Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Poster Abstracts

41-POS Board 21 MECHANICAL FORCE MEASUREMENTS OF TANDEM SOLENOID-REPEAT PROTEINS Khushboo Matwani 1 ; Melanie Weiss 2 ; Pam Rowling 1 ; Maria Zacharopoulou 1 ; Johannes Stigler 2 ; Laura S Itzhaki 1 ; Tandem solenoid-repeat proteins are built from structurally identical α -helical units that stack together to form different architectures. Consensus-designed solenoid-repeat protein scaffolds are ultra-stable and have been used in diverse applications - from biomaterials to therapeutics. Solenoid-repeat proteins also exhibit spring-like behaviour when subjected to force. This property presents the possibility of developing them as FRET-based biosensors to detect forces in the biologically relevant pN range. Mechanical force is an important driver of cellular processes yet is difficult to properly track and quantify. By sandwiching a sensor between domains of a mechanosensing protein of interest, a change in FRET efficiency would provide a direct readout for when it experiences force. A novel tuneable sensor toolkit can be developed using helical repeat proteins since they are amenable to rational design and their force response can be calibrated using optical tweezers. We have characterised the unfolding of two variants of consensus-designed tetratricopeptide repeat proteins comprising two units (CTPR2a and CTPR2rv) and a consensus-designed ankyrin repeat protein comprising three units (DARPin3) for evaluation as sensors. CTPR2a and CTPR2rv unfold at a force of 10 pN and 7 pN, respectively, switching rapidly between the folded and unfolded state. Their unfolding and refolding force-extension profiles were identical indicating no hysteresis. DARPin3 unfolds at a higher force (12 pN), and the unfolding and refolding pathways are different, indicating hysteresis and making them less suitable for use as sensors. DARPin3 displays slower transitions between folded and unfolded states than the CTPRs. More broadly, consensus repeat proteins represent a simplified model system for understanding the complex folding landscape of naturally occurring proteins. 1 University of Cambridge, Pharmacology, Cambridge, United Kingdom 2 Ludwig-Maximilians-Universität, Gene Center, Munich, Germany

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