Bacteriorhodopsin and proteorhodopsin are simple heptahelical proton pumps containing a retinal chromophore covalently bound to helix G via a protonated Schiff base. Following the absorption of a photon, all-trans retinal is isomerized to a 13-cis conformation, initiating a sequence of conformational changes driving vectorial proton transport. In this study we apply time-resolved wide-angle X-ray scattering to visualize in real time the helical motions associated with proton pumping by bacteriorhodopsin and proteorhodopsin. Our results establish that three conformational states are required to describe their photocycles. Significant motions of the cytoplasmic half of helix F and the extracellular half of helix C are observed prior to the primary proton transfer event, which increase in amplitude following proton transfer. These results both simplify the structural description to emerge from intermediate trapping studies of bacteriorhodopsin and reveal shared dynamical principles for proton pumping.

The photoinduced electron transfer reactions in a self-assembled 1:1 complex of zinc(II)tetrasulphonatophenylporphyrin (ZnTPPS(4-)) and methylviologen (MV(2+)) in aqueous solution were investigated with transient absorption spectroscopy. ZnTPPS(4-) was excited either in the Soret or one of the two Q-bands, corresponding to excitation into the S(2) and S(1) states, respectively. The resulting electron transfer to MV(2+) occurred, surprisingly, with the same time constant of τ(FET) = 180 fs from both electronic states. The subsequent back electron transfer was rapid, and the kinetics was independent of the initially excited state (τ(BET) = 700 fs). However, ground state reactants in a set of vibrationally excited states were observed. The amount of vibrationally excited ground states detected increased with increasing energy of the initial excited state, showing that excess excitation energy survived a two-step electron transfer reaction in solution. Differences in the ZnTPSS(•3-)/MV(•+) spectra suggest that the forward electron transfer from the S(2) state at least partially produces an electronically excited charge transfer state, which effectively suppresses the influence of the inverted regime. Other possible reasons for the similar electron transfer rates for the different excited states are also discussed.

Photosynthetic reaction centers convert the energy content of light into a transmembrane potential difference and so provide the major pathway for energy input into the biosphere. We applied time-resolved Laue diffraction to study light-induced conformational changes in the photosynthetic reaction center complex of Blastochloris viridis. The side chain of TyrL162, which lies adjacent to the special pair of bacteriochlorophyll molecules that are photooxidized in the primary light conversion event of photosynthesis, was observed to move 1.3 angstroms closer to the special pair after photoactivation. Free energy calculations suggest that this movement results from the deprotonation of this conserved tyrosine residue and provides a mechanism for stabilizing the primary charge separation reactions of photosynthesis.

The physical properties of amorphous biomolecules are important to the texture and stability of low-moisture foods, the stability of pharmaceuticals, the permeability of edible films, and the viability of organisms during anhydrobiosis. Protein stability is often improved via the inclusion of small-molecule excipients during freeze-drying and organisms overproduce sugars such as sucrose or trehalose during anhydrobiosis. The effect on internal protein dynamics caused by substitution of a protein's surface water molecules with small sugar molecules is unclear. To explore this question, we have analyzed tryptophan phosphorescence decays of human serum albumin (HSA) in the dry amorphous solid state. Phosphorescence is an ideal approach, as the long-lived triplet state of tryptophan is sensitive to the long time-scale molecular motions of proteins in the dry state. Human serum albumin (HSA) was chosen because it contains a single, buried tryptophan residue and thus can provide information on the local dynamics of a specific site in the interior of the protein. Amorphous protein films were prepared by spreading concentrated solutions of HSA with and without sugar onto quartz slides, followed by rapid drying and extensive desiccation. Phosphorescence intensity decays were collected and fit with multiple exponential functions. From the average lifetime of these fits the rates of nonradiative decay (kNR) of the triplet state were calculated; kNR is dependent on the microviscosity of the site and is thus a measure of molecular mobility of the HSA tryptophan site. At all temperatures this measure of molecular mobility was lower in the films containing sucrose. Break-point analysis of a kNR Arrhenius plot revealed two temperature regimes with a transition occurring at the glass transition temperature of sucrose.

Ultrafast time-resolved wide angle x-ray scattering from chemical reactions in solution has recently emerged as a powerful technique for determining the structural dynamics of transient photochemical species. Here we examine the structural evolution of photoexcited CH(2)I(2) in the nonpolar solvent cyclohexane and draw comparisons with a similar study in the polar solvent methanol. As with earlier spectroscopic studies, our data confirm a common initial reaction pathway in both solvents. After photoexcitation, CH(2)I(2) dissociates to form CH(2)I* + I*. Iodine radicals remaining within the solvent cage recombine with a nascent CH(2)I* radical to form the transient isomer CH(2)I-I, whereas those which escape the solvent cage ultimately combine to form I(2) in cyclohexane. Moreover, the transient isomer has a lifetime approximately 30 times longer in the nonpolar solvent. Of greater chemical significance is the property of time-resolved wide angle x-ray diffraction to accurately determine the structure of the of CH(2)I-I reaction intermediate. Thus we observe that the transient iodine-iodine bond is 0.07 A+/-0.04 A shorter in cyclohexane than in methanol. A longer iodine-iodine bond length for the intermediate arises in methanol due to favorable H-bond interaction with the polar solvent. These findings establish that time-resolved x-ray diffraction has sufficient sensitivity to enable solvent dependent structural perturbations of transient chemical species to be accurately resolved.