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The complex kinetics of disease-related amyloid aggregation of proteins such as α-Synuclein (α-Syn) in Parkinson's disease and Aβ42 in Alzheimer's disease include primary nucleation, amyloid fibril elongation and secondary nucleation. The latter can be a key accelerator of the aggregation process. It has been demonstrated that the chaperone domain BRICHOS can interfere with the secondary nucleation process of Aβ42. Here, we explore the mechanism of secondary nucleation inhibition of the BRICHOS domain of the lung surfactant protein (proSP-C) against α-Syn aggregation and amyloid formation. We determine the 3D NMR structure of an inactive trimer of proSP-C BRICHOS and its active monomer using a designed mutant. Furthermore, the interaction between the proSP-C BRICHOS chaperone and a substrate peptide has been studied. NMR-based interaction studies of proSP-C BRICHOS with α-Syn fibrils show that proSP-C BRICHOS binds to the C-terminal flexible fuzzy coat of the fibrils, which is the secondary nucleation site on the fibrils. Super-resolution fluorescence microscopy demonstrates that proSP-C BRICHOS runs along the fibrillar axis diffusion-dependently sweeping off monomeric α-Syn from the fibrils. The observed mechanism explains how a weakly binding chaperone can inhibit the α-Syn secondary nucleation pathway via avidity where a single proSP-C BRICHOS molecule is sufficient against up to ~7-40 α-Syn molecules embedded within the fibrils. Here, the authors probe the mechanism of secondary nucleation inhibition of the BRICHOS domain of the lung surfactant protein (proSP-C) against α-Syn aggregation and amyloid formation using NMR and super-resolution fluorescence microscopy. [ABSTRACT FROM AUTHOR]

Oligomeric species arising during the aggregation of α-synuclein are implicated as a major source of toxicity in Parkinson's disease, and thus a major potential drug target. However, both their mechanism of formation and role in aggregation are largely unresolved. Here we show that, at physiological pH and in the absence of lipid membranes, α-synuclein aggregates form by secondary nucleation, rather than simple primary nucleation, and that this process is enhanced by agitation. Moreover, using a combination of single molecule and bulk level techniques, we identify secondary nucleation on the surfaces of existing fibrils, rather than formation directly from monomers, as the dominant source of oligomers. Our results highlight secondary nucleation as not only the key source of oligomers, but also the main mechanism of aggregate formation, and show that these processes take place under conditions which recapitulate the neutral pH and ionic strength of the cytosol. The formation of protein aggregates is a hallmark of Parkinson's disease, with small oligomeric species implicated as a major source of toxicity. In this work, Xu et al. determine their mechanism of formation and role in aggregation. [ABSTRACT FROM AUTHOR]

Microfluidic diffusional sizing (MDS) is a powerful lab-on-a-chip method for determining the particle sizes, interactions, and association states of fluorescent analytes. MDS requires only microliter volumes and works even in heterogeneous, unpurified samples. Here we describe how MDS can be used to study (i) antibody binding and cellular copy numbers of oncoproteins, (ii) charge-dependent interactions of proteins with lipid membranes, and (iii) the size of native nanodiscs. We also show how single-molecule MDS (smMDS) achieves ultimate sensitivity and superior size resolution through digital counting. [ABSTRACT FROM AUTHOR]

Antimicrobial peptides (AMPs), which combat bacterial infections by disrupting the bacterial cell membrane or interacting with intracellular targets, are naturally produced by a number of different organisms, and are increasingly also explored as therapeutics. However, the mechanisms by which AMPs act on intracellular targets are not well understood. Using machine learning-based sequence analysis, we identified a significant number of AMPs that have a strong tendency to form liquid-like condensates in the presence of nucleic acids through phase separation. We demonstrate that this phase separation propensity is linked to the effectiveness of the AMPs in inhibiting transcription and translation in vitro, as well as their ability to compact nucleic acids and form clusters with bacterial nucleic acids in bacterial cells. These results suggest that the AMP-driven compaction of nucleic acids and modulation of their phase transitions constitute a previously unrecognised mechanism by which AMPs exert their antibacterial effects. The development of antimicrobials that target nucleic acid phase transitions may become an attractive route to finding effective and long-lasting antibiotics. In this work the authors describe antimicrobial peptides (AMPs)-driven phase transitions of intracellular nucleic acids, whereby AMPs induce compaction and phase separation of nucleic acids, resulting in their sequestration and eventual cell death. [ABSTRACT FROM AUTHOR]

