Conical Intersection Accessibility Dictates Brightness in Red Fluorescent Proteins

• DOI: 10.1021/jacs.4c00458
• Publication Date: 2024-06-17T22:24:20Z
• Authors: Elisa Pieri; Alice R. Walker; Mingning Zhu; Todd J. Martínez
• Keywords: Chromophore; Conical intersection; Quantum yield

Red fluorescent protein (RFP) variants are highly sought after for in vivo imaging since longer wavelengths improve depth and contrast fluorescence imaging. However, the lower energy emission wavelength usually correlates with a quantum yield compared to their green emitting counterparts. To guide rational design of bright variants, we have theoretically assessed two (mScarlet mRouge) which reported very different brightness. Using an α-CASSCF QM/MM framework (chromophore all residues within 6 Å it QM region, total more than 450 atoms), identify key points on ground first excited state potential surfaces. The brighter variant mScarlet has rigid scaffold, chromophore stays largely planar state. dimmer mRouge shows flexibility can accommodate pretwisted conformation provides easier access conical intersections. main difference between lies intersection seam regions, appear inaccessible but partially accessible mRouge. This observation is mainly related changes cavity charge distribution, hydrogen-bonding network involving ARG/THR mutation (which both steric hindrance).