DFT/MM description of flavin IR spectra in BLUF domains.

A class of photoreceptors occurring in various organisms consists of domains that are blue light sensing using flavin (BLUF). The vibrational spectra of the flavin chromophore are spectroscopically well characterized for the dark-adapted resting states and for the light-adapted signaling states of BLUF domains in solution. Here we present a theoretical analysis of such spectra by applying density functional theory (DFT) to the flavin embedded in molecular mechanics (MM) models of its protein and solvent environment. By DFT/MM we calculate flavin spectra for seven different X-ray and NMR structures of BLUF domains occurring in the transcriptional antirepressor AppA and in the blue light receptor B (BlrB) of the purple bacterium Rb. Sphaeroides as well as in the phototaxis photoreceptor Slr1694 of the cyanobacterium Synechocystis. By considering the dynamical stabilities of associated all-atom simulation models and by comparing calculated with observed vibrational spectra, we show that two of the considered structures (both AppA) are obviously erroneous and that specific features of two further crystal structures (BlrB and Slr1694) cannot represent the states of the respective BLUF domains in solution. Thereby, the conformational transitions elicited by solvation are identified. In this context we demonstrate how hydrogen bonds of varying strengths can tune in BLUF domains the C═O stretching frequencies of the flavin chromophore. Furthermore we show that the DFT/MM spectra of the flavin calculated for two different AppA BLUF conformations, which are called Trp(in) and Met(in), fit very well to the spectroscopic data observed for the dark and light states, respectively, if (i) polarized MM force fields, which are calculated by an iterative DFT/MM procedure, are employed for the flavin binding pockets and (ii) the calculated frequencies are properly scaled. Although the associated analysis indicates that the Trp(in) conformation belongs to the dark state, no clear light vs dark distinction emerges for the Met(in) conformation. In this connection, a number of methodological issues relevant for such complex computations are thoroughly discussed showing, in particular, why our current descriptions could not decide the light vs dark question for Met(in).

IR spectra of flavins in solution: DFT/MM description of redox effects.

The functional reactions in blue light photoreceptors generally involve transiently reduced flavins exhibiting characteristic infrared (IR) spectra. To approach a theoretical understanding, here we apply density functional theory (DFT) to flavin radicals embedded in a molecular mechanics (MM) model of an aqueous solution. Combining a DFT/MM approach with instantaneous normal-mode analyses (INMA), we compute the IR solution spectra of anionic and neutral flavin radicals. For a set of mid-IR marker bands, we identify those changes of spectral locations, intensities, and widths, which are caused by sequentially adding an electron and a proton to the oxidized flavin. Comparisons with experimental IR solution spectra of flavin radicals show the accuracy of our DFT/MM-INMA approach and allow us to assign the observed bands. The room temperature ensembles of solvent cages required for the INMA calculations of the IR spectra are generated in an MM setting from molecular dynamics (MD) simulations. For the solvated flavin radicals, these MD simulations employ MM force fields derived from DFT/MM calculations.

Density functional theory combined with molecular mechanics: the infrared spectra of flavin in solution.

The photophysics and photochemistry of flavin dyes determine the functional dynamics of a series of blue light photoreceptors that include the so-called BLUF (blue light sensors using flavin) domains. To enable molecular dynamics (MD) simulation studies of such signaling processes, we derived molecular mechanics (MM) models of flavin chromophores from density functional theory (DFT). Two 300 K ensembles of lumiflavin (LF) in aqueous solution were generated by extended MM-MD simulations using different MM potentials for the water. In a DFT/MM hybrid setting, in which LF was treated by DFT and the polarizing environment at atomistic resolution by MM, we applied instantaneous normal mode analyses (INMA) to these ensembles. From these data we determined the inhomogeneously broadened solution spectra as mixtures of Gaussian bands using a novel automated procedure for mode classification. Comparisons with vibrational spectra available in the literature on native and isotopically labeled flavins in aqueous solution serve us to determine suitable frequency scaling factors and to analyze the accuracy of our scaled DFT/MM-INMA approach. We show that our approach not only agrees with established computational descriptions but also extends such methods substantially by giving access to inhomogeneous line widths and band shapes.

Relaxation time prediction for a light switchable peptide by molecular dynamics.

We study a monocyclic peptide called cAPB, whose conformations are light switchable due to the covalent integration of an azobenzene dye. Molecular dynamics (MD) simulations using the CHARMM22 force field and its CMAP extension serve us to sample the two distinct conformational ensembles of cAPB, which belong to the cis and trans isomers of the dye, at room temperature. For gaining sufficient statistics we apply a novel replica exchange technique. We find that the well-known NMR distance restraints are much better described by CMAP than by CHARMM22. In cAPB, the ultrafast cis/trans photoisomerization of the dye elicits a relaxation dynamics of the peptide backbone. Experimentally, we probe this relaxation at picosecond time resolution by IR spectroscopy in the amide I range up to 3 ns after the UV/vis pump flash. We interpret the spectroscopically identified decay kinetics using ensembles of non-equilibrium MD simulations, which provide kinetic data on conformational transitions well matching the observed kinetics. Whereas spectroscopy solely indicates that the relaxation toward the equilibrium trans ensemble is by no means complete after 3 ns, the 20 ns MD simulations of the process predict, independently of the applied force field, that the final relaxation into the trans-ensemble proceeds on a time scale of 23 ns. Overall our explicit solvent simulations cover more than 6 micros.