Nonspecific interactions are a key challenge in the successful development of therapeutic antibodies. The tendency for nonspecific binding of antibodies is often difficult to reduce by rational design, and instead, it is necessary to rely on comprehensive screening campaigns. To address this issue, we performed a systematic analysis of the impact of surface patch properties on antibody nonspecificity using a designer antibody library as a model system and single-stranded DNA as a nonspecificity ligand. Using an in-solution microfluidic approach, we find that the antibodies tested bind to single-stranded DNA with affinities as high as KD = 1 µM. We show that DNA binding is driven primarily by a hydrophobic patch in the complementarity-determining regions. By quantifying the surface patches across the library, the nonspecific binding affinity is shown to correlate with a trade-off between the hydrophobic and total charged patch areas. Moreover, we show that a change in formulation conditions at low ionic strengths leads to DNA-induced antibody phase separation as a manifestation of nonspecific binding at low micromolar antibody concentrations. We highlight that phase separation is driven by a cooperative electrostatic network assembly mechanism of antibodies with DNA, which correlates with a balance between positive and negative charged patches. Importantly, our study demonstrates that both nonspecific binding and phase separation are controlled by the size of the surface patches. Taken together, these findings highlight the importance of surface patches and their role in conferring antibody nonspecificity and its macroscopic manifestation in phase separation. [ABSTRACT FROM AUTHOR]

The detection of proteins is of central importance to biomolecular analysis and diagnostics. Typical immunosensing assays rely on surface-capture of target molecules, but this constraint can limit specificity, sensitivity, and the ability to obtain information beyond simple concentration measurements. Here we present a surface-free, single-molecule microfluidic sensing platform for direct digital protein biomarker detection in solution, termed digital immunosensor assay (DigitISA). DigitISA is based on microchip electrophoretic separation combined with single-molecule detection and enables absolute number/concentration quantification of proteins in a single, solution-phase step. Applying DigitISA to a range of targets including amyloid aggregates, exosomes, and biomolecular condensates, we demonstrate that the assay provides information beyond stoichiometric interactions, and enables characterization of immunochemistry, binding affinity, and protein biomarker abundance. Taken together, our results suggest a experimental paradigm for the sensing of protein biomarkers, which enables analyses of targets that are challenging to address using conventional immunosensing approaches. There are limitations with current protein sensing methods. Here the authors report DigitISA, a digital immunosensor assay based on microchip electrophoretic separation and single-molecule detection that enables quantitation of protein biomarkers in a single, solution-phase step. [ABSTRACT FROM AUTHOR]

The assembly of biomolecules into condensates is a fundamental process underlying the organisation of the intracellular space and the regulation of many cellular functions. Mapping and characterising phase behaviour of biomolecules is essential to understand the mechanisms of condensate assembly, and to develop therapeutic strategies targeting biomolecular condensate systems. A central concept for characterising phase-separating systems is the phase diagram. Phase diagrams are typically built from numerous individual measurements sampling different parts of the parameter space. However, even when performed in microwell plate format, this process is slow, low throughput and requires significant sample consumption. To address this challenge, we present here a combinatorial droplet microfluidic platform, termed PhaseScan, for rapid and high-resolution acquisition of multidimensional biomolecular phase diagrams. Using this platform, we characterise the phase behaviour of a wide range of systems under a variety of conditions and demonstrate that this approach allows the quantitative characterisation of the effect of small molecules on biomolecular phase transitions. A central concept for characterising phase-separating systems is the phase diagram but generation of such diagrams for biomolecular systems is typically slow and low-throughput. Here the authors describe PhaseScan, a combinatorial droplet microfluidic platform for high-resolution acquisition of multidimensional biomolecular phase diagrams. [ABSTRACT FROM AUTHOR]

