title: Library-Aided Probing of Linker Determinants in Hybrid Photoreceptors.
abstract: Signaling proteins comprise interaction and effector modules connected by linkers. Throughout evolution, these recurring modules have multiply been recombined to produce the present-day plethora of signaling proteins. Likewise, modular recombination lends itself to the engineering of hybrid signal receptors, whose functionality hinges on linker topology, sequence, and length. Often, numerous linkers must be assessed to obtain functional receptors. To expedite linker optimization, we devised the PATCHY strategy (primer-aided truncation for the creation of hybrid proteins) for the facile construction of hybrid gene libraries with defined linker distributions. Empowered by PATCHY, we engineered photoreceptors whose signal response was governed by linker length: whereas blue-light-repressed variants possessed linkers of 7n or 7n+5 residues, variants with 7n+1 residues were blue-light-activated. Related natural receptors predominantly displayed linker lengths of 7n and 7n+5 residues but rarely of 7n+1 residues. PATCHY efficiently explores linker sequence space to yield functional hybrid proteins including variants transcending the natural repertoire of signaling proteins.
authors.affiliation: Humboldt-Universität zu Berlin , Institut für Biologie, Biophysikalische Chemie, Invalidenstraße 42, 10115 Berlin, Germany.
authors.forename: Robert
authors.initials: R
authors.lastname: Ohlendorf
authors.affiliation: Humboldt-Universität zu Berlin , Institut für Biologie, Biophysikalische Chemie, Invalidenstraße 42, 10115 Berlin, Germany.
authors.forename: Charlotte Helene
authors.initials: CH
authors.lastname: Schumacher
authors.affiliation: Humboldt-Universität zu Berlin , Institut für Biologie, Biophysikalische Chemie, Invalidenstraße 42, 10115 Berlin, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Richter
authors.affiliation: Humboldt-Universität zu Berlin , Institut für Biologie, Biophysikalische Chemie, Invalidenstraße 42, 10115 Berlin, Germany.|Universität Bayreuth , Lehrstuhl für Biochemie, Universitätsstraße 30, Geb. NW III, 95447 Bayreuth, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Möglich
mesh_terms: D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D000071677:Histidine Kinase / Q000235:genetics / Q000378:metabolism; D008027:Light; D020283:Photoreceptors, Microbial / Q000378:metabolism*; D015202:Protein Engineering; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D015398:Signal Transduction
chemical_list: D029968:Escherichia coli Proteins; D020283:Photoreceptors, Microbial; D000071677:Histidine Kinase
keywords: DNA library; light-oxygen-voltage; protein engineering; sensor histidine kinase; sensory photoreceptor; signal transduction