Biosynthesis of a fluorescent protein with extreme pseudo-Stokes shift by introducing a genetically encoded non-natural amino acid outside the fluorophore

A novel kind of fluorescent protein relying on the intramolecular interplay between two different fluorophores, one of chemical origin and one of biological origin, was developed. The fluorescent non-natural amino acid l-(7-hydroxycoumarin-4-yl)ethylglycine was site-specifically incorporated into the recombinant enhanced cyan fluorescent protein (eCFP) at a permissible surface position ～20 ? away from the protein fluorophore using amber suppression in Escherichia coli with an engineered cognate Methanococcus jannaschii tRNA synthetase. The resulting eCFP(Cou) exhibited almost quantitative intramolecular Fo?rster resonance energy transfer (FRET) between its two fluorophores, showing brilliant cyan emission at 476 nm upon excitation in the near-UV at 365 nm (a wavelength easily accessible via conventional laboratory UV sources), in contrast to its natural counterpart. Thus, this fluorescent protein with unprecedented spectroscopic properties reveals an extreme apparent Stokes shift of ～110 nm between the absorption wavelength of the coumaryl group and the emission wavelength of eCFP.     

Proof complexity of propositional default logic

Default logic is one of the most popular and successful formalisms for non-monotonic reasoning. In 2002, Bonatti and Olivetti introduced several sequent calculi for credulous and skeptical reasoning in propositional default logic. In this paper we examine these calculi from a proof-complexity perspective. In particular, we show that the calculus for credulous reasoning obeys almost the same bounds on the proof size as Gentzen??s system LK. Hence proving lower bounds for credulous reasoning will be as hard as proving lower bounds for LK. On the other hand, we show an exponential lower bound to the proof size in Bonatti and Olivetti??s enhanced calculus for skeptical default reasoning.     

Neurotoxin II bound to acetylcholine receptors in native membranes studied by dynamic nuclear polarization NMR

Methods enabling structural studies of membrane-integrated receptor systems without the necessity of purification provide an attractive perspective in membrane protein structural and molecular biology. This has become feasible in principle since the advent of dynamic nuclear polarization (DNP) magic-angle-spinning NMR spectroscopy, which delivers the required sensitivity. In this pilot study, we observed well-resolved solid-state NMR spectra of extensively (13)C-labeled neurotoxin II bound to the nicotinic acetylcholine receptor (nAChR) in native membranes. We show that TOTAPOL, a biradical required for DNP, is localized at membrane and protein surfaces. The concentration of active, membrane-attached biradical decreases with time, probably because of reactive components of the membrane preparation. An optimal distribution of active biradical has strong effects on the NMR data. The presence of inactive TOTAPOL in membrane-proximal situations but active biradical in the surrounding water/glycerol "glass" leads to well-resolved spectra, yet a considerable enhancement (ε = 12) is observed. The resulting spectra of a protein ligand bound to its receptor are paving the way for further DNP investigations of proteins embedded in native membrane patches.     

Stress functions and internal stresses in linear three-dimensional anisotropic elasticity

A stress function method is presented in order to give a general solution of the incompatibility problem of three-dimensional linear anisotropic elasticity theory. A relation between the internal stress tensor in terms of derivatives of a sixth-order stress function tensor will be derived. Using this formulation the so far open problem of the representation of the second-order stress function tensor by the fourth-order stress function tensor in anisotropic elasticity theory is solved in general.     

Scattering theory for Stark Hamiltonians

We give an introduction to the spectral and scattering theory for Schr?dinger operators. An abstract short range scattering theory is developed. It is applied to perturbations of the Laplacian. Particular attention is paid to the study of Stark Hamiltonians. The main result is an explanation of the discrepancy between the classical and the quantum scattering theory for one-dimensional Stark Hamiltonians.     

Size Measurement of Very Small Spherical Particles by Mie Scattering Imaging (MSI)

The Mie Scattering Imaging method (MSI) gathers out‐of‐focus images of dispersed spherical particles present in a laser light sheet and extracts the individual particle diameter from these images. The general idea of the method has been around for more than a decade and a number of papers has dealt with it over recent years. Our work focuses on small particle sizes from 20 μm down to 2 μm, a range which has not been tackled so far although it is of great importance in particle systems. We present an optical set‐up with a special arrangement of camera lenses that allows to work in this range. An evaluation algorithm based on correlation of the experimental optical information with theoretical Mie scattering was found to give the most accurate results for particle sizing. Besides accuracy measurements on solid spheres the versatility of the method is demonstrated by an example of transient droplet growth between 2–7 μm.