Macromolecular phase separation is thought to be one of the processes that drives the formation of membraneless biomolecular condensates in cells. The dynamics of phase separation are thought to follow the tenets of classical nucleation theory, and, therefore, subsaturated solutions should be devoid of clusters with more than a few molecules. We tested this prediction using in vitro biophysical studies to characterize subsaturated solutions of phase-separating RNA-binding proteins with intrinsically disordered prion like domains and RNA-binding domains. Surprisingly, and in direct contradiction to expectations from classical nucleation theory, we find that subsaturated solutions are characterized by the presence of heterogeneous distributions of clusters. The distributions of cluster sizes, which are dominated by small species, shift continuously toward larger sizes as protein concentrations increase and approach the saturation concentration. As a result, many of the clusters encompass tens to hundreds of molecules, while less than 1% of the solutions are mesoscale species that are several hundred nanometers in diameter. We find that cluster formation in subsaturated solutions and phase separation in supersaturated solutions are strongly coupled via sequence-encoded interactions. We also find that cluster formation and phase separation can be decoupled using solutes as well as specific sets of mutations. Our findings, which are concordant with predictions for associative polymers, implicate an interplay between networks of sequence-specific and solubility-determining interactions that, respectively, govern cluster formation in subsaturated solutions and the saturation concentrations above which phase separation occurs. [ABSTRACT FROM AUTHOR]

Phase-separated biomolecular condensates that contain multiple coexisting phases are widespread in vitro and in cells. Multiphase condensates emerge readily within multicomponentmixtures of biomolecules (e.g., proteins and nucleic acids)when the different components present sufficient physicochemical diversity (e.g., in intermolecular forces, structure, and chemical composition) to sustain separate coexisting phases. Because such diversity is highly coupled to the solution conditions (e.g., temperature, pH, salt, composition), it can manifest itself immediately from the nucleation and growth stages of condensate formation, develop spontaneously due to external stimuli or emerge progressively as the condensates age. Here, we investigate thermodynamic factors that can explain the progressive intrinsic transformation of single-component condensates into multiphase architectures during the nonequilibrium process of aging. We develop a multiscale model that integrates atomistic simulations of proteins, sequence-dependent coarse-grained simulations of condensates, and a minimal model of dynamically aging condensates with nonconservative intermolecular forces. Our nonequilibrium simulations of condensate aging predict that single-component condensates that are initially homogeneous and liquid like can transform into gel-core/liquid-shell or liquid-core/gelshell multiphase condensates as they age due to gradual and irreversible enhancement of interprotein interactions. The type of multiphase architecture is determined by the aging mechanism, the molecular organization of the gel and liquid phases, and the chemical makeup of the protein. Notably, we predict that interprotein disorder to order transitions within the prion-like domains of intracellular proteins can lead to the required nonconservative enhancement of intermolecular interactions. Our study, therefore, predicts a potential mechanism by which the nonequilibrium process of aging results in single-component multiphase condensates. [ABSTRACT FROM AUTHOR]

Periplasmic chaperones 17-kilodalton protein (Skp) and survival factor A (SurA) are essential players in outer membrane protein (OMP) biogenesis. They prevent unfolded OMPs from misfolding during their passage through the periplasmic space and aid in the disassembly of OMP aggregates under cellular stress conditions. However, functionally important links between interaction mechanisms, structural dynamics, and energetics that underpin both Skp and SurA associations with OMPs have remained largely unresolved. Here, using single-molecule fluorescence spectroscopy, we dissect the conformational dynamics and thermodynamics of Skp and SurA binding to unfolded OmpX and explore their disaggregase activities. We show that both chaperones expand unfolded OmpX distinctly and induce microsecond chain reconfigurations in the client OMP structure. We further reveal that Skp and SurA bind their substrate in a fine-tuned thermodynamic process via enthalpy–entropy compensation. Finally, we observed synergistic activity of both chaperones in the disaggregation of oligomeric OmpX aggregates. Our findings provide an intimate view into the multifaceted functionalities of Skp and SurA and the fine-tuned balance between conformational flexibility and underlying energetics in aiding chaperone action during OMP biogenesis. [ABSTRACT FROM AUTHOR]

