Kemp, Philipp ; Weber, Wadim ; Desczyk, Charlotte ; Kaufmann, Marwan ; Panthel, Josefine ; Wörmann, Theresa ; Stein, Viktor (2023)
Dissecting the permeability of the Escherichia coli cell envelope to a small molecule using tailored intensiometric fluorescent protein sensors.
In: ACS omega, 8 (42)
doi: 10.1021/acsomega.3c05405
Artikel, Bibliographie

Kurzbeschreibung (Abstract)

Membranes provide a highly selective barrier that defines the boundaries of any cell while providing an interface for communication and nutrient uptake. However, despite their central physiological role, our capacity to study or even engineer the permeation of distinct solutes across biological membranes remains rudimentary. This especially applies to Gram-negative bacteria, where the outer and inner membrane impose two permeation barriers. Addressing this analytical challenge, we exemplify how the permeability of the cell envelope can be dissected using a small-molecule-responsive fluorescent protein sensor. The approach is exemplified for the biotechnologically relevant macrolide rapamycin, for which we first construct an intensiometric rapamycin detector (iRapTor) while comprehensively probing key design principles in the iRapTor scaffold. Specifically, this includes the scope of minimal copolymeric linkers as a function of topology and the concomitant need for gate post residues. In a subsequent step, we apply iRapTors to assess the permeability of the cell envelope to rapamycin. Despite its lipophilic character, rapamycin does not readily diffuse across the envelope but can be enhanced by recombinantly expressing a nanopore in the outer membrane. Our study thus provides a blueprint for studying and actuating the permeation of small molecules across the prokaryotic cell envelope.

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