A Delocalized Proton-Binding Site within a Membrane Protein

AbstractThe role of protein-bound water molecules in protein function and catalysis is an emerging topic. Here, we studied the solvation of an excess proton by protein-bound water molecules and the contribution of the surrounding amino acid residues at the proton release site of the membrane protein bacteriorhodopsin. It hosts an excess proton within a protein-bound water cluster, which is hydrogen bonded to several surrounding amino acids. Indicative of delocalization is a broad continuum absorbance experimentally observed by time-resolved Fourier transform infrared spectroscopy. In combination with site-directed mutagenesis, the involvement of several amino acids (especially Glu-194 and Glu-204) in the delocalization was elaborated. Details regarding the contributions of the glutamates and water molecules to the delocalization mode in biomolecular simulations are controversial. We carried out quantum mechanics/molecular mechanics (QM/MM) self-consistent charge density functional tight-binding simulations for all amino acids that have been experimentally shown to be involved in solvation of the excess proton, and systematically investigated the influence of the quantum box size. We compared calculated theoretical infrared spectra with experimental ones as a measure for the correct description of excess proton delocalization. A continuum absorbance can only be observed for small quantum boxes containing few amino acids and/or water molecules. Larger quantum boxes, including all experimentally shown involved amino acids, resulted in narrow absorbance bands, indicating protonation of a single binding site in contradiction to experimental results. We conclude that small quantum boxes seem to reproduce representative extreme cases of proton delocalization modes: proton delocalization only on water molecules or only between Glu-194 and Glu-204. Extending the experimental spectral region to lower wave numbers, a water-delocalized proton reproduces the observed continuum absorbance better than a glutamate-shared delocalized proton. However, a full agreement between QM simulations and experimental results on the delocalized excess proton will require a larger quantum box as well as more sophisticated QM/MM methods

Intracellular Lipid Accumulation and Mitochondrial Dysfunction Accompanies Endoplasmic Reticulum Stress Caused by Loss of the Co-chaperone DNAJC3.

Recessive mutations in DNAJC3, an endoplasmic reticulum (ER)-resident BiP co-chaperone, have been identified in patients with multisystemic neurodegeneration and diabetes mellitus. To further unravel these pathomechanisms, we employed a non-biased proteomic approach and identified dysregulation of several key cellular pathways, suggesting a pathophysiological interplay of perturbed lipid metabolism, mitochondrial bioenergetics, ER-Golgi function, and amyloid-beta processing. Further functional investigations in fibroblasts of patients with DNAJC3 mutations detected cellular accumulation of lipids and an increased sensitivity to cholesterol stress, which led to activation of the unfolded protein response (UPR), alterations of the ER-Golgi machinery, and a defect of amyloid precursor protein. In line with the results of previous studies, we describe here alterations in mitochondrial morphology and function, as a major contributor to the DNAJC3 pathophysiology. Hence, we propose that the loss of DNAJC3 affects lipid/cholesterol homeostasis, leading to UPR activation, β-amyloid accumulation, and impairment of mitochondrial oxidative phosphorylation

Molecular pathophysiology of human MICU1 deficiency.

Funder: Ministerium für Innovation, Wissenschaft und Forschung des Landes Nordrhein‐Westfalen; Id: http://dx.doi.org/10.13039/501100009591Funder: Bundesministerium für Bildung und Forschung; Id: http://dx.doi.org/10.13039/501100002347AIMS: MICU1 encodes the gatekeeper of the mitochondrial Ca2+ uniporter, MICU1 and biallelic loss-of-function mutations cause a complex, neuromuscular disorder in children. Although the role of the protein is well understood, the precise molecular pathophysiology leading to this neuropaediatric phenotype has not been fully elucidated. Here we aimed to obtain novel insights into MICU1 pathophysiology. METHODS: Molecular genetic studies along with proteomic profiling, electron-, light- and Coherent anti-Stokes Raman scattering microscopy and immuno-based studies of protein abundances and Ca2+ transport studies were employed to examine the pathophysiology of MICU1 deficiency in humans. RESULTS: We describe two patients carrying MICU1 mutations, two nonsense (c.52C>T; p.(Arg18*) and c.553C>T; p.(Arg185*)) and an intragenic exon 2-deletion presenting with ataxia, developmental delay and early onset myopathy, clinodactyly, attention deficits, insomnia and impaired cognitive pain perception. Muscle biopsies revealed signs of dystrophy and neurogenic atrophy, severe mitochondrial perturbations, altered Golgi structure, vacuoles and altered lipid homeostasis. Comparative mitochondrial Ca2+ transport and proteomic studies on lymphoblastoid cells revealed that the [Ca2+ ] threshold and the cooperative activation of mitochondrial Ca2+ uptake were lost in MICU1-deficient cells and that 39 proteins were altered in abundance. Several of those proteins are linked to mitochondrial dysfunction and/or perturbed Ca2+ homeostasis, also impacting on regular cytoskeleton (affecting Spectrin) and Golgi architecture, as well as cellular survival mechanisms. CONCLUSIONS: Our findings (i) link dysregulation of mitochondrial Ca2+ uptake with muscle pathology (including perturbed lipid homeostasis and ER-Golgi morphology), (ii) support the concept of a functional interplay of ER-Golgi and mitochondria in lipid homeostasis and (iii) reveal the vulnerability of the cellular proteome as part of the MICU1-related pathophysiology

