title: Engineering a nanopore with co-chaperonin function.
abstract: The emergence of an enzymatic function can reveal functional insights and allows the engineering of biological systems with enhanced properties. We engineered an alpha hemolysin nanopore to function as GroES, a protein that, in complex with GroEL, forms a two-stroke protein-folding nanomachine. The transmembrane co-chaperonin was prepared by recombination of GroES functional elements with the nanopore, suggesting that emergent functions in molecular machines can be added bottom-up by incorporating modular elements into preexisting protein scaffolds. The binding of a single-ring version of GroEL to individual GroES nanopores prompted large changes to the unitary nanopore current, most likely reflecting the allosteric transitions of the chaperonin apical domains. One of the GroEL-induced current levels showed fast fluctuations (<1 ms), a characteristic that might be instrumental for efficient substrate encapsulation or folding. In the presence of unfolded proteins, the pattern of current transitions changed, suggesting a possible mechanism in which the free energy of adenosine triphosphate binding and hydrolysis is expended only when substrate proteins are occupied.
authors.affiliation: Department of Chemistry, University of Leuven, Leuven 3001, Belgium.
authors.forename: Ching-Wen
authors.initials: CW
authors.lastname: Ho
authors.affiliation: Department of Chemistry, University of Leuven, Leuven 3001, Belgium.
authors.forename: Veerle
authors.initials: V
authors.lastname: Van Meervelt
authors.affiliation: National Research Institute of Chinese Medicine, Ministry of Health and Welfare, Taipei 11221, Taiwan.
authors.forename: Keng-Chang
authors.initials: KC
authors.lastname: Tsai
authors.affiliation: Electron Microscopy Unit, Veterinary and Agrochemical Research Centre (CODA-CERVA), Brussels 1180, Belgium.
authors.forename: Pieter-Jan
authors.identifier: 0000-0001-5170-9376
authors.initials: PJ
authors.lastname: De Temmerman
authors.affiliation: Electron Microscopy Unit, Veterinary and Agrochemical Research Centre (CODA-CERVA), Brussels 1180, Belgium.
authors.forename: Jan
authors.identifier: 0000-0002-4879-3367
authors.initials: J
authors.lastname: Mast
authors.affiliation: Groningen Biotechnology Institute, University of Groningen, Groningen 9747AG, Netherlands.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Maglia
keywords: GroEL; GroES; Protein folding; loop grafting; nanomachine; single-molecule
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