Biology subjects

Markeeva, E.

Publications and source records attributed to Markeeva, E..

2 recordsLinked to original sources

bioRxiv2025

Discovery of excited state proton transfer in flavin-based fluorescent protein with large Stokes shift

Flavin-binding proteins (flavoproteins) are widespread in nature, revealing versatile oxidation-reduction reactions and photochemistry. Flavoproteins derived from LOV domains are used for engineering of ligh-tresponsive tools in optogenetics, as well as fluorescent markers and photogenerators of reactive oxygen species. Despite extensiev efforts, all currently used LOV-derived proteins have similar absorption spectra with maxima around 275, 35-0375, and 450-485 nm. Here, we describe the discovery of a large Stokes shift flavi-nbased fluorescent protein, LSSFbFP, which can be obtainedin vivo and in vitro, with absorption maxima at 340-350 and 395-405 nm. Fluorescence emission of LSSFbFP mirrors that of classical FbFPs with the maximum at ~500 nm. We sho that the protein binds lumichrome as the chromophore and use low temperature and time-resolved spectroscopy, X-ray crystallography and modeling to prove that the apparent Stokes shift of LSSFbFP occurs due to excited state proton phenomena observed in flavoproteni s and pave the way for engineering of new flavin-based molecular instruments.

biochemistry↗

bioRxiv2023

Reengineering of a flavin-binding fluorescent protein using ProteinMPNN

Recent advances in machine learning techniques have led to development of a number of protein design and engineering approaches. One of them, ProteinMPNN, predicts an amino acid sequence that would fold and match user-defined backbone structure. In this short report, we test whether ProteinMPNN can be used to reengineer a flavin-binding fluorescent protein, CagFbFP. We fixed the native backbone conformation and the identity of 20 amino acids interacting with the chromophore (flavin mononucleotide, FMN), while letting ProteinMPNN predict the rest of the sequence. The software package suggested replacing 36-48 out of the remaining 86 amino acids. The three designs that we tested experimentally displayed different expression levels, yet all were able to bind FMN and displayed fluorescence, thermal stability and other properties similar to those of CagFbFP. Our results demonstrate that ProteinMPNN can be used to generate diverging unnatural variants of fluorescent proteins, and, more generally, to reengineer proteins without losing their ligand-binding capabilities.

bioengineering↗