The UA_handle: a versatile submotif in stable RNA architectures†

Stable RNAs are modular and hierarchical 3D architectures taking advantage of recurrent structural motifs to form extensive non-covalent tertiary interactions. Sequence and atomic structure analysis has revealed a novel submotif involving a minimal set of five nucleotides, termed the UA_handle motif (5′XU/ANnX3′). It consists of a U:A Watson–Crick: Hoogsteen trans base pair stacked over a classic Watson–Crick base pair, and a bulge of one or more nucleotides that can act as a handle for making different types of long-range interactions. This motif is one of the most versatile building blocks identified in stable RNAs. It enters into the composition of numerous recurrent motifs of greater structural complexity such as the T-loop, the 11-nt receptor, the UAA/GAN and the G-ribo motifs. Several structural principles pertaining to RNA motifs are derived from our analysis. A limited set of basic submotifs can account for the formation of most structural motifs uncovered in ribosomal and stable RNAs. Structural motifs can act as structural scaffoldings and be functionally and topologically equivalent despite sequence and structural differences. The sequence network resulting from the structural relationships shared by these RNA motifs can be used as a proto-language for assisting prediction and rational design of RNA tertiary structures

Optimization of process technology and increase in reliability and lifetime of (InAlGa)N-based semiconductor laser diodes

