Conical Intersection Accessibility Dictates Brightness in Red Fluorescent Proteins.

Area of Science:

• Biophysics
• Molecular Biology
• Computational Chemistry

Background:

• Red fluorescent proteins (RFPs) are crucial for in vivo imaging due to enhanced depth and contrast.
• Brighter RFPs are desired, but longer emission wavelengths often correlate with lower quantum yields.
• Understanding structural determinants of RFP brightness is key for rational design.

Purpose of the Study:

• To theoretically investigate the structural differences between two RFP variants, mScarlet and mRouge, with distinct brightness.
• To identify molecular mechanisms responsible for the observed brightness differences.
• To provide insights for designing brighter RFPs.

Main Methods:

• Employed an ab initio quantum mechanics/molecular mechanics (QM/MM) approach, specifically the α-CASSCF method.
• Analyzed ground and first excited state potential energy surfaces of the chromophore and surrounding protein environment.
• Focused on over 450 atoms in the quantum mechanics region.

Main Results:

• The brighter mScarlet variant exhibits a rigid scaffold and a planar chromophore.
• The dimmer mRouge variant shows greater flexibility, allowing pretwisted chromophore conformations and easier access to conical intersections.
• Differences in cavity charge distribution, hydrogen bonding, and an ARG/THR mutation significantly impact intersection seam accessibility.

Conclusions:

• Structural rigidity and chromophore planarity in mScarlet contribute to its higher brightness.
• Chromophore flexibility and accessible conical intersections in mRouge lead to lower brightness.
• Protein environment, including specific mutations and hydrogen bonding, plays a critical role in modulating RFP photophysical properties.