Kazuhiro Fujimoto

Excited states of GFP chromophore and active site studied by the SAC-CI method: Effect of protein-environment and mutations

Excited states of fluorescent proteins were studied using symmetry‐adapted cluster‐configuration interaction (SAC‐CI) method. Protein‐environmental effect on the excitation and fluorescence energies was investigated. In green fluorescent protein (GFP), the overall protein‐environmental effect on the first excitation energy is not significant. However, glutamine (Glu) 94 and arginine (Arg96) have the red‐shift contribution as reported in a previous study (Laino et al., Chem Phys 2004, 298, 17). The excited states of GFP active site (GFP‐W22‐Ser205‐Glu222‐Ser65) were also calculated. Such large‐scale SAC‐CI calculations were performed with an improved code containing a new algorithm for the perturbation selection. The SAC‐CI results indicate that a charge‐transfer state locates at 4.19 eV, which could be related to the channel of the photochemistry as indicated in a previous experimental study. We also studied the excitation and fluorescence energies of blue fluorescent protein, cyan fluorescent protein, and Y66F. The SAC‐CI results are very close to the experimental ones. The protonation state of blue fluorescent protein was determined. Conformation of cyan fluorescent protein indicated by the present calculation agrees to the experimentally observed structure.

Excited states of fluorescent proteins, mKO and DsRed: chromophore-protein electrostatic interaction behind the color variations.

The emitting states of green fluorescent protein (GFP), monomeric Kusabira orange (mKO), and Discosoma red (DsRed) were studied using QM/MM and SAC-CI methods. By comparing the electronic structures among the green-, orange-, and red-emitting states as well as their electrostatic and quantum mechanical interactions within the protein cavity, the basic mechanisms for determining emission colors have been clarified. We found that the orange and red emissions of mKO and DsRed, respectively, result from cancellation between two effects, the pi skeleton extension (red shift) and protein electrostatic potential (blue shift). The extension of the pi skeleton enhances the intramolecular charge-transfer character of the transition, which makes the fluorescence energy more sensitive to the proteins electrostatic potential. On the basis of this mechanism, we predicted amino acid mutations that could red shift the emission energy of DsRed. A novel single amino acid mutation, which was examined computationally, reduced the DsRed emission energy from 2.14 (579 nm) to 1.95 eV (636 nm), which is approaching near-infrared fluorescence.

Electronic Coulombic Coupling of Excitation-Energy Transfer in Xanthorhodopsin

Electronic coupling of excitation-energy transfer (EET) in a retinal (RET) protein, xanthorhodopsin (xR), was studied theoretically. The protein, functioning as a light driven proton pump, contains a carotenoid antenna, salinixanthin (SXN), to collect light energy for an RET chromophore through EET. The pseudo-Coulombic interaction (PCI) between the donor SXN and the acceptor RET molecules was calculated by a transition density fragment interaction (TDFI) method, which overcomes difficulty arising in the evaluation of PCI in xR by a conventional dipole-dipole (dd) method, at the ab initio TDDFT/SAC-CI level of theory. The result nicely agrees with the experimentally observed PCI. To examine the correlation between the SXN-RET alignment and the EET efficiency, we computed PCIs for SXN conformations that are virtually generated around the protein. The calculation shows that the optimal SXN alignment for the maximally tuned efficiency of EET is attained in the native xR. PCI in another retinal protein, archaerhodopsin-2, which also binds a carotenoid but lacks EET activity, was also evaluated. The computed PCI is negligibly small, well explaining the lack of EET efficiency.

Transition-density-fragment interaction approach for exciton-coupled circular dichroism spectra.

A transition-density-fragment interaction (TDFI) method for exciton-coupled circular dichroism (ECCD) spectra is proposed. The TDFI method was previously developed for excitation-energy transfer, which led to the successful estimation of the electronic coupling energy between donor and accepter molecules in xanthorhodopsin [K. J. Fujimoto and S. Hayashi, J. Am. Chem. Soc. 131, 14152 (2009)]. In the present study, the TDFI scheme is extended to the ECCD spectral calculation based on the matrix method and is applied to a dimerized retinal (all-trans N-retinylidene-L-alanine Schiff base) chromophore. Compared with the dipole-dipole and transition charge from ESP methods, TDFI has a much improved description of the electronic coupling. In addition, the matrix method combined with TDFI can reduce the computational costs compared with the full quantum-mechanical calculation. These advantages of the present method make it possible to accurately evaluate the CD Cotton effects observed in experiment.

Theoretical study of the opsin shift of deprotonated retinal schiff base in the M state of bacteriorhodopsin

The origin of the opsin shift, which deprotonated Schiff base (DPSB) shows in the M state of the bacteriorhodopsin (bR) photocycle, was theoretically investigated for the first time using a combined quantum mechanical and molecular mechanical (QM/MM) method. From the QM(SAC-CI)/MM(AMBER99) results, the chromophore conformational effect was found to be the main factor, whereas the Coulombic interaction with the protein environment gave a non-negligible contribution. The present result revised the conclusion drawn by previous studies and provided a new interpretation of the opsin shift mechanism of DPSB. To test the computational models for taking into account the electronic polarization and charge redistribution effects of the surrounding environment, the size of the QM region was expanded up to 5-7 Å from DPSB, which decreased the excitation energies in solution and in protein by 0.08-0.13 eV and 0.21-0.26 eV, respectively. We also found that the rCAM-B3LYP functional significantly improves the B3LYP results when calculating the potential energy curve for the C(6)-C(7) twisting.

A multicore QM/MM approach for the geometry optimization of chromophore aggregate in protein

In this article, we present the multicore (mc) QM/MM method, a QM/MM method that can optimize the structure of chromophore aggregate in protein. A QM region is composed of the sum of the QM subregions that are small enough to apply practical electronic structure calculations. QM/MM energy gradient calculations are performed for each QM subregion. Several benchmark examinations were carried out to figure out availabilities and limitations. In the interregion distances of more than 3.5–4.0 Å, the mcQM/MM energy gradient is very close to that obtained by the ordinary QM/MM method in which all the QM subregions were treated together as a single QM region. In van der Waals complex, the error exponentially drops with the distance, while the error decreases slowly in a hydrogen bonding complex. On the other hand, the optimized structures were reproduced with reasonable accuracy in both cases. The computational efficiency is the best advantage in the mcQM/MM approach, especially when the QM region is significantly large and the QM method used is computationally demanding. With this approach, we could optimize the structures of a bacterial photosynthetic reaction center protein in the ground and excited states, which consists of more than 14,000 atoms.