Modular Thermal Control of Protein Dimerization

Abstract

Protein-protein interactions and protein localization are essential mechanisms of cellular signal transduction. The ability to externally control such interactions using chemical and optogenetic methods has facilitated biological research and provided components for the engineering of cell-based therapies and materials. However, chemical and optical methods are limited in their ability to provide spatiotemporal specificity in light-scattering tissues. To overcome these limitations, we present "thermomers," modular protein dimerization domains controlled with temperature -a form of energy that can be delivered to cells both globally and locally in a wide variety of in vitro and in vivo contexts. Thermomers are based on a sharply thermolabile coiled-coil protein, which we engineered to heterodimerize at a tunable transition temperature within the biocompatible range of 37-42 ºC. When fused to other proteins, thermomers can reversibly control their association, as demonstrated via membrane localization in mammalian cells. This technology enables remote control of intracellular protein-protein interactions with a form of energy that can be delivered with spatiotemporal precision in a wide range of biological, therapeutic and living material scenarios.

Reversible thermal regulation for bifunctional dynamic control of gene expression in Escherichia coli

“…Compared with the chemicaland cell density-response biosensing machinery used for dynamic metabolic control 28 , temperature and light are attractive strategies with reversibility to address these challenges. In particular, thermosensitive genetic tools, which have been engineered for different uses, such as dynamic regulation for defining cell growth and production synthesis phases 11,12 , gene therapy 29 , etc., are more ideal for practical utilizations because of the convenient collocation, low cost, easy operability, and good dispersity of heat-transfer required in varied bioprocesses 30,31 . Therefore, engineering thermal-switchable bioswitch of bifunction can make possible the simultaneous activation and depression of distinct sets of genes in a temperature-dependent manner.…”

Thermal Control of Engineered T-cells

“…In this work, we capitalized on non-thermal pHSP induction by the T-cell receptor pathway to generate sustained killing circuits. In other contexts where the promiscuous responsiveness of pHSPs presents an un-exploitable hindrance, it may be desirable to develop thermal response mechanisms based on orthogonal molecular bioswitches 22,23 .…”

“…This is important because the behavior of pHSPs varies greatly between cell types and cellular states. In this study, we screen a library of pHSPs in primary T-cells and engineer gene circuits providing transient and sustained activation of gene expression in T-cells in response to brief thermal stimuli within the well-tolerated temperature range of 37-42ºC [22][23][24] . Our circuits incorporate feed-forward amplification, positive feedback and recombinase-based state switches.…”

Highly Reversible Tunable Thermal-repressible Split-T7 RNA polymerases (Thermal-T7RNAPs) for dynamic gene regulation

“…We then sought to gain insights on the activation and deactivation kinetics of the systems under the temperature cycling between permissible ON temperature (30 °C) and restrictive OFF temperature (40 °C). While in vitro measurements can directly probe into the coiling/uncoiling of alpha-helix structures (Naik et al, 2001;Piraner et al, 2019) in assisting split-polymerase reconstitution/sequestration but precise and reliable measurements of Thermal-T7RNAP initiating transcription are nonetheless undermined by reporter mRNA instability and slow reporter protein turnover (Motta-Mena et al, 2014). As an alternative, we have adopted a mathematical model from earlier work, which has been previously used to provide information of transcriptional kinetics based on activation/deactivation measurements (Motta-Mena et al, 2014), to obtain similar transcriptional kinetics information of the Thermal-T7RNAP systems during activation (30 °C) and deactivation phases (40 °C).…”