Single-Molecule Studies of Protein Folding with Optical Tweezers.

Manipulation of individual molecules with optical tweezers provides a powerful means of interrogating the structure and folding of proteins. Mechanical force is not only a relevant quantity in cellular protein folding and function, but also a convenient parameter for biophysical folding studies. Optical tweezers offer precise control in the force range relevant for protein folding and unfolding, from which single-molecule kinetic and thermodynamic information about these processes can be extracted. In this review, we describe both physical principles and practical aspects of optical tweezers measurements and discuss recent advances in the use of this technique for the study of protein folding. In particular, we describe the characterization of folding energy landscapes at high resolution, studies of structurally complex multidomain proteins, folding in the presence of chaperones, and the ability to investigate real-time cotranslational folding of a polypeptide.

Next generation single-molecule techniques: Imaging, labeling, and manipulation in vitro and in cellulo.

Owing to their unique abilities to manipulate, label, and image individual molecules in vitro and in cellulo, single-molecule techniques provide previously unattainable access to elementary biological processes. In imaging, single-molecule fluorescence resonance energy transfer (smFRET) and protein-induced fluorescence enhancement in vitro can report on conformational changes and molecular interactions, single-molecule pull-down (SiMPull) can capture and analyze the composition and function of native protein complexes, and single-molecule tracking (SMT) in live cells reveals cellular structures and dynamics. In labeling, the abilities to specifically label genomic loci, mRNA, and nascent polypeptides in cells have uncovered chromosome organization and dynamics, transcription and translation dynamics, and gene expression regulation. In manipulation, optical tweezers, integration of single-molecule fluorescence with force measurements, and single-molecule force probes in live cells have transformed our mechanistic understanding of diverse biological processes, ranging from protein folding, nucleic acids-protein interactions to cell surface receptor function.

The Ribosome Cooperates with a Chaperone to Guide Multi-domain Protein Folding.

Multi-domain proteins, containing several structural units within a single polypeptide, constitute a large fraction of all proteomes. Co-translational folding is assumed to simplify the conformational search problem for large proteins, but the events leading to correctly folded, functional structures remain poorly characterized. Similarly, how the ribosome and molecular chaperones promote efficient folding remains obscure. Using optical tweezers, we have dissected early folding events of nascent elongation factor G, a multi-domain protein that requires chaperones for folding. The ribosome and the chaperone trigger factor reduce inter-domain misfolding, permitting folding of the N-terminal G-domain. Successful completion of this step is a crucial prerequisite for folding of the next domain. Unexpectedly, co-translational folding does not proceed unidirectionally; emerging unfolded polypeptide can denature an already-folded domain. Trigger factor, but not the ribosome, protects against denaturation. The chaperone thus serves a previously unappreciated function, helping multi-domain proteins overcome inherent challenges during co-translational folding.

ClpX(P) generates mechanical force to unfold and translocate its protein substrates.

AAA(+) unfoldases denature and translocate polypeptides into associated peptidases. We report direct observations of mechanical, force-induced protein unfolding by the ClpX unfoldase from E. coli, alone, and in complex with the ClpP peptidase. ClpX hydrolyzes ATP to generate mechanical force and translocate polypeptides through its central pore. Threading is interrupted by pauses that are found to be off the main translocation pathway. ClpX's translocation velocity is force dependent, reaching a maximum of 80 aa/s near-zero force and vanishing at around 20 pN. ClpX takes 1, 2, or 3 nm steps, suggesting a fundamental step-size of 1 nm and a certain degree of intersubunit coordination. When ClpX encounters a folded protein, it either overcomes this mechanical barrier or slips on the polypeptide before making another unfolding attempt. Binding of ClpP decreases the slip probability and enhances the unfolding efficiency of ClpX. Under the action of ClpXP, GFP unravels cooperatively via a transient intermediate.

Studying root-environment interactions in structured microdevices.

