2022-11-12, Vogel, Karin, Bläske, Tobias, Nagel, Marie-Kristin, Globisch, Christoph, Maguire, Shane, Mattes, Lorenz, Kovermann, Michael, Hauser, Karin, Peter, Christine, Isono, Erika

The abundance of plasma membrane-resident receptors and transporters has to be tightly regulated by ubiquitin-mediated endosomal degradation for the proper coordination of environmental stimuli and intracellular signaling. Arabidopsis OVARIAN TUMOR PROTEASE (OTU) 11 and OTU12 are plasma membrane-localized deubiquitylating enzymes (DUBs) that bind to phospholipids through a polybasic motif in the OTU domain. Here we show that the DUB activity of OTU11 and OTU12 towards K63-linked ubiquitin is stimulated by binding to lipid membranes containing anionic lipids. In addition, we show that the DUB activity of OTU11 against K6- and K11-linkages is also stimulated by anionic lipids, and that OTU11 and OTU12 can modulate the endosomal degradation of a model cargo and the auxin efflux transporter PIN2-GFP in vivo. Our results suggest that the catalytic activity of OTU11 and OTU12 is tightly connected to their ability to bind membranes and that OTU11 and OTU12 are involved in the fine-tuning of plasma membrane proteins in Arabidopsis.

2021-08, Scheibe, Christian, Karreman, Christiaan, Schildknecht, Stefan, Leist, Marcel, Hauser, Karin

The 140 amino acid protein α-synuclein (αS) is an intrinsically disordered protein (IDP) with various roles and locations in healthy neurons that plays a key role in Parkinson’s disease (PD). Contact with biomembranes can lead to α-helical conformations, but can also act as s seeding event for aggregation and a predominant β-sheet conformation. In PD patients, αS is found to aggregate in various fibrillary structures, and the shift in aggregation and localization is associated with disease progression. Besides full-length αS, several related polypeptides are present in neurons. The role of many αS-related proteins in the aggregation of αS itself is not fully understood Two of these potential aggregation modifiers are the αS splicing variant αS Δexon3 (Δ3) and the paralog β-synuclein (βS). Here, polarized ATR-FTIR spectroscopy was used to study the membrane interaction of these proteins individually and in various combinations. The method allowed a continuous monitoring of both the lipid structure of biomimetic membranes and the aggregation state of αS and related proteins. The use of polarized light also revealed the orientation of secondary structure elements. While αS led to a destruction of the lipid membrane upon membrane-catalyzed aggregation, βS and Δ3 aggregated significantly less, and they did not harm the membrane. Moreover, the latter proteins reduced the membrane damage triggered by αS. There were no major differences in the membrane interaction for the different synuclein variants. In combination, these observations suggest that the formation of particular protein aggregates is the major driving force for αS-driven membrane damage. The misbalance of αS, βS, and Δ3 might therefore play a crucial role in neurodegenerative disease.

2020-05-15, Stritt, Paul, Jawurek, Michael, Hauser, Karin

The function of membrane proteins is highly impacted by their membrane environment. One suitable approach to get insights into the interaction-induced dynamics of membrane proteins and lipid membranes is time-resolved infrared (IR) spectroscopy. Conclusions about environmental influences to the system can be drawn by correlating the observed kinetics to the well-characterized photocycles of light-driven transmembrane proton pumps like bacteriorhodopsin (BR). For the investigation of photoreceptor-membrane interactions, also minor changes in the absorption spectra must be resolved. Therefore, we applied IR laser spectroscopy using tunable quantum cascade lasers (QCLs) as IR light source. Several QCLs were implemented in a home-built spectrometer and provide a tunability in a broad spectral region covering protein, chromophore and lipid vibrational modes. Kinetics of the BR photocycle were monitored at single wavenumbers. This study demonstrates the high potential of QCL-based spectroscopy for the application to membrane protein studies.

2020-04-06, Avaro, Jonathan Thomas, Wolf, Stefan L. P., Hauser, Karin, Gebauer, Denis

Liquid‐liquid phase separation (LLPS) is an intermediate step during the precipitation of calcium carbonate, and is assumed to play a key role in biomineralization processes. Here, we developed a model where homogeneous phase ion association thermodynamics determines the liquid‐liquid miscibility gap of the aqueous calcium carbonate system, verified experimentally using potentiometric titrations, and kinetic studies based on stopped‐flow ATR‐FTIR spectroscopy. The proposed mechanism explains varying solubilities of solid amorphous calcium carbonates, reconciling previously inconsistent literature values. Accounting for liquid‐liquid amorphous polymorphism, the model also provides clues to the mechanism of polymorph selection. It is general and should be tested for other systems than calcium carbonate, providing a new perspective on the physical chemistry of LLPS mechanisms—based on stable pre‐nucleation clusters rather than un‐/metastable fluctuations—in biomineralization, and beyond.

