Engineering fluorescent proteins to be the customized in vivo labels for monitoring cellular dynamic events is critical in biochemical and biomedical studies. The design and development of novel red fluorescent proteins is one of the most important fronts in this field due to their potential of imaging the entire organism. A recent fluorescent protein mutant eqFP650-67-HqAla with the 8-hydroxyquinolin-imidazolinone (HQI) chromophore has the plausible bathochromic shift of ~30 nm in its emission spectrum wavelength comparing to the parent fluorescent protein eqFP650. However, molecular mechanism of this significant shift remains somewhat obscure. In this study, we carefully benchmarked our computational methods and performed extensive calculations to investigate various structural components' effect on the chromophore's emission energy and decipher the molecular origin of the spectral shift. The influences of conjugation size, substituent group, substituent site as well as the number of substituents have been examined by elaborately designed chromophore derivatives. Accordingly, we proposed several chromophore mutants with dramatic bathochromic shift of up to ~60 nm in their emission spectra. We further evaluated their structural stability in the protein using molecular dynamics simulations. Present theoretical study connects the structural feature of chromophore derivatives in red fluorescent proteins with their splendid performances in shifting the emission frequency and offer the molecular insight. The computational protocol and successive examination procedure to extract the structural effect utilized herein can also be widely applied to other fluorescent proteins in general. © 2018 Wiley Periodicals, Inc.
Keywords: TD-DFT; emission spectrum; fluorescent protein; rational design.
© 2018 Wiley Periodicals, Inc.