The analysis of nanoscopic species, such as proteins and colloidal assemblies, at the single-molecule level has become vital in many areas of fundamental and applied research. Approaches to increase the detection time scales for single molecules in solution without immobilizing them onto a substrate surface and applying external fields are much sought-after. Here, we present an easy-to-implement and versatile nanofluidics-based approach that enables increased observational-time scale analysis of nanoscopic material building blocks such as single biomacromolecules and nanoscale colloids in solution. We use two-photon-based hybrid lithography in conjunction with soft lithography to fabricate nanofluidic devices with nanotrapping geometries down to 100 nm in height. We provide a rigorous description and characterization of the fabrication route that enables the writing of nanoscopic 3D structures directly in photoresist and allows for the integration of nanotrapping and nanochannel geometries within microchannel devices. Using confocal fluorescence burst detection, we validated the functionality of particle confinement in our nanotrap geometries through measurement of particle residence times. All species under study, including nanoscale colloids, α-synuclein oligomers, and double-stranded DNA, showed a 3- to 5-fold increase in average residence time in the detection volume of nanotraps, due to the additional local steric confinement, in comparison to free space diffusion in a nearby microchannel. Our approach thus opens up the possibility for single-molecule studies at prolonged observational-time scales to analyze and detect functionalized nanoparticles and protein assemblies in solution without the need for surface immobilization. [ABSTRACT FROM AUTHOR]

Molecular chaperones contribute to the maintenance of cellular protein homoeostasis through assisting de novo protein folding and preventing amyloid formation. Chaperones of the Hsp70 family can further disaggregate otherwise irreversible aggregate species such as α-synuclein fibrils, which accumulate in Parkinson’s disease. However, the mechanisms and kinetics of this key functionality are only partially understood. Here, we combine microfluidic measurements with chemical kinetics to study α-synuclein disaggregation. We show that Hsc70 together with its co-chaperones DnaJB1 and Apg2 can completely reverse α-synuclein aggregation back to its soluble monomeric state. This reaction proceeds through first-order kinetics where monomer units are removed directly from the fibril ends with little contribution from intermediate fibril fragmentation steps. These findings extend our mechanistic understanding of the role of chaperones in the suppression of amyloid proliferation and in aggregate clearance, and inform on possibilities and limitations of this strategy in the development of therapeutics against synucleinopathies. [ABSTRACT FROM AUTHOR]

Membrane proteins play key roles in cellular signaling and transport, represent the majority of drug targets, and are implicated in many diseases. Their relevance renders them important subjects for structural, biophysical, and functional investigations. However, obtaining membrane proteins in high purities is often challenging with conventional purification steps alone. To address this issue, we present here an approach to increase the purity of α‐helical transmembrane proteins. Our approach exploits the Thioredoxin (Trx) tag system, which is able to confer some of its favorable properties, such as high solubility and thermostability, to its fusion partners. Using Trx fusions of transmembrane helical hairpin constructs derived from the human cystic fibrosis transmembrane conductance regulator (CFTR) and a bacterial ATP synthase, we establish conditions for the successful implementation of the selective heat treatment procedure to increase sample purity. We further examine systematically its efficacy with respect to different incubation times and temperatures using quantitative gel electrophoresis. We find that minute‐timescale heat treatment of Trx‐tagged fusion constructs with temperatures ranging from 50 to 90°C increases the purity of the membrane protein samples from ~60 to 98% even after affinity purification. We show that this single‐step approach is even applicable in cases where regular selective heat purification from crude extracts, as reported for Trx fusions to soluble proteins, fails. Overall, our approach is easy to integrate into existing purification strategies and provides a facile route for increasing the purity of membrane protein constructs after purification by standard chromatography approaches. [ABSTRACT FROM AUTHOR]

