One-pot dual protein labeling 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 Förster resonance energy transfer (FRET) pairs, addresses this issue. 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 labeling 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 labeled with the FRET pair maleimide-Atto 550 and maleimide-Atto 647N, alongside single-stranded methyltetrazine-modified DNA oligonucleotide. Optical tweezers experiments with this labeled 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 complex protein conformational dynamics beyond the limitations of traditional optical tweezers.