separate membrane proteins in their native lipid environment. In recent years, copolymer-encased nanolipoparticles have been proven as suitable protein carriers for various structural biology applications, including cryo-electron microscopy (cryo-EM), small-angle scattering, and conventional and single-molecule X-ray diffraction experiments. Here, we review the current understanding of how such nanolipoparticles are formed and organized at the molecular level with an emphasis on their chemical diversity and factors affecting their size and solubilization efficiency.
... A series of partially-esterified variants of SMA has previously been tested for the solubilisation of plant and cyanobacterial thylakoid membranes [33,[42][43][44]. These polymers, SMA 2625, SMA 1440 and SMA 17352, have various chemical groups attached to some of the maleic acid groups of SMA ( Fig. 1 & Table 1). ...
... All of the polymers were equally capable of solubilising various membrane proteins, from different expression systems (Fig. 3). This contrasts somewhat with previous studies using thylakoid membranes from spinach or from cyanobacteria, where SMA 1440 was considerably more efficient at solubilisation, followed by SMA 30010, SMA 17352, SMA 2625 and then SMA 2000 [42][43][44]. The reasons for this difference are not clear, but the thylakoid membranes are very densely packed and contain more galactolipid, so differences in polymer-lipid interactions may be responsible. ...
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 SMA1440 and SMA17352. SMA2625 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.
... Experimental data shows that SMA2:1 (SMA2000) and 3:1 (SMA3000, SZ25010) with an average molecular mass of 7.5-10 kDa can be equally effective in direct purification of membrane proteins from bacterial membrane and spinach chloroplast thylakoids [59,60]. On the contrary, SMA 1:1 (SMA1000), SMA2021, SMA10235, SMA17352, and SZ09008, SZ09006, SZ40005, SZ42010, SZ33030, SZ28065, SZ28110, and SZ2625 were not as useful. ...
Unlike cytosolic proteins, membrane proteins (MPs) are embedded within the plasma membrane and the lipid bilayer of intracellular organelles. MPs serve in various cellular processes and account for over 65% of the current drug targets. The development of membrane mimetic systems such as bicelles, short synthetic polymers or amphipols, and membrane scaffold proteins (MSP)-based nanodiscs has facilitated the accommodation of synthetic lipids to stabilize MPs, yet the preparation of these membrane mimetics remains detergent-dependent. Bio-inspired synthetic polymers present an invaluable tool for excision and liberation of superstructures of MPs and their surrounding annular lipid bilayer in the nanometric discoidal assemblies. In this article, we discuss the significance of self-assembling process in design of biomimetic systems, review development of multiple series of amphipathic polymers and the significance of these polymeric “belts” in biomedical research in particular in unraveling the structures, dynamics and functions of several high-value membrane protein targets.
... 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. ...
... SMA3000, SMA EF30 and SZ25010 have a styrene:maleic acid ratio of ∼3 : 1 whilst SMA2000 and SZ30010 have a ratio of 2 : 1 and 2.3 : 1, respectively. SMA1440 is a partially esterified variant of SMA[27]. DIBMA is a 1 : 1 alternating polymer of diisobutylene and maleic acid. ...
In the twelve years since styrene maleic acid (SMA) was first used to extract and purify a membrane protein within a native lipid bilayer, this technological breakthrough has provided insight into the structural and functional details of protein–lipid interactions. Most recently, advances in cryo-EM have demonstrated that SMA-extracted membrane proteins are a rich-source of structural data. For example, it has been possible to resolve the details of annular lipids and protein–protein interactions within complexes, the nature of lipids within central cavities and binding pockets, regions involved in stabilising multimers, details of terminal residues that would otherwise remain unresolved and the identification of physiologically relevant states. Functionally, SMA extraction has allowed the analysis of membrane proteins that are unstable in detergents, the characterization of an ultrafast component in the kinetics of electron transfer that was not possible in detergent-solubilised samples and quantitative, real-time measurement of binding assays with low concentrations of purified protein. While the use of SMA comes with limitations such as its sensitivity to low pH and divalent cations, its major advantage is maintenance of a protein's lipid bilayer. This has enabled researchers to view and assay proteins in an environment close to their native ones, leading to new structural and mechanistic insights.