In the present work, reliability relevant aspects of the processing of (InAlGa)N-based laser diodes with an emission wavelength of 400 nm were investigated. The aim was to increase the lifetime of the laser diodes in continuous wave operation with a high chip yield per wafer. For this purpose, individual steps of the chip processing which limit yield and reliability were examined. Optimization of the thinning technology for GaN substrates and the cleavage process for generating the laser facets were crucial for increasing the yield. To increase the lifetime, the p-contact technology was optimized and an efficient activation process of the p-conductivity was established. Then, using the advanced chip process technology, broad-area and ridge-waveguide lasers were fabricated and systematic analyzes of the degradation mechanisms were carried out. This made it possible to identify inhomogeneous p-conductivity and degradation effects of the laser facets as the main causes of the observed aging effects and to describe the processes involved. The yield of working laser chips can be limited both by material loss during the manufacturing process and by process-related total failures at the beginning of operation. During chip processing, the thinning of the GaN-substrates is the most critical step, as a breakage of the wafer leads directly to the loss of wafer material. A two-step thinning process was developed to stabilize this process step. First, the substrate is lapped with a 27 μm silicon carbide grain and a pressure of 25 g/cm2. Afterwards, another short lapping step with a 9 μm boron carbide grain reduces the extent and depth of surface near crystal damage. This process enabled the yield of break-free wafers to be increased from 35 % to 90 % while reducing the target substrate thickness from 200 μm to 150 μm. The separation of the wafer into laser bars is directly related to the thinning process. A laser scribing process was established as a substitute for diamond scribing. In addition to the laser scribed groove depth of approx. 90 μm, a homogeneous scribing depth along the groove was decisive for the later quality of the cleavage surface. This was achieved through a constant pulse overlap during the entire scribing process. Separating trenches, in which the epitaxial structure is etched away, were introduced along the specified cleaving streets to further increase the cleaving accuracy. By combining these technological developments, the yield of correctly cleaved laser bars could be increased to over 90 %. The simultaneous increase in the facet quality resulted in reduced scatter of the laser threshold current density on a wafer from 2 kA/cm2 to 0.5 kA/cm2. The electrical breakdown of the laser diodes during the first electro-optical characterization in pulse or continuous wave operation could be traced back to overhanging metal on the laser facet. The effect created an electrical shunt from the p-contact to the n-GaN. By removing the metallization from the chip edge, this problem was eliminated. Investigations of the p-contacts showed that holes in the Pd contact metal as well as voids at the Pd/p-GaN interface were created after the contact was annealed. An additional Pt covering layer on the Pd contact layer together with an optimized cleaning procedure and annealing process prevented these inhomogeneities and the specific contact resistance was reduced by an order of magnitude to around 3 x 10-3 Ωcm2. During the aging measurements, essentially two lifetime-limiting mechanisms could be identified. The first effect is the coupled voltage output power degradation. In this case, the operating voltage increases in a stepwise manner in continuous wave operation with a simultaneous drop in output power. Systematic variations of individual chip processing steps and operating conditions, together with electroluminescence examinations of the active region, led to a model of this degradation mechanism. An operation-induced change in the hole current to the active region is the cause of this effect. As a result, the current-carrying area of the active region decreases. Optimized activation of the p-type conductivity in a nitrogen-oxygen atmosphere (ratio of 7:3) could increase the ratio of Mg to H concentration from 3 to 20 and thus shift this effect to higher current densities or longer operation times. A minimal ridge etching depth down to the electron blocking layer together with the use of a low-hydrogen containing SiO2 insulator further stabilized the operating voltage. In this way, ridge waveguide lasers with threshold current densities and voltages of 2.5 kA/cm2 and 5.5 V with lifetimes of several 100 hours at output powers up to 20 mW at room temperature and a stable operating voltage were achieved. Facet degradation was identified as a second aging mechanism. In a laboratory atmosphere, continuous wave operation leads to the formation of a SiOx layer on the laser facets. This effect changes the facet reflectivity. The consequence is an instability of the laser power. Since the laboratory atmosphere could be identified as the source for the deposits, hermetic enclosures for the lasers in defined atmospheres should solve this problem. The investigations and developments presented here allowed to increase the lifetime of ridge waveguide lasers from a few hours to several 100 hours and simultaneously to increase the yield of laser diodes capable of continuous wave operation. In addition, optimization potentials in epitaxy and facet technology were shown and recommendations for future developments were given.In der vorliegenden Arbeit wurden zuverlässigkeitsrelevante Aspekte der Chipprozessierung von (InAlGa)N-basierten Rippenwellenleiterlaserdioden mit einer Emissionswellenlänge von 400 nm untersucht. Als Ziel sollte bei einer hohen Chipausbeute pro