| Autor: |
Pieri E; Department of Chemistry and The PULSE Institute, Stanford University, Stanford, California 94305, United States.; SLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, California 94025, United States.; Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599, United States., Walker AR; Department of Chemistry and The PULSE Institute, Stanford University, Stanford, California 94305, United States.; SLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, California 94025, United States.; Department of Chemistry, Wayne State University, Detroit, Michigan 48202, United States., Zhu M; Department of Chemistry and The PULSE Institute, Stanford University, Stanford, California 94305, United States.; SLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, California 94025, United States., Martínez TJ; Department of Chemistry and The PULSE Institute, Stanford University, Stanford, California 94305, United States.; SLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, California 94025, United States. |
| Abstrakt: |
Red fluorescent protein (RFP) variants are highly sought after for in vivo imaging since longer wavelengths improve depth and contrast in fluorescence imaging. However, the lower energy emission wavelength usually correlates with a lower fluorescent quantum yield compared to their green emitting counterparts. To guide the rational design of bright variants, we have theoretically assessed two variants (mScarlet and mRouge) which are reported to have very different brightness. Using an α-CASSCF QM/MM framework (chromophore and all protein residues within 6 Å of it in the QM region, for a total of more than 450 QM atoms), we identify key points on the ground and first excited state potential energy surfaces. The brighter variant mScarlet has a rigid scaffold, and the chromophore stays largely planar on the ground state. The dimmer variant mRouge shows more flexibility and can accommodate a pretwisted chromophore conformation which provides easier access to conical intersections. The main difference between the variants lies in the intersection seam regions, which appear largely inaccessible in mScarlet but partially accessible in mRouge. This observation is mainly related with changes in the cavity charge distribution, the hydrogen-bonding network involving the chromophore and a key ARG/THR mutation (which changes both charge and steric hindrance). |
