Optimized and far-red-emitting variants of fluorescent protein eqFP611

Fluorescent proteins (FPs) emitting in the far-red region of the spectrum are highly advantageous for whole-body imaging applications because scattering and absorption of long-wavelength light is markedly reduced in tissue. We characterized variants of the red fluorescent protein eqFP611 with bright fluorescence emission shifted up to 639 nm. The additional red shift is caused by a trans-cis isomerization of the chromophore. The equilibrium between the trans and cis conformations is strongly influenced by amino acid residues 143 and 158. Pseudo monomeric tags were obtained by further genetic engineering. For the red chromophores of eqFP611 variants, molar extinction coefficients of up to approximately 150,000 were determined by an approach that is not affected by the presence of molecules with nonfunctional red chromophores. The bright fluorescence makes the red-shifted eqFP611 variants promising lead structures for the development of near-infrared fluorescent markers. The red fluorescent proteins performed well in cell biological applications, including two-photon imaging.     

Carbon chains of type C2n+1N(-) (n=2-6): A theoretical study of potential interstellar anions

Linear anions of type C(2n+1)N(-) (n=2-6), which are expected to be good candidates for experimental investigation by microwave spectroscopy and radio astronomy, were studied by means of the coupled cluster variant CCSD(T). Making use of corrections taken over from HC(3)NC(3)N(-) and HC(5)N, accurate equilibrium structures ( approximately 0.0005 A accuracy in bond lengths) have been established for all five anions. The electric dipole moments increase strongly with increasing chain length. For C(13)N(-), a very large equilibrium dipole moment of 16.53 D (with respect to center-of-mass coordinate system, negative end of dipole at terminal carbon site) is predicted. The lowest vertical detachment energies, leading to (2)Sigma states of the radicals for C(3)N(-) and C(5)N(-) and to (2)Pi states in the case of the larger anions, are calculated to lie in the range of 4.40-4.63 eV. The ground-state rotational and quartic centrifugal distortion constants of C(5)N(-) are predicted to be 1389.4 MHz and 33.8 Hz, respectively. All anions studied appear to be fairly normal semirigid linear molecules. Throughout, good agreement with available matrix isolation IR spectroscopic data is obtained and many predictions of spectroscopic properties are made.     

Synthesis and photoinduced intramolecular processes of fulleropyrrolidine-oligothienylenevinylene-ferrocene triads

Two new triads based on N-methylfulleropyrolidine, oligothienylenevinylenes (nTV) and ferrocene (Fc), namely C(60)-nTV-Fc (n=2, 4) have been synthesized. A HOMO-LUMO gap as low as 1.09-1.11 eV was experimentally determined by cyclic voltammetry. In both polar and nonpolar solvents, photoinduced charge-separation (CS) processes in C(60)-nTV-Fc predominantly take place from the singlet excited states of C(60) and nTV; this result was indicated by steady and time-resolved emission spectroscopy. In the case of C(60)-4TV-Fc, the CS state was indicated by the nanosecond transient absorption spectra. In C(60)-2TV-Fc, although the CS process was also confirmed by the fluorescence quenching in nonpolar and polar solvents, the lifetimes of the CS states were shorter than those of C(60)-4TV-Fc. It was revealed that the introduction of Fc donor moiety at the end of the longer nTV chain in the C(60)-nTV dyad systems effectively increases the CS efficiency and the lifetimes of CS states.     

Synthesis, structural characterization, catalytic properties, and theoretical study of compounds containing an Al-O-M (M = Ti, Hf) core

Two single oxygen-bridged heterobimetallic oxides of Al(III) with group 4 metals (Ti, Hf) have been prepared. The reaction of LAlMeOH (1) [L = CH(N(Ar)(CMe))2, Ar = 2,6-iPr2C6H3] with dimethylmetallocenes of Ti and Hf in toluene (80 degrees C) and ether (room temperature), respectively, resulted in the formation of LAl(Me)(mu-O)M(Me)Cp2 [M = Ti (2), Hf (3)] in moderate to good yield. Compounds 2 and 3 were characterized by elemental analysis, IR, NMR (1H and 13C), EI-MS, and single-crystal X-ray structural analysis. Furthermore, compound 2 showed good catalytic activity in ethylene and styrene homopolymerization, while compound 3 is less active in ethylene polymerization. The styrene polymerization yields atactic polystyrene.     

Dual triggering of DNA binding and fluorescence via photoactivation of a dinuclear ruthenium(II) arene complex

The dinuclear RuII arene complexes [{(eta6-arene)RuCl}2(mu-2,3-dpp)](PF6)2, arene=indan (1), benzene (2), p-cymene (3), or hexamethylbenzene (4) and 2,3-dpp=2,3-bis(2-pyridyl)pyrazine, have been synthesized and characterized. Upon irradiation with UVA light, complexes 1 and 2 readily underwent arene loss, while complexes 3 and 4 did not. The photochemistry of 1 was studied in detail. In the X-ray structure of [{(eta6-indan)RuCl}2(mu-2,3-dpp)](PF6)2 (1), 2,3-dpp bridges two RuII centers 6.8529(6) A apart. In water, aquation of 1 in the dark occurs with replacement of chloride with biexponential kinetics and decay constants of 100+/-1 min-1 and 580+/-11 min-1. This aquation was suppressed by 0.1 M NaCl. UV or visible irradiation of 1 in aqueous or methanolic solution led to arene loss. The fluorescence of the unbound arene is approximately 40 times greater than when it is complexed. Irradiation of 1 also had a significant effect on its interactions with DNA. The DNA binding of 1 is increased after irradiation. The non-irradiated form of 1 preferentially formed DNA adducts that only weakly blocked RNA polymerase, while irradiation of 1 transformed the adducts into stronger blocks for RNA polymerase. The efficiency of irradiated 1 to form DNA interstrand cross-links was slightly greater than that of cisplatin in both 10 mM NaClO4 and 0.1 M NaCl. In contrast, the interstrand cross-linking efficiency of non-irradiated 1 in 10 mM NaClO4 was relatively low. An intermediate amount of cross-linking was observed when the sample of DNA already modified by non-irradiated 1 was irradiated. DNA unwinding measurements supported the conclusion that both mono- and bifunctional adducts with DNA can form. These results show that photoactivation of dinuclear RuII arene complexes can simultaneously produce a highly reactive ruthenium species that can bind to DNA and a fluorescent marker (the free arene). Importantly, the mechanism of photoreactivity is also independent of oxygen. These complexes, therefore, have the potential to combine both photoinduced cell death and fluorescence imaging of the location and efficiency of the photoactivation process.     

