DOI: 10.5281/zenodo.5561937
title: Membrane Protein Channels Equipped With a Cleavable Linker for Inducing Catalysis Inside Nanocompartments

description: Data underlying the figures in the publication “Membrane protein channels equipped with a cleavable linker for inducing catalysis inside nanocompartments”, published in <em>J. Mater. Chem. B, </em><em><strong>2021</strong></em><em>.</em> <em>https://pubs.rsc.org/en/content/articlelanding/2021/TB/D1TB01463C</em> Table of contents: <strong>1. Figure 1</strong>: Experimental data for <em>Figure 1</em>. OmpF structure and linker design. <em>(A)</em> 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. <em>(B)</em> The linker length corresponds to the distance between the two opposing Cys of OmpF-M, as estimated by PyMol modeling. <em>(C)</em> Chemical structure of the designed linker terminal maleimide groups, fluorophore side chains (pink) and the diol unit (blue). <strong>2. Figure 2</strong>: Experimental data for <em>Figure 2</em>. Ultrastructural morphology of <em>(A)</em> CNCs without OmpF, <em>(B)</em> CNCs with linker-OmpF-M inserted in the membrane, <em>(C)</em> CNCs with OmpF-M, and <em>(D)</em> OmpF wild-type. Scale bars: 200 nm. <em>(E)</em> 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). <strong>3. Figure 3</strong>: Experimental data for <em>Figure 3</em>. Linker-based control of laccase activity in CNCs in response to NaIO4. <em>(A)</em> Schematic representation of periodate-induced linker cleavage, activating in situ catalysis. Addition of NaIO<sub>4</sub> 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. <em>(B)</em> An increase in absorbance at 470 nm reflects laccase-catalyzed DMP conversion in NaIO<sub>4</sub>-treated CNC-linker-OmpF-M. Laccase activity was minimal in CNCs lacking OmpF or in CNC-linker OmpF-M in the absence of NaIO<sub>4</sub> (closed pores). Measurements were carried out at pH 7.4 for 9 hours at RT. <strong>4. Figure 4</strong>: Experimental data for <em>Figure 4</em>. Absorbance intensity of the laccase product was measured at 470 nm. Measurements were carried out in triplicate (SD) at pH 7.4 and RT. <em>(A)</em> Activity of free laccase in PBS compared to laccase encapsulated in CNC-linker-OmpF in the presence of NaIO<sub>4</sub> over 20 h. <em>(B)</em> 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 (<em>E</em> and <em>F</em>, respectively). Scale bars: 200 nm. <strong>5. Figure 5</strong>: Experimental data for <em>Figure 5</em>. Longevity of CNC morphology. TEM micrographs of CNCs encapsulating laccase with <em>(A)</em> unmodified OmpF-M inserted in the compartment membrane, and <em>(B)</em> linker-OmpF-M reveal an intact morphology after 11 months at 4 1C. Scale bars: 500 nm. <strong>6. Figure S5</strong>: Experimental data for <em>Figure S5</em>. <strong>7. Figure S8</strong>: Experimental data for <em>Figure S8</em>. <strong>8. Figure S9</strong>: Experimental data for <em>Figure S9</em>. <strong>9. Figure S10</strong>: Experimental data for <em>Figure S10</em>. <strong>10. Figure S12</strong>: Experimental data for <em>Figure S12</em>.