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 (sigma TPA ) for the S0 -> S1 transition in GFPs. Furthermore, it seems that details of h-bonds network in the CRO's vicinity influences sigma TPA response to CRO - environment electrostatic interactions in YFPs. We postulate that engineering FPs with more hydrophilic CRO's environment can lead to greater sigma TPA . 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.

Automated summary

The spectral properties of fluorescent proteins are influenced by their protein environment. By studying the interactions between the chromophore and the environment, researchers have found that modifying hydrogen bonds and electrostatic interactions can enhance the absorption spectra of fluorescent proteins. Additionally, creating a more hydrophilic environment for the chromophore may lead to improved spectral characteristics.