Fig. 6: Type II Zorya defense proteins interact with cardiolipin-rich lipid membranes.

a ZorA, ZorB, and ZorE were synthesized via the T7 transcriptional activation cascade (plasmid P70a-T7rnap, 0.15 nM, linear T7p14-zorA, T7p14-zorB, T7p14-zorC, 10 nM). The TXTL reactions were immediately pushed into the QCMD modules with SLBs on the sensor. b Adsorption kinetics of the blank (P70a-T7rnap, 0.15 nM) and ZorA (P70a-T7rnap, 0.15 nM and T7p14-zorA, 10 nM) conditions with either a pure DOPE or DOPE + 5.1% CL SLBs. c The adsorption kinetics of the blank (P70a-T7rnap, 0.15 nM), ZorA alone (P70a-T7rnap, 0.15 nM and T7p14-zorA, 10 nM), ZorB alone (P70a-T7rnap, 0.15 nM and T7p14-zorB, 10 nM), and ZorA and ZorB together (P70a-T7rnap, 0.15 nM, T7p14-zorA, 10 nM and T7p14-zorB, 10 nM) with a DOPE + 6.7% CL SLB. d The frequency changes after 8 hours of TXTL incubation, for the cases described in c. The bar plot level and error bars represent the mean and standard deviation of at least 3 replicates. e The adsorption kinetics of the blank (P70a-T7rnap, 0.15 nM) and ZorE (P70a-T7rnap, 0.15 nM and T7p14-zorE, 10 nM) conditions with either a pure DOPC or pure DOPE SLBs. f The adsorption kinetics of the blank (P70a-T7rnap, 0.15 nM) and ZorE (P70a-T7rnap, 0.15 nM and T7p14-zorE, 10 nM) conditions with either a pure DOPE or DOPE + 6.7% CL SLBs. g The adsorption kinetics of the blank (P70a-T7rnap, 0.15 nM), ZorAB (P70a-T7rnap, 0.15 nM, T7p14-zorA, 10 nM, and T7p14-zorB, 10 nM), and ZorABE (P70a-T7rnap, 0.15 nM, T7p14-zorA, 10 nM, T7p14-zorB, 10 nM, and T7p14-zorE, 5 nM) conditions with a DOPE + 6.7% CL SLB. h An illustration of the hypothetical localization of the Zorya proteins in the host E. coli inner membrane. All Zorya proteins preferentially interact with the CL-rich polar regions of the inner membrane. In addition, ZorA likely has a weak interaction with the equatorial region of the inner membrane and ZorE likely has a reversible interaction with the equatorial region of the inner membrane.