Mann, S. I. | bioRxiv Science

bioRxiv2025

De novo designed bright, hyperstable rhodamine binders for fluorescence microscopy

De novo protein design has emerged as a powerful strategy with the promise to create new tools. The practical performance of designed fluorophore binders, however, has remained far from meeting fluorescence microscopy demands. Here, we design de novo Rhodamine Binder (Rhobin) tags that combine ideal properties including size, brightness, and now adding hyperstability. Rhobin allows live and fixed cell imaging of a wide range of subcellular targets in mammalian cells. Its reversible fluorophore binding further enables live super-resolution STED microscopy with low photobleaching, as well as PAINT-type single-molecule localization microscopy. We showcase Rhobin in the extremophile Sulfolobus acidocaldarius living at 75{degrees}C, an application previously inaccessible by existing tags. Rhobin will serve as the basis for a new class of live cell fluorescent tags and biosensors.

bioengineering↗

bioRxiv2024

De novo design of proteins that bind naphthalenediimides, powerful photooxidants with tunable photophysical properties

De novo protein design provides a framework to test our understanding of protein function and to build proteins with cofactors and functions not found in nature. Here, we report the design of proteins designed to bind powerful photooxidants and the evaluation of the use of these proteins to generate diffusible small molecule reactive species for applications in proximity labeling. Because excited state dynamics are influenced by the dynamics and hydration of a photo-oxidants environment, it was important to not only design a binding site, but also to evaluate its dynamic properties. Thus, we used computational design in conjunction with molecular dynamics (MD) simulations to design a protein, designated NBP (NDI Binding Protein) that held a naphthalenediimide (NDI), a powerful photooxidant, in a programable molecular environment. Solution NMR confirmed the structure of the complex. We evaluated two NDI cofactors in this de novo protein, using ultra-fast pump-probe spectroscopy to evaluate light-triggered intra- and intermolecular electron transfer function. Moreover, we demonstrated the utility of this platform to activate multiple molecular probes for protein proximity labeling.

biophysics↗