Urs P. Wild

Coupled rotation within single F0F1 enzyme complexes during ATP synthesis or hydrolysis

F0F1 ATP synthases are the smallest rotary motors in nature and work as ATP factories in bacteria, plants and animals. Here we report on the first observation of intersubunit rotation in fully coupled single F0F1 molecules during ATP synthesis or hydrolysis. We investigate the Na+‐translocating ATP synthase of Propionigenium modestum specifically labeled by a single fluorophore at one c subunit using polarization‐resolved confocal microscopy. Rotation during ATP synthesis was observed with the immobilized enzyme reconstituted into proteoliposomes after applying a diffusion potential, but not with a Na+ concentration gradient alone. During ATP hydrolysis, stepwise rotation of the labeled c subunit was found in the presence of 2 mM NaCl, but not without the addition of Na+ ions. Moreover, upon the incubation with the F0‐specific inhibitor dicyclohexylcarbodiimide the rotation was severely inhibited.

Direct observation of the triplet lifetime quenching of single dye molecules by molecular oxygen

The influence of oxygen on the photophysical properties of individual DiI18 molecules has been investigated by means of both wide field and confocal scanning optical fluorescence microscopy. Excited close to saturation intensity, single-molecule fluorescence detected in wide field by a charge-coupled device camera showed a dramatic increase at exposure to air compared to the fluorescence when the sample was protected from oxygen by nitrogen flush. The change of the triplet lifetime of individual dye molecules due to oxygen quenching was measured in real time by means of time resolved single photon counting. In the presence of oxygen, the triplet state lifetime decreases from several tens of milliseconds down to fractions of a millisecond, whereas no changes of the intersystem crossing quantum yield and the fluorescence lifetime are observed. The triplet lifetimes in the presence and absence of oxygen, respectively, are anti-correlated indicative of heterogeneity of the polymer surrounding the dye molecules.

On the origin of the red emission of light adapted purple membrane of Halobacterium halobium.

The light induced reaction cycle of bacteriorhodopsin (bR) in the light adapted (la.) purple membrane of Halobacterium halobium is described in the literature as consisting of a primary photochemical event followed by several thermal steps [ 1,2] . The initial chromophore, all-trans-retinal, is converted within 6 ps [3] after excitation by light into a product with an absorption maximum at about 630 nm [4,5]. This primary photoproduct is referred to as batho-form or K-form. A red luminescence centered around 700 nm has been observed from 1.a. purple membrane [6,7]. Studying this luminescence from bR in aqueous suspensions of purple membrane at 77% and room temperature we have obtained results which show that there exists a second primary photoproduct of bR. This is stable at 77% and has an excitation spectrum with a maximum at 597 i: 3 nm. In view of this finding, we discuss some results taken from the literature for which no satisfactory explanation has yet been given.

Theoretical study on geometry and spectroscopic properties of 1,1′-binaphthyl in the electronic ground and first excited singlet states

Abstract Equilibrium geometries for the electronic ground and first excited singlet states of 1,1-binaphthyl have been calculated by minimization of the total energy with respect to all internal coordinates. Using these results, an interpretation of the fluorescence S 1 → S 0 and absorption spectra S m ← S 0 and S n ← S 1 in rigid and fluid solutions is given. For the first time the equilibrium geometry of the first excited singlet state of 1, 1′-binaphthyl has been calculated. On excitation to the S 1 state the dihedral angle θ between the two naphthalene moieties is de- creased from 61 ° to 41 °. A detailed survey of CH bond lengths in the S 0 and S 1 states has been given. This result should be of particular importance for the theoretical treatment of radiationless transitions. Using equilibrium geometries for the S 0 and S 1 states a satisfactory interpretation of the S m ← S 0 and S n ← S 1 absorption spectra as well as of the fluorescence spectra in fluid and rigid solutions can be given. Concerning the S n ← S 1 absorption spectrum in fluid solution, the calculations predict a strong absorption (A ← B transition) in the still uninvestigated region of energies lower than 11000 cm −1 . From the results of this paper and of other calculations it can be concluded that the Warshel-Karplus method yields reliable equilibrium geometries for electronic ground and excited states of unsaturated hydrocarbons [22,23].

Quantum beats of prompt fluorescence in tetracene crystals

Abstract We have measured the quantum beats in the decay of pulse-excited “prompt” fluorescence in tetracene crystals as a function of magnetic field. We have demonstrated that counting the beats is a very simple and reliable method to study a possible influence of triplet-triplet interactions. In all so far performed experiments in tetracene crystals no influence has been found.

The citrate carrier CitS probed by single-molecule fluorescence spectroscopy.

