Two-Photon Absorption Properties of Gold Fluorescent Protein: A Combined Molecular Dynamics and Quantum Chemistry Study

Abstract

Molecular dynamics (MD) simulations were carried out to obtain the conformational changes of the chromophore in the gold fluorescent protein (PDB ID: 1OXF ). To obtain two-photon absorption (TPA) cross-sections, time dependent density functional theory (TD-DFT) computations were performed for chromophore geometries sampled along the trajectory. The TD-DFT computations used the CAM-B3LYP functional and 6-31+G(d) basis set. Results showed that two dihedral angles change remarkably over the simulation time. TPA cross-sections were found to average 13.82 GM for the excitation to S computed from the equilibrium geometries; however, extending the structures with a water molecule and GLU residue, which make H bonds with the chromophore molecule, increased excitation energies and TPA cross-sections significantly. Besides the effects of the surrounding residues and the dihedrals on the spectroscopic properties, some bond lengths affected the excitation energies and the TPA cross-sections significantly (up to ±25-30%), while the effects of the bond angles were smaller (±5%). Overall the present results provide insight into the effects of the conformational flexibility on TPA (with gold fluorescent protein as a specific example) and suggest that further experimental measurements of TPA for the gold fluorescent protein should be undertaken.

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

“…[30] Moreover, we base our conclusions on 21 snapshots from QM/MM MD structure refinement instead of only one structure for DsRed [22] while it is well established that conformational sampling and structure quality are important for quantitatively correct absorption spectra of photoactive proteins. [33,34,80,81] It is noteworthy that regardless of quite crude sampling in the present contribution and known β-barrel stiffness, [1,82] we observe significant range of s TPA values for only 5.0 ps of QM/MM MD. For instance, s TPA is 0.1-27.0 GM and f is more compact in range of 1.008-1.016 for EGFP and M3 model.…”

“…To achieve this goal, we utilise three FPs’ models differing in composition of TDDFT‐treated FP part (QM subsystem) and whether or not the electrostatic interactions between QM subsystem and remaining a.a. residues and water molecules is accounted for. The spectra of FPs are generally not well represented by a single structure owing to a significant and spread of 0.3–0.5 eV and 6–14 GM, respectively, even for short molecular dynamics (MD) that allow only for a crude local conformational space sampling [33,34] . Hence, to account for an ensemble of structures in solution, we utilise 21 snapshots from hybrid QM/MM MD simulation.…”

“…The spectra of FPs are generally not well represented by a single structure owing to a significant DE and s TPA spread of 0.3-0.5 eV and 6-14 GM, respectively, even for short molecular dynamics (MD) that allow only for a crude local conformational space sampling. [33,34] Hence, to account for an ensemble of structures in solution, we utilise 21 snapshots from hybrid QM/MM MD simulation. This also allows to account for finite-temperature broadening of absorption bands.…”

Quantum mechanical/molecular mechanical studies of photophysical properties of fluorescent proteins

“…Other studies have used constrained models, often referred to as “clusters,” containing the chromophore and a select set of neighboring covalently and non‐covalently bonded amino acids, as well as water molecules. For these models, both pure QM 16,62–64 and QM/MM 65–67 methods have been employed. Within the QM/MM studies, a diverse selection of FPs have had 1PA features computed, for example, GFP, 65 mStrawberry and mCherry from the fruit series, 66 and mPlum 67 .…”

“…Altogether, they are now part of a wide palette of colors that range across the whole visible spectrum. Among the different photophysical properties of FPs, multi‐photon absorption (MPA), particularly two‐photon absorption (2PA), has gained great relevance and has been widely explored using experimental 5 and computational means 8–19 . 2PA microscopy has notable advantages over its one‐photon absorption (1PA) counterpart including: better‐focus, less auto‐bleaching, and a lower risk of photo‐damage in the tissue in the context of in vivo imaging.…”