Reverse Engineering of a Thermosensing Regulator Switch.

Affiliation

Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Suipacha 531, Universidad Nacional de Rosario, Argentine National Research Council (CONICET), Rosario, Argentina. Electronic address: [Email]

Abstract

To address the mechanism of thermosensing and its implications for molecular engineering, we previously deconstructed the functional components of the bacterial thermosensor DesK, a histidine kinase with a five-span transmembrane domain that detects temperature changes. The system was first simplified by building a sensor that consists of a single chimerical transmembrane segment that retained full sensing capacity. Genetic and biophysical analysis of this minimal sensor enabled the identification of three modular components named determinants of thermodetection (DOTs). Here we combine and tune the DOTs to determine their contribution to activity. A transmembrane zipper represents the master DOT that drives a reversible and activating dimerization through the formation of hydrogen bonds. Our findings provide the mechanism and insights to construct a synthetic transmembrane helix based on a poly-valine scaffold that harbors the DOTs and regulates the activity. The construct constitutes a modular switch that may be exploited in biotechnology and genetic circuitry.

Keywords

histidin-kinase,reverse engineer,serine zipper,transmembrane signaling,

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