A prominent region of the Na(+)-dependent citrate carrier (CitS) from Klebsiella pneumoniae is the highly conserved loop X-XI, which contains a putative citrate binding site. To monitor potential conformational changes within this region by single-molecule fluorescence spectroscopy, the target cysteines C398 and C414 of the single-Cys mutants (CitS-sC398, CitS-sC414) were selectively labeled with the thiol-reactive fluorophores AlexaFluor 546/568 C(5) maleimide (AF(546), AF(568)). While both single-cysteine mutants were catalytically active citrate carriers, labeling with the fluorophore was only tolerated at C398. Upon citrate addition to the functional protein fluorophore conjugate CitS-sC398-AF(546), complete fluorescence quenching of the majority of molecules was observed, indicating a citrate-induced conformational change of the fluorophore-containing domain of CitS. This quenching was specific for the physiological substrate citrate and therefore most likely reflecting a conformational change in the citrate transport mechanism. Single-molecule studies with dual-labeled CitS-sC398-AF(546/568) and dual-color detection provided strong evidence for a homodimeric association of CitS.

High resolution luminescence spectrometer. 2:data treatment and corrected spectra.

In Part 1 [Appl. Opt. 12, 1286 (1973)] we described the design of a high resolution luminescence spectrometer for research purposes. In this paper the computer processing of its digital output is discussed. The data treatment is based upon statistical concepts. Subroutines for wavelength calibration, dark count correction, data smoothing, and curve presentation have been developed. Corrected emission and excitation spectra may be obtained with additional calibration data, and relative quantum yields may be determined. The final luminescence spectra are plotted with their corresponding standard deviations.

A low temperature investigation of the intermediates of the photocycle of light-adapted bacteriorhodopsin. Optical absorption and fluorescence measurements☆

Optical absorption and emission measurements have been made on samples of light-adapted purple membrane of Halobacterium halobium at temperatures ranging from 77 K to room temperature. As a result of these experiments a set of equations is given which described thermal and photochemical reactions interrelating various intermediates of the reaction cycle of the chromophore of light-adapted bacteriorhodopsin (BR). Further some specific problems connected to these intermediates have been investigated. Thus the room temperature emission spectrum of bacteriorhodopsin has been found to exhibit a Stokes shift of 3430 cm-1 only, if low excitation intensities are used. The recently detected intermiediate P-BR can be shown to convert thermally into bacteriorhodopsin following a first-order decay with the activation energy delta E = 2.4 +/- 0.2 kcal/mol. The thermal decay of K-BR consists of two exponentials if measured on purple membrane suspensions in a mixture of H2O and glycerol (1 : 1, v/v). A simple procedure is given for trapping the intermediate L-BR at 170 K in a very pure form. M-BR is shown to consist of two species, MI-BR and MII-BR. They are characterized by similar optical absorption spectra but different thermal stability. Further the oscillator strengths corresponding to the long wavelength absorption bands of the intermediates bacteriorhodopsin, K-, L, MI- and MII-BR have been calculated. They have been discussed with respect to the question which of the corresponding absorption spectra show the characteristics of isomerism of the chromophore or simply solvatochromism.

Ultraviolet spectra and electronic structure of nitroethylene

Abstract The u.v. spectrum of nitroethylene has been measured and interpreted with the aid of a Pariser-Parr-Pople SCF-MO calculation. A line shape analysis has been performed. Absorption bands at 49,400, 41,300, and 33,900 cm −1 have been observed. The first two bands are in good agreement with the calculated π—π ∗ transitions. The weak band at 33,900 cm −1 is assigned to a n — π ∗ transition. Ultraviolet spectra have also been taken of trans -1-nitro-1-propene, nitromethane, and 3-nitro-1-propene. The spectrum of the first compound which also allows resonance between the π-systems of the ethylene and the nitro-group is very similar to the spectrum of nitroethylene. The spectrum of 3-nitro-1-propene resembles that of nitromethane.

Phase behavior and anisotropic optical properties of photoluminescent polarizers

The phase behavior and anisotropic optical properties of tensile deformed blends of a photoluminescent polymer guest in an ultra-high molecular weight polyethylene matrix were studied on the level of single molecules by means of scanning confocal optical microscopy. It is shown that upon tensile deformation of the blends, the system transforms from a phase-separated system into a quasi-molecular solid solution. The influence of this phase transition on the anisotropic optical properties of oriented blend films was also investigated with polarized steady-state photoluminescence spectroscopy. We show that well-dissolved guest molecules tend to reach higher degrees of orientation at lower draw ratios of the blend films compared to guests that phase-separate from the matrix polymer. Dichroic ratios in emission in the range of 50 were observed in optimized blend films based on photoluminescent oligomers and linear low density polyethylene.