Equilibrium binding constants (Kb) between chemical compounds and target proteins or between interacting proteins provide a quantitative understanding of biological interaction mechanisms. Reported uncertainties of measured experimental parameters are critical for decision-making in many scientific areas, e.g., in lead compound discovery processes and in comparing computational predictions with experimental results. Uncertainties in measured Kb values are commonly represented by a symmetric normal distribution, often quoted in terms of the experimental value plus–minus the standard deviation. However, in general, the distributions of measured Kb (and equivalent Kd) values and the corresponding free energy change ΔGb are all asymmetric to varying degree. Here, using a simulation approach, we illustrate the effect of asymmetric Kb distributions within the realm of isothermal titration calorimetry (ITC) experiments. Further we illustrate the known, but perhaps not widely appreciated, fact that when distributions of any of Kb, Kd and ΔGb are transformed into each other, their degree of asymmetry is changed. Consequently, we recommend that a more accurate way of expressing the uncertainties of Kb, Kd, and ΔGb values is to consistently report 95% confidence intervals, in line with other authors' suggestions. The ways to obtain such error ranges are discussed in detail and exemplified for a binding reaction obtained by ITC. [ABSTRACT FROM AUTHOR]

Intracellular phase separation of proteins into biomolecular condensates is increasingly recognized as a process with a key role in cellular compartmentalization and regulation. Different hypotheses about the parameters that determine the tendency of proteins to form condensates have been proposed, with some of them probed experimentally through the use of constructs generated by sequence alterations. To broaden the scope of these observations, we established an in silico strategy for understanding on a global level the associations between protein sequence and phase behavior and further constructed machinelearning models for predicting protein liquid-liquid phase separation (LLPS). Our analysis highlighted that LLPS-prone proteins are more disordered, less hydrophobic, and of lower Shannon entropy than sequences in the Protein Data Bank or the Swiss- Prot database and that they show a fine balance in their relative content of polar and hydrophobic residues. To further learn in a hypothesis-free manner the sequence features underpinning LLPS, we trained a neural network-based language model and found that a classifier constructed on such embeddings learned the underlying principles of phase behavior at a comparable accuracy to a classifier that used knowledge-based features. By combining knowledge-based features with unsupervised embeddings, we generated an integrated model that distinguished LLPS-prone sequences both from structured proteins and from unstructured proteins with a lower LLPS propensity and further identified such sequences from the human proteome at a high accuracy. These results provide a platform rooted in molecular principles for understanding protein phase behavior. [ABSTRACT FROM AUTHOR]

Liquid–liquid phase separation of proteins underpins the formation of membraneless compartments in living cells. Elucidating the molecular driving forces underlying protein phase transitions is therefore a key objective for understanding biological function and malfunction. Here we show that cellular proteins, which form condensates at low salt concentrations, including FUS, TDP-43, Brd4, Sox2, and Annexin A11, can reenter a phase-separated regime at high salt concentrations. By bringing together experiments and simulations, we demonstrate that this reentrant phase transition in the high-salt regime is driven by hydrophobic and non-ionic interactions, and is mechanistically distinct from the low-salt regime, where condensates are additionally stabilized by electrostatic forces. Our work thus sheds light on the cooperation of hydrophobic and non-ionic interactions as general driving forces in the condensation process, with important implications for aberrant function, druggability, and material properties of biomolecular condensates. Elucidating the molecular driving forces underlying liquid–liquid phase separation is a key objective for understanding biological function and malfunction. Here the authors show that a wide range of cellular proteins, including FUS, TDP-43, Brd4, Sox2, and Annexin A11, which form condensates at low salt concentrations, can reenter a phase-separated regime at high salt concentrations. [ABSTRACT FROM AUTHOR]

