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. 2021 May 18;11:10536. doi: 10.1038/s41598-021-90039-3

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Reconstitution of WT opsin and TM5 mutants into liposomes. (A) Coomassie-stained SDS-PAGE of purified WT opsin and the F1 (F228A), F2 (F221A, F228A) and F3 (F220C, F221A, F228A) opsin constructs. Molecular weight markers are indicated. (B) Protein dependence of the reconstitution of vesicles with scramblase activity. NBD-PC-containing vesicles were reconstituted with different amounts of the indicated opsin construct and the extent of fluorescence reduction on dithionite treatment was determined by curve-fitting. The data were processed to calculate the probability "p(≥ 1) scramblase" that an individual vesicle in the ensemble possesses a functional scramblase. The graph shows "p(≥ 1) scramblase" versus the corrected protein/phospholipid ratio (PPR*) of the sample (the correction eliminates the contribution of vesicles that are refractory to reconstitution). (C) Ratio of mono-exponential fit constants (α) obtained from analyses of the reconstitution of WT and mutant opsins similar to (and including) the analysis shown in (B). The ratio α_construct/α_WT ~ 1 for F1 and F2 opsins, indicating that these proteins reconstitute similarly to WT opsin as dimers or higher order multimers. However, α_F3/α_WT ~ 0.4, consistent with reconstitution of F3 as a monomer (α_WT = 0.25 ± 0.07, mean ± S.D.). The bar chart is constructed from the results of three independent experiments, each involving 4–5 reconstitutions at different protein/phospholipid ratios (*, p = 0.03, unpaired t-test (two-tail)).