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

Abstract

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 timedependent density functional theory (TDDFT), we calculate oneand 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 crosssection (s TPA ) for the S 0 ! S 1 transition in GFPs. Furthermore, it seems that details of h-bonds network in the CRO's vicinity influences s TPA response to CRO -environment electrostatic interactions in YFPs. We postulate that engineering FPs with more hydrophilic CRO's environment can lead to greater s 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.

Quantum chemistry study of the multiphoton absorption in enhanced green fluorescent protein at the single amino acid residue level

“…how the 1PA and 2PA spectra respond to electrostatic field created by the CRO's protein environment. [16] In case of 5 GFPs representatives, however, the h-bonding to the CRO's phenolate and distant electrostatic fields seem to cooperate in enhancing 2PA process while simultaneously slightly reducing the 1PA intensity for the S 0 ! S 1 transition.…”

“…Generally speaking, the electrostatic interactions play a fundamental role in FPs spectral tuning. [16][17][18][19][20][21][22][23][24][25][26][27][28][29][30] Based on the quadratic Stark effect, Drobizhev and co-workers [22,29] developed a model to rationalize one-photon excitation energy (DE 1PA ) variation in GFPs and RFPs (red fluorescent proteins) in terms of electric field produced by the CRO's protein environment. They connected DE 1PA to the permanent dipole moment change (Δμ) upon the excitation to the lowest-lying singlet excited state (S 1 ).…”

“…S 1 transition. [16] The more energetic absorption bands may again respond differently to specific CRO -environment interactions. We also propose that creating more hydrophilic CRO's environment, by e. g. accomodating more water molecules, can lead to electric field shielding that would favour greater s 2PA values.…”

“…We also identified Y145 and T203 a.a. residues as important h-bond donors to the CRO's phenolate that are responsible for s 2PA damping for excitations higher in energy than the S 1 state. [16] According to the early studies, the famous T203Y mutation is crucial for developing YFPs from GFPs. It effectively removes the T203-CRO h-bond (Figure S1 in the Supporting Information) and results in ca.…”

“…In case of FPs, this often leads to multiphoton absorption peaks lying in the biological spectral window and, hence, advantageous deeptissue imaging. [13,[50][51][52] In our previous work, [16] we described general impact of CRO's environment as a whole on 1PA and 2PA spectra in chosen GFPs and YFPs to guide development of novel markers, first and foremost with enhanced 2PA process. In the present contribution, we extend these efforts by describing impact of specific a.a. residues on 1PA, 2PA, and 3PA in enhanced green fluorescent protein (EGFP).…”