A FRET-Based Biosensor for the Src N-Terminal Regulatory Element

Abstract

In signaling proteins, intrinsically disordered regions often represent regulatory elements, which are sensitive to environmental effects, ligand binding, and post-translational modifications. The conformational space sampled by disordered regions can be affected by environmental stimuli and these changes trigger, vis a vis effector domain, downstream processes. The disordered nature of these regulatory elements enables signal integration and graded responses but prevents the application of classical approaches for drug screening based on the existence of a fixed three-dimensional structure. We have designed a genetically encodable biosensor for the N-terminal regulatory element of the c-Src kinase, the first discovered protooncogene and lead representative of the Src family of kinases. The biosensor is formed by two fluorescent proteins forming a FRET pair fused at the two extremes of a construct including the SH4, unique and SH3 domains of Src. An internal control is provided by an engineered proteolytic site allowing the generation of an identical mixture of the disconnected fluorophores. We show FRET variations induced by ligand binding. The biosensor has been used for a high-throughput screening of a library of 1669 compounds with seven hits confirmed by NMR.

Development of FRET Biosensor to Characterize CSK Subcellular Regulation

“…Therefore, the EIY version was chosen for subsequent research in this work. 25,20,19,22,23). In cells transfected with Src-tail, Fyn-tail, and EIY CSK-FRET (1.0 µg of DNA on a 24-well plate), the ECFP/FRET ratio values (mean ± S.D.)…”

“…Although membrane localization is important in the regulation of CSK's function, there is a lack of imaging tools that can directly visualize CSK's activity at the plasma membrane and its submembrane microdomains. Fluorescence resonance energy transfer (FRET) technology and genetically encoded FRET biosensors provide powerful tools for the monitoring of biochemical activity and visualization of signaling molecules in vitro and in living cells [21][22][23]. The FRET donor and acceptor pair, consisting of enhanced cyan fluorescent protein (ECFP) and a variant of yellow fluorescent protein (YPet), has been optimized for FRET biosensor engineering; it can form weak dimers that enhance the efficiency and sensitivity of FRET [24].…”

Targeting the Src N-terminal regulatory element in cancer

“…Targeting of proteins' intrinsically disordered regions was identified as one of the 10 top emerging technologies in 2019 by the World Economic Forum and it has been proven in other disordered cancer targets [87]. Our group has designed a screening system to search chemical libraries for binders of the Src SNRE [88] and we are currently following up a promising lead.…”