Building a model of the blue cone pigment based on the wild type rhodopsin structure with QM/MM methods.

Understanding the mechanism of color tuning of the retinal chromophore by its host protein became one of the key issues in the research on rhodopsins. While early mutation studies addressed its genetic origin, recent studies advanced to investigate its structural origin, based on X-ray crystallographic structures. For the human cone pigments, no crystal structures have been produced, and homology models were employed to elucidate the origin of its blue-shifted absorption. In this theoretical study, we take a different route to establish a structural model for human blue. Starting from the well-resolved structure of bovine rhodopsin, we derive multiple mutant models by stepwise mutation and equilibration using molecular dynamics simulations in a hybrid quantum mechanics/molecular mechanics framework. Our 30fold mutant reproduces the experimental UV-vis absorption shift of 0.45 eV and provides new insights about both structural and genetic factors that affect the excitation energy. Electrostatic effects of individual amino acids and collaborative structural effects are analyzed using semiempirical (OM2/MRCI) and ab initio (SORCI) multireference approaches.

Color tuning in binding pocket models of the chlamydomonas-type channelrhodopsins.

We examined the shift of absorption maxima between the chlamydomonas-type channelrhodopsins (ChRs) and bacteriorhodopsin (BR). Starting from the BR X-ray structure, we modeled the color tuning in the binding pockets of the ChRs by mutating up to 28 amino acids in the vicinity of the chromophore. By applying the efficient self-consistent charge density functional tight binding (SCC-DFTB) method in a quantum mechanical/molecular mechanical (QM/MM) framework, including explicit polarization and calculating excitation energies with the semiempirical OM2/MRCI method and the ab initio SORCI method, we have shown that multiple mutations in the binding pocket of BR causes large hypsochromic shifts that are of the same order as the experimentally observed shifts of the absorption maxima between BR and the ChRs. This study further demonstrates that mutations in the proximity of the Schiff base and complex counterion lead to a stronger but more flexible interaction with the retinal, which could serve as a possible explanation for the spectral patterns found in the ChRs.

The protonation state of Glu181 in rhodopsin revisited: interpretation of experimental data on the basis of QM/MM calculations.

The structure and spectroscopy of rhodopsin have been intensely studied in the past decade both experimentally and theoretically; however, important issues still remain unresolved. Of central interest is the protonation state of Glu181, where controversial and contradictory experimental evidence has appeared. While FTIR measurements indicate this residue to be unprotonated, preresonance Raman and UV-vis spectra have been interpreted in favor of a protonated Glu181. Previous computational approaches were not able to resolve this issue, providing contradicting data as well. Here, we perform hybrid QM/MM calculations using DFT methods for the electronic ground state, MRCI methods for the electronically excited states, and a polarization model for the MM part in order to investigate this issue systematically. We constructed various active-site models for protonated as well as unprotonated Glu181, which were evaluated by computing NMR, IR, Raman, and UV-vis spectroscopic data. The resulting differences in the UV-vis and Raman spectra between protonated and unprotonated models are very subtle, which has two major consequences. First, the common interpretation of prior Raman and UV-vis experiments in favor of a neutral Glu181 appears questionable, as it is based on the assumption that a charge at the Glu181 location would have a sizable impact. Second, also theoretical results should be interpreted with care. Spectroscopic differences between the structural models must be related to modeling uncertainties and intrinsic methodological errors. Despite a detailed comparison of various rhodopsins and mutants and consistently favorite results with charged Glu181 models, we find merely weak evidence from UV-vis and Raman calculations. On the contrary, difference FTIR and NMR chemical shift measurements on Rh mutants are indicative of the protonation state of Glu181. Supported by our results, they provide strong and independent evidence for a charged Glu181.

Long-distance proton transfer with a break in the bacteriorhodopsin active site.

Bacteriorhodopsin is a proton-pumping membrane protein found in the plasma membrane of the archaeon Halobacterium salinarium. Light-induced isomerization of the retinal chromophore from all-trans to 13-cis leads to a sequence of five conformation-coupled proton transfer steps and the net transport of one proton from the cytoplasmic to the extracellular side of the membrane. The mechanism of the long-distance proton transfer from the primary acceptor Asp85 to the extracellular proton release group during the O --> bR is poorly understood. Experiments suggest that this long-distance transfer could involve a transient state [O] in which the proton resides on the intermediate carrier Asp212. To assess whether the transient protonation of Asp212 participates in the deprotonation of Asp85, we performed hybrid Quantum Mechanics/Molecular Mechanics proton transfer calculations using different protein structures and with different retinal geometries and active site water molecules. The structural models were assessed by computing UV-vis excitation energies and C=O vibrational frequencies. The results indicate that a transient [O] conformer with protonated Asp212 could indeed be sampled during the long-distance proton transfer to the proton release group. Our calculations suggest that, in the starting proton transfer state O, the retinal is strongly twisted and at least three water molecules are present in the active site.