2022-07-14, King, Michael, Avaro, Jonathan Thomas, Peter, Christine, Hauser, Karin, Gebauer, Denis

In experimental studies, heavy water (D₂O) is employed, e.g., so as to shift the spectroscopic solvent background, but any potential effects of this solvent exchange on reaction pathways are often neglected. While the important role of light water (H₂O) during the early stages of calcium carbonate formation has been realized, studies into the actual effects of aqueous solvent exchanges are scarce. Here, we present a combined computational and experimental approach to start to fill this gap. We extended a suitable force field for molecular dynamics (MD) simulations. Experimentally, we utilised advanced titration assays and time-resolved attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy. We find distinct effects in various mixtures of the two aqueous solvents, and in pure H₂O or D₂O. Disagreements between the computational results and experimental data regarding the stabilities of ion associates might be due to the unexplored role of HDO, or an unprobed complex phase behaviour of the solvent mixtures in the simulations. Altogether, however, our data suggest that calcium carbonate formation might proceed “more classically” in D₂O. Also, there are indications for the formation of new structures in amorphous and crystalline calcium carbonates. There is huge potential towards further improving the understanding of mineralization mechanisms by studying solvent-mediated isotope effects, also beyond calcium carbonate. Last, it must be appreciated that H₂O and D₂O have significant, distinct effects on mineralization mechanisms, and that care has to be taken when experimental data from D₂O studies are used, e.g., for the development of H₂O-based computer models.

2021-01-06, Siu, Ho-Wah, Heck, Benjamin, Kovermann, Michael, Hauser, Karin

Expanded polyglutamine (polyQ) sequences cause numerous neurodegenerative diseases which are accompanied by the formation of polyQ fibrils. The unique role of glutamines in the aggregation onset is undoubtedly accepted and a lot structural data of the fibrils have been acquired, however side-chain specific structural dynamics inducing oligomerization are not well understood yet. To analyze spectroscopically the nucleation process, we designed various template-assisted glutamine-rich β-hairpin monomers mimicking the structural motif of a polyQ fibril. In a top-down strategy, we use a template which forms a well-defined stable hairpin in solution, insert polyQ-rich sequences into each strand and monitor the effects of individual glutamines by NMR, CD and IR spectroscopic approaches. The design was further advanced by alternating glutamines with other amino acids (T, W, E, K), thereby enhancing the solubility and increasing the number of cross-strand interacting glutamine side chains. Our spectroscopic studies reveal a decreasing hairpin stability with increased glutamine content and demonstrate the enormous impact of only a few glutamines – far below the disease threshold – to destabilize structure. Furthermore, we could access sub-ms conformational dynamics of monomeric polyQ-rich peptides by laser-excited temperature-jump IR spectroscopy. Both, the increased number of interacting glutamines and higher concentrations are key parameters to induce oligomerization. Concentration-dependent time-resolved IR measurements indicate an additional slower kinetic phase upon oligomer formation. The here presented peptide models enable spectroscopic molecular analyses to distinguish between monomer and oligomer dynamics in the early steps of polyQ fibril formation and in a side-chain specific manner.

2020-05-01, Krüger, Annika, Bürkle, Alexander, Hauser, Karin, Mangerich, Aswin

Poly-ADP-ribosylation (PARylation) is a fully reversible post-translational modification with key roles in cellular physiology. Due to the multi-domain structure of poly(ADP-ribose) polymerase-1 (PARP1) and the highly dynamic nature of the PARylation reaction, studies on the biochemical mechanism and structural dynamics remain challenging. Here, we report label-free, time-resolved monitoring of PARP1-dependent PARylation using ATR-FTIR spectroscopy. This includes PARP1 activation by binding to DNA strand break models, NAD+ substrate binding, PAR formation, and dissociation of automodified PARP1 from DNA. Analyses of PARP1 activation at different DNA models demonstrate a strong positive correlation of PARylation and PARP1 dissociation, with the strongest effects observed for DNA nicks and 3’ phosphorylated ends. Moreover, by examining dynamic structural changes of PARP1, we reveal changes in the secondary structure of PARP1 induced by NAD+ and PARP inhibitor binding. In summary, this approach enables holistic and dynamic insights into PARP1-dependent PARylation with molecular and temporal resolution.

