Fig. 4. Tse5-CT contains transmembrane regions that allow it to insert into lipid monolayers and the cytoplasmic membrane of E. coli cells.

a Representative Langmuir–Blodgett balance experiment showing the lateral pressure increase on lipid monolayers after the addition of Tse5-CT at time 0. Initial lateral pressures (Π₀) in mN m⁻¹ for representative experiments are indicated above each curve. b The plot of lateral pressure increases as a function of initial lateral pressure for every single experiment (n = 11). A maximal insertion pressure (MIP) of 34.99 mN m⁻¹ has been determined by extrapolating the fitted curve to ΔΠ = 0. The dotted line indicates the threshold value of lateral pressure consistent with unstressed biological membranes. The equation obtained from the linear regression analysis is y = −0.3258x + 11,401 with an R-squared of 0.96. c Tse5-CT propensity to contain transmembrane (blue) and amphipathic (green) helices as predicted by MemBrain 3.1. d Representation not to scale of constructs containing the dual reporter PhoA-LacZα (abbreviated P-L in the figure) at different C-terminal fusion points of Tse5-CT: K1229, A1269, A1281, K1300 and Q1317 (full-length Tse5-CT). All constructs start at Ile1169, and the truncation points are indicated by the length of the coloured bars and the residue number shown below. The dotted boxes indicate the regions that have been deleted. The predicted transmembrane propensity of Tse5-CT residues is also shown in the background. All fusion constructs contain a signal peptide (SP) at the N-terminal. Bars are colour coded based on the experimental results. Thus, a blue bar corresponds to periplasmic localisation of PhoA-LacZα; a red bar corresponds to a cytoplasmic localisation; a purple bar corresponds to localisation in a TM region; and a red and purple bar (A1281) corresponds to a borderline result, where it is not clear if it localises in the cytosol or a TM region. e E. coli DH5α cells transformed with spTse5-CT-PhoA-LacZα fusion proteins growing on dual reporter agar plates. f Measures of the PhoA-LacZ enzymatic activities for each spTse5-CT-PhoA-LacZα fusion protein. Red and blue bars indicate LacZ and PhoA enzymatic activities, respectively. Calculated NAR values and the location of the dual reporter fusion points are indicated below the graph. All enzymatic activities were measured in triplicate (n = 3), and the graph shows the mean values and standard deviations (SD).