Red/Green Color Tuning of Visual Rhodopsins: Electrostatic Theory Provides a Quantitative Explanation.

We present a structure-based theory of the long-wavelength (red/green) color tuning in visual rhodopsins and its application to the analysis of site-directed mutagenesis experiments. Using a combination of electrostatic and molecular-mechanics methods, we explain the measured mutant-minus-wild-type absorption shifts and conclude that the dominant mechanism of the color tuning in these systems is electrostatic pigment-protein coupling. An important element of our analysis is the independent determination of protonation states of titratable residues in the wild type and the mutant protein as well as the self-consistent reoptimization of hydrogen atom positions, which includes the relaxation of the hydrogen bonding network and the reorientation of water molecules. On the basis of this analysis, we propose a "dipole-orientation rule" according to which both the position and the orientation of a polar group introduced in the protein environment determine the direction of the transition energy shift of the retinal chromophore.

Revealing the functional states in the active site of BLUF photoreceptors from electrochromic shift calculations.

Photoexcitation with blue light of the flavin chromophore in BLUF photoreceptors induces a switch into a metastable signaling state that is characterized by a red-shifted absorption maximum. The red shift is due to a rearrangement in the hydrogen bond pattern around Gln63 located in the immediate proximity of the isoalloxazine ring system of the chromophore. There is a long-lasting controversy between two structural models, named Q63A and Q63J in the literature, on the local conformation of the residues Gln63 and Tyr21 in the dark state of the photoreceptor. As regards the mechanistic details of the light-activation mechanism, rotation of Gln63 is opposed by tautomerism in the Q63A and Q63J models, respectively. We provide a structure-based simulation of electrochromic shifts of the flavin chromophore in the wild type and in various site-directed mutants. The excellent overall agreement between experimental and computed data allows us to evaluate the two structural models. Compelling evidence is obtained that the Q63A model is incorrect, whereas the Q63J is fully consistent with the present computations. Finally, we confirm independently that a keto-enol tautomerization of the glutamine at position 63, which was proposed as molecular mechanism for the transition between the dark and the light-adapted state, explains the measured 10 to 15 nm red shift in flavin absorption between these two states of the protein. We believe that the accurateness of our results provides evidence that the BLUF photoreceptors absorption is fine-tuned through electrostatic interactions between the chromophore and the protein matrix, and finally that the simplicity of our theoretical model is advantageous as regards easy reproducibility and further extensions.

Mixing behavior and interphase formation in the diethylene triamine-water system studied by optical imaging and spatially resolved Brillouin scattering.

The injection of water beneath liquid diethylene triamine in a glass cuvette leads to an unexpected phase evolution behavior of the two liquids. The space and time dependent developments of the molecular structure and the underlying transport associated with mixing of the two liquids are monitored by optical imaging and scanning Brillouin microscopy. Apparently, results obtained by either experimental technique lead to disparate interpretations. Whereas optical imaging suggests the existence of a two phase structure, which disappears within a few hours, acoustic microscopy indicates the evolution of a more gradually evolving and longer-lived three phase structure. According to molecular acoustics, the transport of diethylene triamine into water and vice versa behaves strongly asymmetric in time. An attempt is made to reconcile the observed optical and acoustic manifestations of the mixing process on the basis of molecular complex formation.