2022-05, Siu, Ho-Wah, Stritt, Paul, Zhao, Heng, Hauser, Karin

Polyglutamine (polyQ) model peptides are ideally suited to analyze the involvement of glutamines in the disease-related aggregation onset. Here we use a template-assisted design of polyQ-rich hairpin peptides (Trpzip-Q_n) to monitor structural stability with fluorescence spectroscopy. The hairpin model imitates the monomeric motif of a polyQ fibril and is stabilized by hydrophobic interactions of two cross-strand pairs of tryptophans (Trps) which are used as fluorophores to report on structural changes. The Trps also frame the polyQ repeats located on each hairpin strand with a different number of glutamines (Q_n). Single-stranded sequences mimic the unfolded state and were used as references to differentiate the intrinsic fluorescence signal from the spectral effect caused by structural changes. Temperature-induced hairpin unfolding was monitored by the spectral shift of the Trp fluorescence signal and transition temperatures were determined. The magnitude of the spectral shift indicates the degree of structural disorder. We observed that a longer polyQ repeat is more disordered and weakens the cross-strand Trp-Trp interactions resulting in a decrease of the spectral shift. Aggregation to a fibrillar and more ordered structure shows an increase of the spectral shift. In addition, a band at 280 nm occurs in the spectrum which clearly correlates with the turbidity of the sample and is attributed to scattering of larger aggregated structures. Our study reveals that the number of glutamines, pH and temperature affect structural stability and aggregation of polyQ repeats.

2020-05-15, Hinze, Wolfgang G., Fallah, Mohammad A., Hauser, Karin

In many scientific fields there is a high interest to study molecular adsorption processes on surfaces. The adsorbed molecule can have significant impact on the properties of the material under study, for example protein adsorption to inorganic material can enhance its biocompatibility. Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy is a suitable method to monitor such adsorption processes close to a surface. In this study, ZnO films were synthesized on silicon ATR substrates via a mild hydrothermal reaction. The films were then characterized by scanning electron microscopy (SEM) and FTIR microscopy. Chemical imaging with FTIR microscopy allowed to analyze the composition of the heterogeneous film samples. ATR-FTIR spectroscopy was then applied to investigate the adsorption properties of the ZnO films. Protein solutions of bovine serum albumine (BSA) were circulated in a closed cycle over the ZnO film and IR spectra were recorded during the adsorption process. A stronger protein adsorption was observed for silicon substrates coated with ZnO than for plain silicon. Furthermore, subsequent flushing with pure water and desorption measurements indicated a stronger protein binding to ZnO than to plain silicon.

2020-04-06, Avaro, Jonathan Thomas, Wolf, Stefan L. P., Hauser, Karin, Gebauer, Denis

Flüssig‐flüssig‐Phasenseparation (liquid‐liquid phase separation, LLPS) ist eine Zwischenstufe während der Fällung von Calciumcarbonat und man vermutet, dass sie eine entscheidende Rolle in Biomineralisationsprozessen spielt. In diesem Artikel stellen wir ein Modell vor, in dem die Flüssig‐flüssig‐Mischungslücke im wässrigen Calciumcarbonatsystem durch die Thermodynamik der Ionenassoziation in der homogenen Phase bestimmt wird, was experimentell durch potentiometrische Titrationen und kinetische Studien mittels ATR‐FTIR‐Spektroskopie bestätigt wird. Der vorgeschlagene Mechanismus erklärt die variable Löslichkeit von amorphen Calciumcarbonaten und räumt Widersprüche in Literaturdaten aus. Da flüssig‐flüssig amorphe Polymorphie berücksichtigt wird, liefert das Modell Hinweise zum Mechanismus der Polymorphselektion. Es ist allgemein und sollte für andere Systeme als Calciumcarbonat überprüft werden. Ausgehend von stabilen Pränukleationsclustern anstelle von in‐ oder metastabilen Fluktuationen ergibt sich ein neuer Blickwinkel auf die physikalische Chemie der Flüssig‐flüssig‐Phasenseparationen in Biomineralisationsprozessen und darüber hinaus.