Surface tension and capillary waves at the nematic-isotropic interface in ternary mixtures of liquid crystal, colloids, and impurities

In mixtures of thermotropic liquid crystals with spherical poly(methyl methacrylate) particles, self-supporting networklike structures are formed during slow cooling past the isotropic-to-nematic phase transition. Experimental results support the hypothesis that a third component, alkane remnants slowly liberated from the particles, plays a crucial role. A theoretical model, based on the phenomenological Landau-de Gennes, Carnahan-Starling, and hard-sphere crystal theories, is developed to describe the continuous phase separation in a ternary nematic-impurity-colloid mixture. The interfacial tension and the dispersion relation of the surface modes of the nematic-isotropic interface are determined. The colloids decrease the interfacial tension and the damping rate of surface waves, whereas impurities act in an opposite way. This should strongly influence the formation of abovementioned networklike structures and could help explain some of their rheological properties.     

Patterned burst-pulse vocalizations of the northern right whale dolphin, Lissodelphis borealis

Vocalizations from the northern right whale dolphin, Lissodelphis borealis, were recorded during a combined visual and acoustic shipboard survey of cetacean populations off the west coast of the United States. Seven of twenty single-species schools of L. borealis produced click and pulsed vocalizations. No whistles were detected during any of the encounters. Clicks associated with burst-pulse vocalizations were lower in frequency and shorter in duration than clicks associated with echolocation. All burst-pulse sounds were produced in a series containing 6-18 individual burst-pulses. These burst-pulse series were stereotyped and repeated. A total of eight unique burst-pulse series were detected. Variation in the temporal characteristics of like units compared across repeated series was less than variation among all burst-pulses. These stereotyped burst-pulse series may play a similar communicative role as do stereotyped whistles found in other delphinid species.     

Photoconvertible fluorescent protein EosFP: biophysical properties and cell biology applications

EosFP is a fluorescent protein from the coral Lobophyllia hemprichii that changes its fluorescence emission from green to red upon irradiation with near-UV light. Here we present the spectroscopic properties of wild-type EosFP and a variety of monomeric and dimeric mutants and provide a structural interpretation of its oligomerization and photoconversion, which is based on X-ray structure analysis of the green and red species that we reported recently. Because functional expression of the monomeric EosFP variant is limited to temperatures of 30 degrees C, we have developed a tandem dimer. This construct, in which two EosFP subunits are connected by a flexible 12 amino acid linker, expresses well after fusion with the androgen and endothelin A receptors at 37 degrees C. A variety of applications in cellular imaging, developmental biology and automated high-content screening applications are presented, which demonstrate that EosFP is a powerful tool for in vivo monitoring of cellular processes.     

Fringed sound fields and their interaction with liquid-solid interfaces

On the one hand, it is well known that Gaussian beams possess the ability to stimulate Rayleigh waves, resulting in the Schoch effect, a lateral beam displacement. This effect, often characterized by a reflected sound pattern consisting of two anti-phase beams, is due to the re-radiation of sound because of the stimulation of leaky Rayleigh waves. On the other hand, fringed sound beams are characterized by the fact that they consist of a number of neighboring anti-phase narrow beams. They are a first approximation of a sound field originating from a phased array of harmonic vibrating crystals in which each crystal vibrates in anti-phase compared to its neighbor. The individual lobes within the fringed sound pattern diverge much less than standard Gaussian beams of the same size. The current study investigates the interaction of fringed beams with a liquid-solid interface. It is found that under certain conditions, a fringed beam, incident at the Rayleigh angle, produces a reflected sound pattern that contains a wide lobe that is not fringed. It is also shown that under other conditions, contrary to the famous forward displacement of the reflected sound for incident Gaussian beams, a strong backward displacement occurs for fringed beams.     

Faceting and stability of smectic A droplets on a solid substrate

It is shown that a smectic A droplet deposited on a solid substrate treated for strong homeotropic anchoring is faceted at the top in spite of the fact that there are no steps at the free surface, but instead edge dislocations in the bulk. The radius of the facet and the full profile of the curved part of the droplet are determined as a function of the temperature in the vicinity of a nematic-smectic A phase transition. It is shown that the observed profiles do not correspond to the actual equilibrium shape, but to metastable configurations close to their point of marginal stability. In addition, we predict that the profiles must be different for a given temperature depending on whether the droplet has been heated or cooled down to reach this temperature. Finally, we discuss the problem of the formation of giant dislocations in big droplets (Grandjean terraces).