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eMap is a web-based platform for identifying and visualizing electron or hole transfer pathways in proteins based on their crystal structures. The underlying model can be viewed as a coarse-grained version of the Pathways model, where each tunneling step between hopping sites represented by electron transfer active (ETA) moieties is described with one effective decay parameter that describes protein-mediated tunneling. ETA moieties include aromatic amino acid residue side chains and aromatic fragments of cofactors that are automatically detected, and, in addition, electron/hole residing sites that can be specified by the users. The software searches for the shortest paths connecting the user-specified electron/hole source to either all surface-exposed ETA residues or the user-specified target. The identified pathways are ranked according to their effective length. The pathways are visualized in 2D as a graph, in which each node represents an ETA site, and in 3D using available protein visualization tools. Here, we present the capability and user interface of eMap 1.0, which is available at https://emap.bu.edu .

Kindling fluorescent protein (KFP), one of the chronologically first members of photoswitchable colored proteins from the green fluorescent protein (GFP) family, increasingly attracts efforts from experimental and theoretical sides. Ambiguous conclusions in solving puzzles of photochemistry of KFP and of its parent natural protein asFP595 are partially explained by lack of reliable theoretical data on chromophore properties in the electronically excited state. We report the results of state-of-the-art quantum chemistry calculations of the structure and energy of the KFP chromophore, 2-acetyl-,4-(p-hydroxybenzylidene)-1-methyl-5-imidazolone (AHBMI), both in the ground and excited states. Ground state equilibrium structures of anionic and zwitterionic protonation states of AHBMI were computed by the conventional MP2 method while excited state structures were characterized by the extended multireference perturbation theory method XMCQDPT2 including optimization of geometry parameters at this level. In particular, the computational results demonstrate that the basicity of the N2 nitrogen atom of the imidazolinone ring should noticeably increase upon electronic excitation, thus affecting excited state proton transfer events in proteins. The results of these simulations as well as of quantum mechanical-molecular mechanical (QM/MM) calculations for model protein systems evidence that KFP conformations with the zwitterionic chromophore are hardly expected to occur in the ground state, but may be populated upon excitation.

Red fluorescent proteins (RFPs) are indispensable tools for deep-tissue imaging, fluorescence resonance energy transfer applications, and super-resolution microscopy. Using time-resolved optical spectroscopy this study investigated photoinduced dynamics of three RFPs, KillerRed, mRFP, and DsRed. In all three RFPs, a new transient absorption intermediate was observed, which decays on a microsecond-millisecond time scale. This intermediate is characterized by red-shifted absorption at 1.68-1.72 eV (λmax = 720-740 nm). On the basis of electronic structure calculations, experimental evidence, and published literature, the chemical nature of the intermediate is assigned to an unusual open-shell dianionic chromophore (dianion-radical) formed via photoreduction. A doubly charged state that is not stable in the isolated (gas phase) chromophore is stabilized by the electrostatic field of the protein. Mechanistic implications for photobleaching, blinking, and phototoxicity are discussed.

The effect of the protein environment on the electronic structure of the green fluorescent protein (GFP) chromophore is investigated by QM/MM (quantum mechanics/molecular mechanics) calculations. The protein has very small effect on the excitation energy of the bright absorbing and the lowest triplet states of the anionic GFP chromophore, deprotonated 4-hydroxybenzylidene-2,3-dimethylimidazolinone (HBDI) anion, however, it increases vertical detachment energy from 2.5 eV (gas-phase deprotonated HBDI anion) to 5.0 eV (solvated protein). We also investigated possible existence of the charge-transfer-to-solvent (CTTS) states associated with the GFP chromophore. Although precursors of such states appear in cluster calculations, a tightly packed structure of the protein prevents the formation of the CTTS states in this system. Motivated by a recently discovered new type of photoconversion, oxidative redding, we characterized the redox properties of GFP. The computed standard reduction potential of the anionic form of GFP is 0.47 V (for the GFP(•) + 1e → GFP(-) reaction), and the reduction potential at physiological conditions (pH 7, T = 25 °C) is 0.06 V.