Wafer die Lebensdauer der Laserdioden im Dauerstrichbetrieb gesteigert werden. Hierfür wurden zunächst einzelne Prozessschritte hinsichtlich ausbeute- und zuverlässigkeitslimitierender Aspekte untersucht. Entscheidend für eine Erhöhung der Ausbeute waren die Optimierungen der Abdünntechnologie für GaN-Substrate sowie des Spaltprozesses zur Erzeugung der Laserfacetten. Zur Erhöhung der Lebensdauer wurde die p-Kontakttechnologie optimiert und ein effizienterer Aktivierungsprozess der p-Leitfähigkeit etabliert. Anschließend wurden mithilfe der weiterentwickelten Prozesstechnologie Breitstreifen- und Rippenwellenleiterlaser hergestellt und systematische Analysen der Degradationsmechanismen durchgeführt. Dadurch konnten eine inhomogene p-Leitfähigkeit und Degradationseffekte der Laserfacetten als Hauptursachen für die beobachteten Alterungseffekte identifiziert und die dabei ablaufenden Prozesse beschrieben werden. Die Ausbeute an betriebsfähigen Laserchips kann sowohl durch Materialverlust während des Herstellungsprozesses als auch durch prozessbedingte Totalausfälle zu Beginn des Betriebs begrenzt werden. Während der Prozessierung ist das Abdünnen der GaN-Substrate der kritischste Schritt, da ein Bruch des Wafers direkt zum Verlust von Wafermaterial führt. Zur Stabilisierung dieses Prozessschritts wurde ein zweistufiger Abdünnprozess entwickelt. Hierbei wird zunächst mit einem 27 μm großen Siliziumcarbidkorn und einem Anpressdruck von 25 g/cm2 das Substrat geläppt. Abschließend erfolgt ein weiterer kurzer Läppschritt mit einem 9 μm großen Borcarbidkorn zur Erhöhung der Oberflächengüte und Reduzierung der eingebrachten Störschichtdicke. Durch diesen Prozess konnte, bei gleichzeitiger Reduktion der Substratzieldicke von 200 μm auf 150 μm, die Ausbeute an bruchfreien Wafern von 35 % auf 90 % gesteigert werden. In direktem Zusammenhang mit dem Abdünnprozess steht das Vereinzeln des Wafers in Laserriegel. Hierfür wurde anstelle des Diamantritzprozesses ein Laserritzprozess etabliert. Entscheidend für die spätere Spaltqualität war neben der absoluten Ritztiefe von ca. 90 μm deren Homogenität entlang des Ritzgrabens. Diese wurde durch einen konstanten Pulsüberlapp während des gesamten Ritzprozesses erzielt. Zur zusätzlichen Erhöhung der Spalttreue wurden Trenngräben, in denen die Epitaxiestruktur weggeätzt wird, entlang der vorgegebenen Ritzstraßen eingeführt. Durch die Kombination dieser Technologieentwicklungen konnte die Ausbeute an korrekt gespaltenen Laserriegeln auf über 90 % erhöht werden. Die gleichzeitige Erhöhung der Facettenqualität spiegelte sich in der reduzierten Streuung der Laserschwellenstromdichte auf einem Wafer von 2 kA/cm2 auf 0,5 kA/cm2 wider. Das elektrische Durchbrechen der Laserdioden bei der ersten elektro-optischen Charakterisierung im Puls- oder Dauerstrichbetrieb ließ sich auf Metallüberhänge an der Laserfacette zurückführen. Diese erzeugten einen elektrischen Nebenschluss vom p-Kontakt zum n-GaN. Durch das Zurückziehen der Metallisierung von der Chipkante konnte dieses Problem vollständig beseitigt werden. Untersuchungen der p-Kontakte ergaben, dass Löcher im Pd-Kontaktmetall sowie Hohl-räume an der Pd/p-GaN-Grenzfläche nach der Formierung des Kontakts entstanden. Durch eine zusätzliche Pt-Schicht oberhalb der Pd-Kontaktschicht zusammen mit einer optimierten Reinigungsprozedur und Formierung konnten diese Inhomogenitäten verhindert und der spezifische Kontaktwiderstand um eine Größenordnung auf etwa 3 x 10-3 Ωcm2 redu-ziert werden. Im Rahmen der Alterungsuntersuchungen konnten im Wesentlichen zwei lebensdauerbegrenzende Mechanismen identifiziert werden. Der erste Effekt ist die gekoppelte Spannung-Lichtleistungsdegradation. Hierbei steigt im Dauerstrichbetrieb bei gleichzeitigem Abfall der Lichtleistung die Betriebsspannung stufenförmig an. Systematische Variationen von einzelnen Prozessschritten und Betriebsbedingungen führten zusammen mit Elektro-lumineszenzuntersuchungen der aktiven Zone zu einem Modell dieses Degradationsmechanismus. So ist eine betriebsinduzierte Veränderung des Löcherstroms zur aktiven Zone, wodurch die stromdurchflossene Fläche und somit der gepumpte Bereich innerhalb der aktiven Zone verringert werden, ursächlich für diesen Effekt. Eine optimierte Aktivierung der p-Leitfähigkeit unter Stickstoff-Sauerstoffatmosphäre (Verhältnis 7:3) konnte das Verhältnis von Mg- zu H-Konzentration von 3 auf 20 erhöhen und somit diesen Effekt zu höheren Stromdichten bzw. größeren Alterungsdauern verschieben. Eine minimale Rip-penätztiefe bis in die Elektronenblockierschicht zusammen mit der Verwendung eines wasserstoffarmen SiO2-Isolators führten zu einer weiteren Stabilisierung der Betriebsspannung. So konnten Rippenwellenleiterlaser mit Schwellenstromdichten und -spannungen von 2,5 kA/cm2 und 5,5 V hergestellt werden, welche bei Raumtemperatur und Ausgangsleistungen bis ca. 20 mW Lebensdauern von mehreren 100 h bei stabiler Betriebsspannung aufwiesen. Als zweiter Alterungsmechanismus wurde die Facettendegradation identifiziert. So führt unter Laboratmosphäre der Dauerstrichbetrieb zur Bildung einer SiOx-Schicht auf den Laserfacetten. Dies sorgt für eine Modulation der Facettenreflektivität. Die Folge sind Instabilitäten der Laserleistung. Da als Quelle für die Ablagerungen die Laboratmosphäre identifiziert werden konnte, sollten hermetische Einhausungen der Laser in definierten Atmosphären dieses Problem lösen. Die hier vorgestellten Untersuchungen und Weiterentwicklungen führten zu einer Erhöhung der Lebensdauer von Rippenwellenleiterlasern von wenigen Stunden auf mehrere 100 h bei einer gleichzeitig deutlich gestiegenen Ausbeute an dauerstrichfähigen Laserdioden. Zusätzlich konnten Optimierungspotenziale in der Epitaxie und Facettentechnologie aufgezeigt und Empfehlungen für zukünftige Entwicklungen gegeben werden

