The Role of Hydrogen Bonds and Electrostatic Interactions in Enhancing Two-Photon Absorption in Green and Yellow Fluorescent Proteins.

The spectral properties of fluorescent proteins (FPs) depend on the protein environment of the chromophore (CRO). A deeper understanding of the CRO - environment interactions in terms of FPs' spectral characteristics will allow for a rational design of novel markers with desired properties. Here, we are taking a step towards achieving this important goal. With the time-dependent density functional theory (TDDFT), we calculate one- and two-photon absorption (OPA and TPA) spectra for 5 green FPs (GFPs) and 3 yellow FPs (YFPs) differing in amino acid sequence. The goal is to reveal the roles of: (i) electrostatic interactions, (ii) hydrogen-bonds (h-bonds) and (iii) h-bonds together with distant electrostatic field in absorption spectra tuning. Our results point to design hypothesis towards FPs optimised for TPA-based applications. Both h-bonds and electrostatic interactions co-operate in enhancing TPA cross-section ( σ T P A ) for the S 0 → S 1 transition in GFPs. Furthermore, it seems that details of h-bonds network in the CRO's vicinity influences σ T P A response to CRO - environment electrostatic interactions in YFPs. We postulate that engineering FPs with more hydrophilic CRO's environment can lead to greater σ T P A . We also find that removing h-bonds formed with the CRO's phenolate leads to TPA enhancement for transition to higher excited states than S ₁ . Particularly Y145 and T203 residues are important in this regard.
(© 2022 Wiley-VCH GmbH.)