... Based on the efficacy of both protein and chlorophyll extraction, we have found five SMA copolymers: SMA ® 1440, XIRAN ® 25010, XIRAN ® 30010, SMA ® 17352, and SMA ® PRO 10235, that show promise in extracting supramolecular protein complexes from spinach TMs. The current study extends earlier works that demonstrated the application of SMA copolymers for solubilization of higher plant [13,17] and cyanobacterial [18] thylakoids. To advance the application of SMA copolymers for studying TM organization [17] we examined the correlation between physicochemical properties of five SMA copolymers and composition of isolated photosynthetic supramolecular protein complexes extracted from two biological membranesspinach and pea TMs. ...
... For this study, commercially hydrolyzed SMA copolymerspoly(styrene-co-maleic acid) ammonium or sodium salts without additional purification were used. PRO 10235 supplied as anhydride was converted to ammonium salt by refluxing the polymer with continuous stirring in the presence of ammonium hydroxide at 75 °C for 5 h [13]. To avoid photooxidative degradation of copolymers due to photo-crosslinking and chromophore formation [26], aqueous polymer solutions with mass fraction 10 % were stored away from direct sunlight at room temperature. ...
... Mature spinach leaves were purchased locally and stored overnight in the dark at 4 °C. Total TMs from intact chloroplasts were isolated from spinach leaves or pea seedlings according to a previously described method [13] adapted from Bruce et al. [27]. All preparative procedures were carried out on ice in dim lightning to minimize light-associated degradation of chlorophyll-containing proteins. ...
Derivatives of poly(styrene-co-maleic acid) (pSMA), have recently emerged as effective reagents for extracting membrane protein complexes from biological membranes. Despite recent progress in using SMAs to study artificial and bacterial membranes, very few reports have addressed their use in studying the highly abundant and well characterized thylakoid membranes. Recently, we tested the ability of twelve commercially available SMA copolymers with different physicochemical properties to extract membrane protein complexes (MPCs) from spinach thylakoid membrane. Based on the efficacy of both protein and chlorophyll extraction, we have found five highly efficient SMA copolymers: SMA® 1440, XIRAN® 25010, XIRAN® 30010, SMA® 17352, and SMA® PRO 10235, that show promise in extracting MPCs from chloroplast thylakoids. To further advance the application of these polymers for studying biomembrane organization, we have examined the composition of thylakoid supramolecular protein complexes extracted by the five SMA polymers mentioned above. Two commonly studied plants, spinach (Spinacia oleracea) and pea (Pisum sativum), were used for extraction as model biomembranes. We found that the pSMAs differentially extract protein complexes from spinach and pea thylakoids. Based on their differential activity, which correlates with the polymer chemical structure, pSMAs can be divided into two groups: unfunctionalized polymers and ester derivatives.
... In our previous study, we focused on 1:1 styrene:maleic amide molar ratio (St:MA) zSMAs with molecular weight greater than 10 kDa 19 . In reference to studies on traditional SMAs that solubilize membranes into nanodiscs, oftentimes the commercially-available SMAs with molecular weight smaller than 10 kDa were used [20][21][22][23][24][25][26][27][28] , and the best membrane solubilization was reported with 2:1 St:MA SMAs 23,27,29 . Here, we compared the extraction/ reconstitution of membrane proteins into LNDs by a series of zSMAs with different St:MAs (i.e., 1:1, 2:1, and 3:1), molecular weight, and polydispersity indices (i.e., in-house prepared zSMAs via controlled/"living" polymerization vs zSMAs derived from commercial SMAs). ...
