One-Pot Dual Protein Labelling for Simultaneous Mechanical and Fluorescent Readouts in Optical Tweezers

Optical tweezers are widely used in the study of biological macromolecules but are limited by their one-directional probing capability, potentially missing critical conformational changes. Combining fluorescence microscopy with optical tweezers, employing Forster resonance energy transfer (FRET) pairs, addresses this issue. Moreover, attaching one FRET probe to a tethered protein and the other to a protein in solution allows precise localisation of interaction sites, while probing mechanical properties. When integrating fluorescence microscopy with optical tweezers, orthogonal protein conjugation methods are needed to enable simultaneous, site-specific attachment of fluorophores and DNA handles, commonly used to apply force to molecules of interest. In this study, we utilized commercially available reagents for dual site-specific labelling of the homodimeric heat shock protein 90 (Hsp90) using thiol-maleimide and inverse electron demand Diels-Alder cycloaddition (IEDDAC) bioorthogonal reactions. In a one-pot approach, Hsp90 modified with a cysteine mutation and the non-canonical amino acid cyclopropene-L-lysine (CpK) was labelled with the FRET pair maleimide-Atto550 and maleimide-Atto647N, alongside single- stranded methyltetrazine-modified DNA oligonucleotide. Optical tweezers experiments with this labelled Hsp90 construct revealed structural transitions consistent with previous studies, validating the approach. Fluorescence measurements confirmed the proximity of FRET pairs in the N-terminally closed state of Hsp90 in this experimental setup. This integrative method provides a powerful tool for probing protein conformational dynamics and protein interactions beyond the limitations of traditional optical tweezers. StatementThe developed method combines fluorescence microscopy with optical tweezers, enhancing single-molecule protein studies by overcoming the limitations of one- directional mechanical probing. Utilizing two orthogonal protein conjugation methods for one-pot dual labelling, the heat shock protein 90 was labelled with a FRET pair and single-stranded DNA oligonucleotides. Validated by comparison with published conformational changes, mechanical unfolding signatures, and FRET pair distances, this approach provides a powerful tool to explore single-molecule conformational dynamics and protein interactions.