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Membrane Protein Channels Equipped With a Cleavable Linker for Inducing Catalysis Inside Nanocompartments

Data underlying the figures in the publication “Membrane protein channels equipped with a cleavable linker for inducing catalysis inside nanocompartments”, published in J. Mater. Chem. B, 2021. <a href="https://pubs.rsc.org/en/content/articlelanding/2021/TB/D1TB01463C">https://pubs.rsc.org/en/content/articlelanding/2021/TB/D1TB01463C</a> Table of contents: 1. Figure 1: Experimental data for Figure 1. OmpF structure and linker design. (A) PyMol ribbon representation of OmpF-M secondary structure reveals a distance of about 30 Å between the side groups of the two amino acids replaced by Cys in OmpF-M. (B) The linker length corresponds to the distance between the two opposing Cys of OmpF-M, as estimated by PyMol modeling. (C) Chemical structure of the designed linker terminal maleimide groups, fluorophore side chains (pink) and the diol unit (blue). 2. Figure 2: Experimental data for Figure 2. Ultrastructural morphology of (A) CNCs without OmpF, (B) CNCs with linker-OmpF-M inserted in the membrane, (C) CNCs with OmpF-M, and (D) OmpF wild-type. Scale bars: 200 nm. (E) FCS autocorrelation curves (solid line) and raw data (dots) of PBS solutions of the linker (black), standalone linker-OmpF-M in 1% OG (blue), and CNCs with linker-OmpF-M inserted in the membrane (red). 3. Figure 3: Experimental data for Figure 3. Linker-based control of laccase activity in CNCs in response to NaIO4. (A) Schematic representation of periodate-induced linker cleavage, activating in situ catalysis. Addition of NaIO4 cleaves the linker and thereby unblocks the pore. In the open state, substrate enters the compartment where it is oxidized to a detectable product by the confined laccase. (B) An increase in absorbance at 470 nm reflects laccase-catalyzed DMP conversion in NaIO4-treated CNC-linker-OmpF-M. Laccase activity was minimal in CNCs lacking OmpF or in CNC-linker OmpF-M in the absence of NaIO4 (closed pores). Measurements were carried out at pH 7.4 for 9 hours at RT. 4. Figure 4: Experimental data for Figure 4. Absorbance intensity of the laccase product was measured at 470 nm. Measurements were carried out in triplicate (SD) at pH 7.4 and RT. (A) Activity of free laccase in PBS compared to laccase encapsulated in CNC-linker-OmpF in the presence of NaIO4 over 20 h. (B) Activity measured for one week of free laccase in PBS compared to CNCs with and without periodate-cleaved pores or CNCs lacking pores. Periodate treatment had no effect on the ultrastructural morphology of (C) CNClinker-OmpF-M compared to (D) CNC-no OmpF after 1 week nor after 3 weeks (E and F, respectively). Scale bars: 200 nm. 5. Figure 5: Experimental data for Figure 5. Longevity of CNC morphology. TEM micrographs of CNCs encapsulating laccase with (A) unmodified OmpF-M inserted in the compartment membrane, and (B) linker-OmpF-M reveal an intact morphology after 11 months at 4 1C. Scale bars: 500 nm. 6. Figure S5: Experimental data for Figure S5. 7. Figure S8: Experimental data for Figure S8. 8. Figure S9: Experimental data for Figure S9. 9. Figure S10: Experimental data for Figure S10. 10. Figure S12: Experimental data for Figure S12.

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