... In any case, increasing the temperature from RT to 37 °C has only minor effects on solubilization, and therefore solubilization by zSMA and M zSMA copolymers can be carried out at RT or above without compromising efficiency. Increasing ionic strength is believed to improve solubilization by traditional SMAs due to a decrease of the electrostatic repulsion between the negatively charged membranes and maleic acid groups, which facilitates SMA binding, the first step in membrane solubilization 26,27,29 . Generally, the presence of 100-200 mM NaCl is sufficient, and high salt concentration can be detrimental because the negative charges on traditional SMAs are important to keep the copolymers soluble in aqueous solution 27 . ...
... Of these, the 2:1 zSMA showed the best performance, with solubilization of ~60% of the MsbA in crude membranes, ~3.5-folds the extraction efficiency by the detergent mix. An increase in solubilization efficiency by SMAs with St:MA molar ratios 2:1 vs 1:1 has been reported 23,27,29 , and our data suggest that this improved efficiency depends, at least in part, on the target protein (Fig. 2). We also noticed that zSMAs outperformed M zSMAs by ~2-folds under otherwise similar conditions. ...
Membrane proteins can be reconstituted in polymer-encased nanodiscs for studies under near-physiological conditions and in the absence of detergents, but traditional styrene-maleic acid copolymers used for this purpose suffer severely from buffer incompatibilities. We have recently introduced zwitterionic styrene-maleic amide copolymers (zSMAs) to overcome this limitation. Here, we compared the extraction and reconstitution of membrane proteins into lipid nanodiscs by a series of zSMAs with different styrene:maleic amide molar ratios, chain sizes, and molecular weight distributions. These copolymers solubilize, stabilize, and support membrane proteins in nanodiscs with different efficiencies depending on both the structure of the copolymers and the membrane proteins.
... In the present study, we focus on the action of a specific SMA formulation, SMA 1440 (Cray Valley, now part of Polyscope), which has been shown to be effective in extracting pigment protein complexes from galactolipid-rich TM in spinach and cyanobacteria. 17,18 SMA 1440 has a styrene-tomaleic acid ratio (S/MA) of 1.5:1 and is also functionalized with butoxyethanol to increase its hydrophobicity. This butoxyethanol group has also been shown to cause a significant increase in the size of SMA 1440 aggregates, compared to nonfunctionalized polymer by small-angle X-ray scattering. ...
... This discrepancy may be attributed to the high pH subphase (pH 9.5, 50 mM Tris-Cl, and 125 mM KCl), which was chosen based on the working extraction condition of SMA 1440 copolymers in photosystems. 17,18 For the galactolipids, although several works earlier reported Π−A isotherms of the pure MGDG and DGDG, 33−35 we could not fabricate stable monolayers of any galactolipids with reproducible Π−A isotherms under high pH subphase conditions. The instability of pure galactolipid monolayers may be attributed to a predominantly nonlamellar phase as already observed for saturated MGDG. ...
Styrene-maleic acid (SMA) copolymers have recently gained attention for their ability to facilitate the detergent-free solubilization of membrane protein complexes and their native boundary lipids into polymer-encapsulated, nano-sized Lipid Particles, referred to as SMALPs. However, the interfacial interactions between SMA and lipids, which dictate the mechanism, efficiency, and selectivity of lipid and membrane protein extraction, are barely understood. Our recent finding has shown that SMA 1440, a chemical derivative of SMA family with a functionalized butoxyethanol group, was most active in galactolipid-rich membranes, as opposed to phospholipid membranes. In present work, we have performed X-ray reflectometry (XRR) and neutron reflectometry (NR) on the lipid monolayers at the liquid-air interface following by the SMA copolymer adsorption. XRR and Langmuir $\Pi - A$ isotherms captured the fluidifying effect of galactolipids, which made SMA copolymers easily infiltrate into the lipid membranes. NR results revealed the detailed structural arrangement of SMA 1440 copolymers within the membranes and highlighted the partition of the butoxyethanol group into the lipid tail region. This work allows us to propose a possible mechanism for the membrane solubilization by SMA.
