Evaluation of commercially available styrene-co-maleic acid polymers for the extraction of membrane proteins from spinach chloroplast thylakoids

Korotych, Olena; Mondal, Jyotirmoy; Gattás‐Asfura, Kerim M.; Hendricks, Jessica

doi:10.1016/j.eurpolymj.2018.10.035

Cited by 23 publications

(23 citation statements)

References 46 publications

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“…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).…”

Section: Solubilization Of Hr and Msba In Crude Membranes By Zsmassupporting

confidence: 80%

“…The overall conclusion is that solubilization by the new 1:1 and 2:1 zSMA, and 1:1, 2:1 and 3:1 M zSMAs to a large extent, is quite forgiving and better than that by 2:1 SMA, a benchmark for the SMALP studies due to the recognition that the solubilization efficiency by the 2:1 SMAs is better than that of the 1:1 SMAs 23,27,29 . This is important because, in principle, solubilization conditions already established for a membrane protein using SMAs or detergents can be used with few or no changes to solubilize them into zSMALPs with reasonable results, minimizing the need for extensive trials.…”

Section: Discussionmentioning

confidence: 93%

“…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 .…”

Section: Solubilization Of Hr and Msba In Crude Membranes By Zsmasmentioning

confidence: 99%

“…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).…”

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confidence: 99%

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Extraction and reconstitution of membrane proteins into lipid nanodiscs encased by zwitterionic styrene-maleic amide copolymers

Fiori

Zheng

Kamilar

et al. 2020

Sci Rep

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0.04% sodium cholate. For the measurements, MsbA-loaded nanodiscs were enriched based on the affinity of the His-tagged MsbA for Ni 2+. The supernatant from the solubilization of proteoliposomes was mixed with Ni-NTA beads (Thermo Fisher Scientific) at a ratio of 100 µL resin/mL solubilized protein, and after incubation at 4 °C overnight with gentle rotation the samples were transferred to a gravity flow column. The resin was washed with 10 column volumes of 100 mM NaCl and 20 mM Tris/HCl, with 0.1 mM TCEP and 20 mM imidazole, pH 7.4, and elution was achieved by increasing imidazole to 200 mM. Eluted fractions were analyzed on gels (16% SDS-PAGE) stained with Instant Blue (Expedeon) and used for the ATPase measurements. Estimation of nanodiscs size by dynamic light scattering (DLS). Measurements were performed at 22 °C on a Zetasizer Nano ZSP (Malvern Instruments, Westborough, MA) using 40-µL disposable microcuvettes. Size-number distributions were generated using the Zetasizer software version 7.11 and were analyzed using the protein analysis distribution. Statistics. Statistical comparisons were performed by the Student's t test for paired or unpaired data, or one-way analysis of variance, as appropriate. P < 0.05 in a two-tailed analysis was considered significant. The number of experiments given in the main text and figure legends corresponds to independent measurements.

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Section: Solubilization Of Hr and Msba In Crude Membranes By Zsmassupporting

confidence: 80%

Section: Discussionmentioning

confidence: 93%

Section: Solubilization Of Hr and Msba In Crude Membranes By Zsmasmentioning

confidence: 99%

mentioning

confidence: 99%

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Extraction and reconstitution of membrane proteins into lipid nanodiscs encased by zwitterionic styrene-maleic amide copolymers

Fiori

Zheng

Kamilar

et al. 2020

Sci Rep

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“…The chemical basis for this inactivity is not known, yet we have also conrmed decreased solubilization efficacy of these SMAs on protein extraction from spinach chloroplast thylakoid membranes. 45 Therefore, PSI-SMALP encapsulated using SMA 1440 was used exclusively for subsequent experiments in this study. It is worth noting, Te thylakoid membranes solubilized with DDM liberate more chlorophyll containing protein complexes than SMA 1440 in all trials.…”

Section: Resultsmentioning

confidence: 99%