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Page 8 of 17 Mezhyrova et al. Microbiome Res Rep 2023;2:28 https://dx.doi.org/10.20517/mrr.2023.28

Figure 1. Effect of DnaJ on the co-translational solubilization and membrane insertion of MS2-L. All expressions were carried out in
BW25113ΔDnaJ lysate (A) MS2-L was P-CF expressed in the presence of increasing DnaJ concentrations (0, 10, 30, and 100 µM).
Solubilization efficiencies were determined by densitometry from immunoblots of pellet and supernatant fractions. Combined signals of
pellet and supernatant were normalized to 1. Error bars represent the SEM of n = 6 (n = 5 for 100 µM DnaJ) biological replicates;
(B) MS2-L was either P-CF or L-CF (10 µM DMPG NDs) synthesized in the presence or absence of 100 µM DnaJ. Solubilized MS2-L was
quantified as described in (A). Error bars indicate the SEM of n = 6 (n = 5 for P-CF + 100 µM DnaJ) biological replicates; (C) Complex
formation of DnaJ with D-CF synthesized MS2-L. After expression in the presence of Brij78 and 30 µM DnaJ, the toxin was StrepII-
purified from the RM. As verified by SDS-PAGE (left panel) and immunoblot (right panel), the chaperone (containing a His -tag) co-
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purified with MS2-L. D-CF: Detergent-based cell-free expression; DMPG: 1,2-dimyristoyl-sn-glycero-3-phospho-(1’-rac-glycerol); ND:
nanodisc; P-CF: precipitate forming cell-free expression; RM: reaction mix.
in the D-CF mode. After expression and subsequent purification, the solubilized MS2-L derivatives were
immobilized via the C-terminal StrepII tags on magnetic beads. Purified DnaJ was added as prey and
putative complexes were eluted after removal of unbound DnaJ by washing. All MS2-L constructs
synthesized in the presence of Brij78 captured DnaJ, as verified by positive pulldown results [Figure 2A].
Some reduced but still detectable DnaJ binding was observed with the shortest construct MS2-Lp .
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Accordingly, all analyzed MS2-L constructs inserted into NDs (DMPG) did also result in a pulldown of
DnaJ, indicating that the relevant binding site remains accessible in the membrane inserted MS2-L and its
derivatives [Figure 2B]. The reduced binding of the construct MS2-Lp to DnaJ furthermore suggests that
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the binding site is not exclusively located within the soluble domain of the toxin, but may as well cover parts
of the trans-membrane domain.

Formation of oligomeric MS2-L complexes in lipid and detergent environments
Native LILBID mass spectrometry is an established tool to analyze the oligomeric state of membrane
proteins in NDs and other environments [17,26,28] . By tuning the intensity level of the laser pulses, the detection
of complete or partially dissociated sample complexes dependent on their stability can be achieved. MS2-L
samples were first synthesized in the standard S30 lysate either in the presence of 60 µM NDs (DMPG) or in
the D-CF mode in the presence of Brij78. In order to reveal the effects of the N-terminal soluble domain on
MS2-L oligomerization, full-length MS2-L and the truncated constructs containing deletions of the
N-terminal domain were analyzed [Figure 3, left panels]. High oligomeric assemblies containing at least 10
monomers were detected with ND samples containing full-length MS2-L as well as with all five MS2-Lp
derivatives [Figure 3, left panel, black trace]. Overall, the stoichiometry of the oligomeric assemblies in lipid
environments appears to be similar within all of the six constructs. It is important to note that high
oligomeric complexes of the corresponding MS2-L derivative are not just held together by the scaffold
proteins, but form stable homomeric complexes. This is visible by the detection of complexes from which
the MSP1E3D1 scaffold proteins were stripped off during the LILBID process.