Exploration and characterisation of the human proteome is a key objective enabling a heightened understanding of biological function, malfunction and pharmaceutical design. Since proteins typically exhibit their behaviour by binding to other proteins, the challenge of probing protein-protein interactions has been the focus of new and improved experimental approaches. Here, we review recently developed microfluidic techniques for the study and quantification of protein–protein interactions. We focus on methodologies that utilise the inherent strength of microfluidics for the control of mass transport on the micron scale, to facilitate surface and membrane-free interrogation and quantification of interacting proteins. Thus, the microfluidic tools described here provide the capability to yield insights on protein–protein interactions under physiological conditions. We first discuss the defining principles of microfluidics, and methods for the analysis of protein–protein interactions that utilise the diffusion-controlled mixing characteristic of fluids at the microscale. We then describe techniques that employ electrophoretic forces to manipulate and fractionate interacting protein systems for their biophysical characterisation, before discussing strategies that use microdroplet compartmentalisation for the analysis of protein interactions. We conclude by highlighting future directions for the field, such as the integration of microfluidic experiments into high-throughput workflows for the investigation of protein interaction networks. [ABSTRACT FROM AUTHOR]

In rational drug design, it is important to determine accurately and with high precision the binding constant (the affinity or the change in Gibbs energy, ∆G), the change in enthalpy (ΔH), and the entropy change upon small molecule drug binding to a disease-related target protein. These thermodynamic parameters of the protein-ligand association reaction are usually determined by isothermal titration calorimetry (ITC). Here, the repeatability, precision, and accuracy of the measurement of the affinity and the change in enthalpy upon acetazolamide (AZM) interaction with human carbonic anhydrase II (CA II) are discussed based on the measurements using several ITC instruments. The AZM-CA II reaction was performed at decreasing protein-ligand concentrations until the determination of ∆G and ΔH was not possible, indicating a lower limit for accuracy. To obtain the confidence intervals (CI) of the ∆G and ΔH of AZM binding to CA II, the binding reaction was repeated numerous times at the optimal concentration of 10 µM and 25 °C temperature. The CI (at a confidence level α = 0.95) for ΔH = − 51.2 ± 1.0 kJ/mol and ∆G = − 45.4 ± 0.5 kJ/mol was determined by averaging the results of multiple repeats. [ABSTRACT FROM AUTHOR]

Thermodynamic properties of single molecules including enthalpic and entropic contributions are often determined from experiments by a direct control and precise measurement of the local temperature. However, common temperature monitoring techniques using, for example, ultrafine temperature probes can lead to uncertainties as the probe cannot be placed in the vicinity of the molecule of interest. Here, we devised an approach to measure the local temperature in freely diffusing confocal single-molecule Förster Resonance Energy Transfer (smFRET) experiments in situ by directly adding the temperature-sensitive fluorescent dye Rhodamine B, whose fluorescence lifetime serves as a probe of the local temperature in the confocal volume. We demonstrate that the temperature and FRET efficiencies of static and dynamic molecules can be extracted within one measurement simultaneously, without the need of a reference chamber. We anticipate this technique to be particularly useful in the physicochemical analyses of temperature-dependent biomolecular processes from single-molecule measurements. [ABSTRACT FROM AUTHOR]

