QM/MM studies of structural and energetic properties of the far-red fluorescent protein HcRed

The far-red fluorescent protein HcRed was investigated using molecular dynamics (MD) and combined quantum mechanics/molecular mechanics (QM/MM) calculations. Three models of HcRed (anionic chromophore) were considered, differing in the protonation states of nearby Glu residues (A: Glu214 and Glu146 both protonated; B: Glu214 protonated and Glu146 deprotonated; C: Glu214 and Glu146 both deprotonated). SCC-DFTB/MM MD simulations of model B yield good agreement with the available crystallographic data at ambient pH. Bond lengths in the QM region are well reproduced, with a root mean square (rms) deviation between experimental and average MD data of 0.079 A; the chromophore is almost co-planar, which is consistent with experimental observation; and the five hydrogen bonds involving the chromophore are conserved. QM/MM geometry optimizations were performed on representative snapshot structures from the MD simulations for each model. They confirm the structural features observed in the MD simulations. According to the DFT(B3LYP)/MM results, the cis-conformation of the chromophore is more stable than the trans-form by 9.1-12.9 kcal mol(-1) in model B, and by 12.4-19.9 kcal mol(-1) in model C, consistent with the experimental preference for the cis-isomer. However, in model A when both Glu214 and Glu146 are protonated, the stability is inverted with the trans-form being favored. The different protonation states of the titratable active-site residues Glu214 and Glu146 thus critically influence the manner in which the relative stability and degree of planarity of the cis- and trans-conformers vary with pH. Coupled with the known correlation of chromophore conformation with fluorescence efficiency, this work provides a detailed structural basis for the observed phenomenon that red fluorescent proteins such as HcRed, mKate and Rtms5 show bright fluorescence at high pH.