De Novo Design of Flavoproteins with Controlled Excited State Dynamics
- 1University of Bristol
Abstract
Designing photochemical properties in proteins is a frontier challenge in biomolecular design, which, when realised, will open new avenues in catalysis and biomolecular electronics. Here we demonstrate an integrated computational design framework, supported by ultrafast spectroscopy, combining protein design, quantum mechanical/molecular mechanics molecular dynamics (QM/MM MD) and electronic structure calculations, to control flavin excited states in de novo proteins. Iterative design yields FC-4D2, a flavocytochrome with covalently bound flavin and non-covalently bound heme B and Fl-4D2, where the heme-binding site is removed. Both proteins are highly thermostable, and access all physiologically relevant flavin redox and protonation states, while high-resolution X-ray crystallography confirms the near-[A]ngstrom-level accuracy in design. Incorporating QM/MM MD and linear-response time-dependent density functional theory (LR-TDDFT) into the design workflow enables engineering of a flavin-tryptophan charge-transfer variant, Fl-4D2CT, that modifies the flavin absorption spectrum, substantially alters its redox properties and suppresses fluorescence. Ultrafast studies reveal how protein design reshapes the excited state relaxation timescale and mechanism. Our study establishes a modular strategy for programming flavin photochemistry in designed proteins.
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BibTeX
@misc{anderson2026novo,
title = {{De Novo Design of Flavoproteins with Controlled Excited State Dynamics}},
author = {R. Anderson and E. Bungay and J. Underhill and M. Liu and A. Stuart and C. Back and J. Berrones-Reyes and P. Dubiel and J. Birrell and A. G. Rao and C. Williams and R. Barringer and P. Hinchliffe and B. Lichtenstein and B. Curchod and J. Butt and T. Oliver and A. J. Mulholland},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.08.757584},
url = {https://www.biorxiv.org/content/10.64898/2026.10.08.757584v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - De Novo Design of Flavoproteins with Controlled Excited State Dynamics
AU - R. Anderson
AU - E. Bungay
AU - J. Underhill
AU - M. Liu
AU - A. Stuart
AU - C. Back
AU - J. Berrones-Reyes
AU - P. Dubiel
AU - J. Birrell
AU - A. G. Rao
AU - C. Williams
AU - R. Barringer
AU - P. Hinchliffe
AU - B. Lichtenstein
AU - B. Curchod
AU - J. Butt
AU - T. Oliver
AU - A. J. Mulholland
PY - 2026
DA - 2026/10/08
PB - bioRxiv
DO - 10.64898/2026.10.08.757584
UR - https://www.biorxiv.org/content/10.64898/2026.10.08.757584v1
AB - Designing photochemical properties in proteins is a frontier challenge in biomolecular design, which, when realised, will open new avenues in catalysis and biomolecular electronics. Here we demonstrate an integrated computational design framework, supported by ultrafast spectroscopy, combining protein design, quantum mechanical/molecular mechanics molecular dynamics (QM/MM MD) and electronic structure calculations, to control flavin excited states in de novo proteins. Iterative design yields FC-4D2, a flavocytochrome with covalently bound flavin and non-covalently bound heme B and Fl-4D2, where the heme-binding site is removed. Both proteins are highly thermostable, and access all physiologically relevant flavin redox and protonation states, while high-resolution X-ray crystallography confirms the near-[A]ngstrom-level accuracy in design. Incorporating QM/MM MD and linear-response time-dependent density functional theory (LR-TDDFT) into the design workflow enables engineering of a flavin-tryptophan charge-transfer variant, Fl-4D2CT, that modifies the flavin absorption spectrum, substantially alters its redox properties and suppresses fluorescence. Ultrafast studies reveal how protein design reshapes the excited state relaxation timescale and mechanism. Our study establishes a modular strategy for programming flavin photochemistry in designed proteins.
ER -