Funktionselemente von Proteinen am Beispiel von Bakteriorhodopsin

Die Beschreibung von Proteinen als eine Zusammensetzung einzelner Funktionselemente wird anhand des Membranproteins Bakteriorhodopsin als Modellsystem vorgestellt. Dabei werden sowohl aus der Literatur bekannte Elemente behandelt als auch zwei neue Funktionselemente vorgestellt; eine Protonendiode, die ähnlich einer Diode aus der Elektronik eine Ladungsbewegungsrichtung vorgibt, und ein transienter Protonenleiter, der durch die Umpositionierung proteininterner Wassermoleküle zu einer linearen Kette aufgebaut wird. Das Funktionselement des Retinals in seiner Bindetasche beispielsweise findet sich in der gesamten Gruppe der Retinalproteine, für die es als Namensgeber dient. Zum Teil haben sich die Retinalproteine konvergent entwickelt und weisen trotz deutlich unterschiedlicher Herkunft analoge Funktionselemente auf. Deshalb wird davon ausgegangen, dass man weitere Elemente aus dem Modellsystem ebenfalls in anderen Proteinen wiederfindet und so einfacher auf deren Funktion schließen kann

SERS Background Imaging – a Versatile Tool Towards More Reliable SERS Analytics

Surface-enhanced Raman scattering (SERS) is a highly selective and sensitive straightforward analytical method, which is however not yet established in routine analysis due to a lack of reliability and reproducibility. Here we utilise the broad SERS continuum background (SERS-BG) accompanying every SERS measurement as a versatile tool towards more reliable SERS analytics. We apply a heterogeneous gold SERS substrate immersed with an adenosine triphosphate solution to show that the integrated SERS-BG distinctly correlates with the intensity of the analyte signals in the SERS spectrum. Based on this relationship we introduce an easy-to-handle, automatable and more reliable SERS measurement procedure starting with fast and high-contrast imaging of the SERS substrate followed by hot spot localisation and recording of highly enhanced SERS spectra at the centre of the diffraction-limited spot. We further demonstrate the applicability of SERS-BG imaging by combining it with other optical modalities and electron microscopy to assess structure-property relationships. Additionally, we perform Monte-Carlo simulations to evaluate the sampling error in SERS experiments highlighting the advantages of our method over conventional SERS experiments.</div

Chemical fingerprinting of single glandular trichomes of Cannabis sativa by Coherent anti-Stokes Raman scattering (CARS) microscopy

Abstract Background Cannabis possesses a rich spectrum of phytochemicals i.e. cannabinoids, terpenes and phenolic compounds of industrial and medicinal interests. Most of these high-value plant products are synthesised in the disk cells and stored in the secretory cavity in glandular trichomes. Conventional trichome analysis was so far based on optical microscopy, electron microscopy or extraction based methods that are either limited to spatial or chemical information. Here we combine both information to obtain the spatial distribution of distinct secondary metabolites on a single-trichome level by applying Coherent anti-Stokes Raman scattering (CARS), a microspectroscopic technique, to trichomes derived from sepals of a drug- and a fibre-type. Results Hyperspectral CARS imaging in combination with a nonlinear unmixing method allows to identify and localise Δ9-tetrahydrocannabinolic acid (THCA) in the secretory cavity of drug-type trichomes and cannabidiolic acid (CBDA)/myrcene in the secretory cavity of fibre-type trichomes, thus enabling an easy discrimination between high-THCA and high-CBDA producers. A unique spectral fingerprint is found in the disk cells of drug-type trichomes, which is most similar to cannabigerolic acid (CBGA) and is not found in fibre-type trichomes. Furthermore, we differentiate between different cell types by a combination of CARS with simultaneously acquired two-photon fluorescence (TPF) of chlorophyll a from chloroplasts and organic fluorescence mainly arising from cell walls enabling 3D visualisation of the essential oil distribution and cellular structures. Conclusion Here we demonstrate a label-free and non-destructive method to analyse the distribution of secondary metabolites and distinguish between different cell and chemo-types with high spatial resolution on a single trichome. The record of chemical fingerprints of single trichomes offers the possibility to optimise growth conditions as well as guarantee a direct process control for industrially cultivated medicinal Cannabis plants. Moreover, this method is not limited to Cannabis related issues but can be widely implemented for optimising and monitoring all kinds of natural or biotechnological production processes with simultaneous spatial and chemical information