Changes in molecular conformations are one of the major driving forces of complex biological processes. Many studies based on single-molecule techniques have shed light on conformational dynamics and contributed to a better understanding of living matter. In particular, single-molecule FRET experiments have revealed unprecedented information at various time scales varying from milliseconds to seconds. The choice and the attachment of fluorophores is a pivotal requirement for single-molecule FRET experiments. One particularly well-studied millisecond conformational change is the opening and closing of DNA hairpin structures. In this study, we addressed the influence of base- and terminal-labeled fluorophores as well as the fluorophore DNA interactions on the extracted kinetic information of the DNA hairpin. Gibbs free energies varied from ΔG0 = −3.6 kJ/mol to ΔG0 = −0.2 kJ/mol for the identical DNA hairpin modifying only the labeling scheme and design of the DNA sample. In general, the base-labeled DNA hairpin is significantly destabilized compared to the terminal-labeled DNA hairpin and fluorophore DNA interactions additionally stabilize the closed state of the DNA hairpin. Careful controls and variations of fluorophore attachment chemistry are essential for a mostly undisturbed measurement of the underlying energy landscape of biomolecules. [ABSTRACT FROM AUTHOR]

In the western part of the Koralmbahn railway line, tests were carried out as part of planned specialised civil engineering works in silty to sandy still water sediments in order to find out about the interaction of jet grouting and dewatering measures with this ground, which is considered to be as geotechnically relatively unfavourable. Small-scale tunnelling work was also carried out to gain experience for the later tunnel construction work in this area. The most important discoveries were: that the jet grouting trials achieved jet grout columns with a diameter of 1.5 m within the still water sediments; and the simulation of a tunnel drive was able to demonstrate that stable conditions at the face can be achieved with the chosen advance support measures. The evaluated results made it possible to optimise the design of the tunnel structures and the forecast performance rates for the grouting works. Im westlichen Bereich der Koralmbahn sind im Rahmen geplanter Spezialtiefbaumaßnahmen in schluffigen bis sandigen Stillwassersedimenten Versuche durchgeführt worden, um Erkenntnisse über die Interaktion von DSV- und Entwässerungsmaßnahmen mit diesem geotechnisch als relativ ungünstig geltenden Gebirge zu erlangen. Zusätzlich wurden durch kleinmaßstäbliche Vortriebsarbeiten Erfahrungen für die späteren Tunnelbauarbeiten in diesem Bereich gewonnen. Als wichtigste Erkenntnisse können genannt werden: Bei den Versuchen zu DSV-Arbeiten waren in den Stillwassersedimenten DSV-Säulen mit einem Durchmesser von 1,5 m zu erreichen; durch die Simulation eines Tunnelvortriebs konnte demonstriert werden, dass mit den gewählten Vorraussicherungsmaßnahmen stabile Verhältnisse im Bereich der Ortsbrust erzielbar sind. Basierend auf den ermittelten Ergebnissen können die bisherige Planung der Tunnelbauwerke sowie die prognostizierten Leistungsansätze bei den DSV-Maßnahmen optimiert werden. [ABSTRACT FROM AUTHOR]

Our meagre understanding of CFTR misfolding and its reversal by small-molecule correctors hampers the development of mechanism-based therapies of cystic fibrosis. Here we exploit a helical-hairpin construct—the simplest proxy of membrane-protein tertiary contacts—containing CFTR's transmembrane helices 3 and 4 and its corresponding disease phenotypic mutant V232D to gain molecular-level insights into CFTR misfolding and drug rescue by the corrector Lumacaftor. Using a single-molecule FRET approach to study hairpin conformations in lipid bilayers, we find that the wild-type hairpin is well folded, whereas the V232D mutant assumes an open conformation in bilayer thicknesses mimicking the endoplasmic reticulum. Addition of Lumacaftor reverses the aberrant opening of the mutant hairpin to restore a compact state as in the wild type. The observed membrane escape of the V232D hairpin and its reversal by Lumacaftor complement cell-based analyses of the full-length protein, thereby providing in vivo and in vitro correlates of CFTR misfolding and drug-action mechanisms. Georg Krainer and Antoine Treff et al. use a helical-hairpin construct derived from the cystic fibrosis transmembrane conductance regulator (CFTR) to investigate misfolding caused by the disease-linked V232D mutation. Using single-molecule FRET, they show that the V232D hairpin assumes an open conformation in lipid bilayers, which is reversed by the pharmacological corrector Lumacaftor. [ABSTRACT FROM AUTHOR]