Engineering a pH responsive pore forming protein.

Autor:	Kisovec M; Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia., Rezelj S; Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia., Knap P; Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia., Cajnko MM; Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia., Caserman S; Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia., Flašker A; Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia., Žnidaršič N; Department of Biology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia., Repič M; Department of Computational Biochemistry and Drug Design, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia., Mavri J; Department of Computational Biochemistry and Drug Design, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia., Ruan Y; U1006 Institut National de la Santé et de la Recherche Médicale (INSERM), Université Aix-Marseille, Parc Scientifique et Technologique de Luminy, 163 avenue de Luminy, 13009, Marseille, France., Scheuring S; U1006 Institut National de la Santé et de la Recherche Médicale (INSERM), Université Aix-Marseille, Parc Scientifique et Technologique de Luminy, 163 avenue de Luminy, 13009, Marseille, France., Podobnik M; Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia., Anderluh G; Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Hajdrihova 19, 1000 Ljubljana, Slovenia.
Jazyk:	angličtina
Zdroj:	Scientific reports [Sci Rep] 2017 Feb 08; Vol. 7, pp. 42231. Date of Electronic Publication: 2017 Feb 08.
DOI:	10.1038/srep42231
Abstrakt:	Listeriolysin O (LLO) is a cytolysin capable of forming pores in cholesterol-rich lipid membranes of host cells. It is conveniently suited for engineering a pH-governed responsiveness, due to a pH sensor identified in its structure that was shown before to affect its stability. Here we introduced a new level of control of its hemolytic activity by making a variant with hemolytic activity that was pH-dependent. Based on detailed structural analysis coupled with molecular dynamics and mutational analysis, we found that the bulky side chain of Tyr406 allosterically affects the pH sensor. Molecular dynamics simulation further suggested which other amino acid residues may also allosterically influence the pH-sensor. LLO was engineered to the point where it can, in a pH-regulated manner, perforate artificial and cellular membranes. The single mutant Tyr406Ala bound to membranes and oligomerized similarly to the wild-type LLO, however, the final membrane insertion step was pH-affected by the introduced mutation. We show that the mutant toxin can be activated at the surface of artificial membranes or living cells by a single wash with slightly acidic pH buffer. Y406A mutant has a high potential in development of novel nanobiotechnological applications such as controlled release of substances or as a sensor of environmental pH. Competing Interests: The authors declare no competing financial interests.
Databáze:	MEDLINE
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