Recently, the first known light-driven sodium pumps, from the microbial rhodopsin family, were discovered. We have solved the structure of one of them, Krokinobacter eikastus rhodopsin 2 (KR2), in the monomeric blue state and in two pentameric red states, at resolutions of 1.45 Å and 2.2 and 2.8 Å, respectively. The structures reveal the ion-translocation pathway and show that the sodium ion is bound outside the protein at the oligomerization interface, that the ion-release cavity is capped by a unique N-terminal α-helix and that the ion-uptake cavity is unexpectedly large and open to the surface. Obstruction of the cavity with the mutation G263F imparts KR2 with the ability to pump potassium. These results pave the way for the understanding and rational design of cation pumps with new specific properties valuable for optogenetics.

Individual multichromophoric dendrimer molecules, bearing eight perylenecarboximide chromophores at the rim, immobilized in a thin polyvinylbutyral (PVB) film were studied by far-field fluorescence microscopy. Fluorescence intensity trajectories as a function of time (transients), spectra, and decay traces were recorded separately or simultaneously. For comparison, similar measurements have been performed on a model compound containing one perylenecarboximide chromophore. Collective on/off jumps of the fluorescence intensity were observed for single dendrimer molecules, resembling previously reported collective jumps for the emission of single light-harvesting antenna systems. Spectra and decays of both non-interacting and dimer-like interacting chromophoric sites could be distinguished within an individual dendrimer. Transitions between the different spectral forms and decay times, observed for a single molecule, underline the dynamic character of the interactions among the chromophores. Evidence for a stepwise bleaching process of the multichromophoric system was found. Furthermore, the single-molecule data incontestably prove the assumptions stated in the ensemble model.

Photoactive Yellow Protein ( PYP), discovered almost 20 years ago in Ectothiorhodospira (Halorhodospira) halophila, 1 is a 4-hydroxycinnamic acid-containing protein that functions as a blue-light photoreceptor in a behavioral avoidance response in this organism. During the past 10 years, PYP has become a model system for studies in photochemistry and protein folding, to the extent that it has become competitive with the rhodopsins. This is because PYP is small and very water-soluble, forms crystals readily (diffracting to high resolution), and shows excellent chemical-and photo-stability. These overall characteristics have allowed the application of an array of physicochemical techniques to analyze the biological function of PYP, i.e., the translation of a change of the configuration of its 4-hydroxycinnamic acid chromophore into an altered conformation of the surrounding protein. This has led to detailed insight into this process, both temporally and spatially, with respect to the structure of the transient intermediates involved, although we are still quite far from being able to track the position of all atoms in space, upon light activation of the protein in the relevant time domain. Nevertheless, the data already obtained may function as a calibration set in future work, to extend the time span of molecular dynamics simulations of conformational transitions in proteins to the time scale relevant for catalytic turnover. Occasionally, the application of multiple biophysical techniques has led to (seemingly) conflicting results. In one example, this has revealed the fact that the light-induced conformational transitions in this photoreceptor protein can become restricted by the mesoscopic context, e.g., via a crystal lattice. Other inconsistencies, such as those regarding the radius of gyration of the protein, still remain to be explained. Below, we discuss the spatial and temporal details of the series of steps initiated in PYP by a short pulse of blue light, as revealed with this array of biophysical techniques, thereby highlighting contributions from our own group.

Wild type green fluorescent protein (GFP) from Aequorea victoria absorbs predominantly at 398 nm. Illumination with UV (254 nm) or visible (390 nm) light transforms this state (GFP(398)) into one absorbing at 483 nm (GFP(483)). Here we show that this photoconversion of GFP is a one-photon process that is paralleled by decarboxylation of Glu 222. We propose a mechanism in which decarboxylation is due to electron transfer between the gamma-carboxylate of Glu 222 and the p-hydroxybenzylidene-imidazolidinone chromophore of GFP, followed by reverse transfer of an electron and a proton to the remaining carbon side chain atom of Glu 222. Oxidative decarboxylation of a gamma-carboxylate represents a new type of posttranslational modification that may also occur in enzymes with high-potential reaction intermediates.

Two procedures based on the weighted least-squares (LS) and the maximum likelihood estimation (MLE) method to confidently analyze single-molecule (SM) fluorescence decays with a total number (N) of 2,500-60,000 counts have been elucidated and experimentally compared by analyzing measured bulk and SM decays. The key observation of this comparison is that the LS systematically underestimates the fluorescence lifetimes by approximately 5%, for the range of 1,000-20,000 events, whereas the MLE method gives stable results over the whole intensity range, even at counts N less than 1,000, where the LS analysis delivers unreasonable values. This difference can be attributed to the different statistics approaches and results from improper weighting of the LS method. As expected from theory, the results of both methods become equivalent above a certain threshold of N detected photons per decay, which is here experimentally determined to be approximately 20,000. In contrast to the bulk lifetime distributions, the SM fluorescence lifetime distributions exhibit standard deviations that are sizably larger than the statistically expected values. This comparison proves the strong influence of the inhomogenuous microenvironment on the photophysical behavior of single molecules embedded in a 10-30-nm thin polymer layer.

In this study photophysical characteristics of LOV-based fluorescent proteins which are essential for analytic methods as well as imaging approaches have been comparatively analyzed in detail.

The generation of an excitatory receptor current in mammalian olfactory sensory neurons (OSNs) involves the sequential activation of two distinct types of ion channels: cAMP-gated Ca 2ϩ -permeable cation channels and Ca

Higher generations of poly(propylene imine) dendrimers functionalized with aliphatic chains form large micrometer-sized spherical objects in aqueous solution below pH 8. These spheres are giant vesicles with a multilaminar onion-like structure. The size distribution and the structure of the vesicles depend on the pH of the solution and the endgroups at the periphery of the dendrimer. The vesicles containing azobenzene units (2 and 3) fluoresce with a maximum at λ max ) 600 nm. This emission can be attributed to the dense and ordered arrangement of the azobenzene chromophores in the bilayer structure. Laser irradiation of a small area of giant vesicles of 2 or 3 with 1064 and/or 420 nm light leads to changes in the morphology of the vesicles. Infrared light induces a rearrangement, whereas the azobenzene units isomerize under the influence of 420 nm light. Both irradiations lead to a change in refractive index in the illuminated area. Irradiation using 420 nm light is accompanied by an increase in the emission intensity. In aqueous solutions at pH 1, the increase in fluorescence intensity is concurrent with a blue shift of the emission maximum to 540 nm. This blue shift is not observed when the experiment is performed in Milli Q water (pH 5.5). The enhanced fluorescence can be attributed to reorganization of the chromophores within the giant vesicle. The increase in emission proves that the giant vesicle is a kinetically formed system that reaches a thermodynamically more relaxed state after light-induced isomerization.