Axel Gansmüller

Double-Quantum 13C Nuclear Magnetic Resonance of Bathorhodopsin, the First Photointermediate in Mammalian Vision

The 13C chemical shifts of the primary visual photointermediate bathorhodopsin have been observed by performing double-quantum magic-angle-spinning NMR at low temperature in the presence of illumination. Strong isomerization shifts have been observed upon the conversion of rhodopsin into bathorhodopsin.

Towards an interpretation of 13C chemical shifts in bathorhodopsin, a functional intermediate of a G-protein coupled receptor.

Photoisomerization of the membrane-bound light receptor protein rhodopsin leads to an energy-rich photostate called bathorhodopsin, which may be trapped at temperatures of 120 K or lower. We recently studied bathorhodopsin by low-temperature solid-state NMR, using in situ illumination of the sample in a purpose-built NMR probe. In this way we acquired (13)C chemical shifts along the retinylidene chain of the chromophore. Here we compare these results with the chemical shifts of the dark state chromophore in rhodopsin, as well as with the chemical shifts of retinylidene model compounds in solution. An earlier solid-state NMR study of bathorhodopsin found only small changes in the (13)C chemical shifts upon isomerization, suggesting only minor perturbations of the electronic structure in the isomerized retinylidene chain. This is at variance with our recent measurements which show much larger perturbations of the (13)C chemical shifts. Here we present a tentative interpretation of our NMR results involving an increased charge delocalization inside the polyene chain of the bathorhodopsin chromophore. Our results suggest that the bathochromic shift of bathorhodopsin is due to modified electrostatic interactions between the chromophore and the binding pocket, whereas both electrostatic interactions and torsional strain are involved in the energy storage mechanism of bathorhodopsin.

Light penetration and photoisomerization in rhodopsin studied by numerical simulations and double-quantum solid-state NMR spectroscopy

The penetration of light into optically thick samples containing the G-protein-coupled receptor rhodopsin is studied by numerical finite-element simulations and double-quantum solid-state NMR experiments. Illumination with white light leads to the generation of the active bathorhodopsin photostate in the outer layer of the sample but generates a large amount of the side product, isorhodopsin, in the sample interior. The overall yield of bathorhodopsin is improved by using monochromatic 420 nm illumination and by mixing the sample with transparent glass beads. The implications of these findings on the interpretation of previously published rhodopsin NMR data are discussed.

Windowed R-PDLF recoupling: A flexible and reliable tool to characterize molecular dynamics

This work focuses on the improvement of the R-PDLF heteronuclear recoupling scheme, a method that allows quantification of molecular dynamics up to the microsecond timescale in heterogeneous materials. We show how the stability of the sequence towards rf-imperfections, one of the main sources of error of this technique, can be improved by the insertion of windows without irradiation into the basic elements of the symmetry-based recoupling sequence. The impact of this modification on the overall performance of the sequence in terms of scaling factor and homonuclear decoupling efficiency is evaluated. This study indicates the experimental conditions for which precise and reliable measurement of dipolar couplings can be obtained using the popular R18(1)(7) recoupling sequence, as well as alternative symmetry-based R sequences suited for fast MAS conditions. An analytical expression for the recoupled dipolar modulation has been derived that applies to a whole class of sequences with similar recoupling properties as R18(1)(7). This analytical expression provides an efficient and precise way to extract dipolar couplings from the experimental dipolar modulation curves. We hereby provide helpful tools and information for tailoring R-PDLF recoupling schemes to specific sample properties and hardware capabilities. This approach is particularly well suited for the study of materials with strong and heterogeneous molecular dynamics where a precise measurement of dipolar couplings is crucial.

Structure and dynamics of guest molecules confined in a mesoporous silica matrix: Complementary NMR and PDF characterisation

We present an experimental approach to study the structure and dynamics of molecular functional complexes in porous host matrices. Combining the results of Solid-State NMR and pair distribution function analysis based on total X-ray scattering data the structural arrangement and dynamical behaviour of Na2[Fe(CN)5NO]·2H2O (SNP) embedded in amorphous SiO2 matrix is investigated. We show that the SNP complexes are embedded as isolated complexes within the SiO2 pores and propose a structural model for the cation and anion arrangement. Additionally the NMR results demonstrate the rotational dynamics of the SNP complexes.

Membrane fluidity does not explain how solvents act on the middle-ear reflex

Some volatile aromatic solvents have similar or opposite effects to anesthetics in the central nervous system. Like for anesthetics, the mechanisms of action involved are currently the subject of debate. This paper presents an in vivo study to determine whether direct binding or effects on membrane fluidity best explain how solvents counterbalance anesthesias depression of the middle-ear reflex (MER). Rats were anesthetized with a mixture of ketamine and xylazine while also exposed to solvent vapors (toluene, ethylbenzene, or one of the three xylene isomers) and the amplitude of their MER was monitored. The depth of anesthesia was standardized based on the magnitude of the contraction of the muscles involved in the MER, determined by measuring cubic distortion product oto-acoustic emissions (DPOAEs) while triggering the bilateral reflex with contralateral acoustic stimulation. The effects of the aromatic solvents were quantified based on variations in the amplitude of the DPOAEs. The amplitude of the alteration to the MER measured in anesthetized rats did not correlate with solvent lipophilocity (as indicated by logKow values). Results obtained with the three xylene isomers indicated that the positions of two methyl groups around the benzene ring played a determinant role in solvent/neuronal cell interaction. Additionally, Solid-state Nuclear Magnetic Resonance (NMR) spectra for brain microsomes confirmed that brain lipid fluidity was unaffected by solvent exposure, even after three days (6h/day) at an extremely high concentration (3000ppm). Therefore, aromatic solvents appear to act directly on the neuroreceptors involved in the acoustic reflex circuit, rather than on membrane fluidity. The affinity of this interaction is determined by stereospecific parameters rather than lipophilocity.

Multiscale structure-properties analysis of photoactive nanocomposite materials

In the last decades, the confinement of various types of functional material in mesoporous silica matrices has been used to design hybrid organic-inorganic nanocomposites with unique and fascinating properties. Such nanocomposites have attracted considerable interest owing to their potential applications in various domains [1-2], while reports with precise structural information of such molecular nanomaterials are still rather scarce and quite disparate. However, in order to be able to derive a structure-functionality relationship of such hybrid complexes, a detailed description of the structural organisation of the guest species and of their immediate surrounding is absolutely mandatory. We show in this contribution that detailed structural information can be obtained by using an appropriate multiscale approach combining various experimental techniques such as X-ray total scattering coupled to atomic pair distribution function (PDF) and solid-state NMR spectroscopy. This multiscale approach does provide more extensive and accurate structural information [3]. The PDF approach has allowed the identification of the nature of the incorporated species and their arrangement as well as the distinction of the various existing phases: isolated molecules and nanoparticles. The multi-nuclei Solid State NMR investigation has provided information on both the amorphous host and the molecular guest and adds a dynamic dimension to the classical static structural characterisation. We also discuss the influence of the structural changes on the physical properties of the investigated materials