When interacting with the environment, plant roots integrate sensory information over space and time in order to respond appropriately under non-uniform conditions. The complexity and dynamic properties of soil across spatial and temporal scales pose a significant technical challenge for research into the mechanisms that drive metabolism, growth, and development in roots, as well as on inter-organismal networks in the rhizosphere. Synthetic environments, combining microscopic access and manipulation capabilities with soil-like heterogeneity, are needed to elucidate the intriguing antagonism that characterizes subsurface ecosystems. Microdevices have provided opportunities for innovative approaches to observe, analyse, and manipulate plant roots and advanced our understanding of their development, physiology, and interactions with the environment. Initially conceived as perfusion platforms for root cultivation under hydroponic conditions, microdevice design has, in recent years, increasingly shifted to better reflect the complex growth conditions in soil. Heterogeneous micro-environments have been created through co-cultivation with microbes, laminar flow-based local stimulation, and physical obstacles and constraints. As such, structured microdevices provide an experimental entry point into the complex network behaviour of soil communities.

Co-translational folding of nascent polypeptides: Multi-layered mechanisms for the efficient biogenesis of functional proteins.

The coupling of protein synthesis and folding is a crucial yet poorly understood aspect of cellular protein folding. Over the past few years, it has become possible to experimentally follow and define protein folding on the ribosome, revealing principles that shape co-translational folding and distinguish it from refolding in solution. Here, we highlight some of these recent findings from biochemical and biophysical studies and their potential significance for cellular protein biogenesis. In particular, we focus on nascent chain interactions with the ribosome, interactions within the nascent protein, modulation of translation elongation rates, and the role of mechanical force that accompanies nascent protein folding. The ability to obtain mechanistic insight in molecular detail has set the stage for exploring the intricate process of nascent protein folding. We believe that the aspects discussed here will be generally important for understanding how protein synthesis and folding are coupled and regulated.

Monitoring Distracted Driving Behaviours with Smartphones: An Extended Systematic Literature Review.

Driver behaviour monitoring is a broad area of research, with a variety of methods and approaches. Distraction from the use of electronic devices, such as smartphones for texting or talking on the phone, is one of the leading causes of vehicle accidents. With the increasing number of sensors available in vehicles, there is an abundance of data available to monitor driver behaviour, but it has only been available to vehicle manufacturers and, to a limited extent, through proprietary solutions. Recently, research and practice have shifted the paradigm to the use of smartphones for driver monitoring and have fuelled efforts to support driving safety. This systematic review paper extends a preliminary, previously carried out author-centric literature review on smartphone-based driver monitoring approaches using snowballing search methods to illustrate the opportunities in using smartphones for driver distraction detection. Specifically, the paper reviews smartphone-based approaches to distracted driving behaviour detection, the smartphone sensors and detection methods applied, and the results obtained.

[A naturalistic study on the effects of antidepressants (e. g. SSRI) and acetyl salicylic acid (ASA) on the self-rated perception of the psychotherapeutic process of inpatient psychosomatic treatment and its results: Could ASA be beneficial?] / Eine naturalistische Untersuchung zur Auswirkung häufiger antidepressiver Medikationen (wie SSRI) und der Acetylsalicylsäure (ASS) auf die Selbstbeurteilungen der Wahrnehmung und des Ergebnisses stationärer Psychotherapie: Könnte ASS förderlich sein?

Objectives: Psychic perceptions are at the core of psychotherapeutic processes and modifiable by certain psychopharmacologic agents including antidepressants and cyclooxygenase (COX) inhibitors like acetylsalicylic acid (ASA). Methods: We analyzed the medical records of 208 participants, and used the weekly mean dosages and the number of weeks in therapy to predict ward experience (Stationserfahrungsbogen) and symptom burden (symptom-check list 90-R) by means of linear regression analyses and four repeated measures. Results: Time predicted symptom relief. ASA signified a more favorable ward experience and a trend towards less suffering. Antidepressants did not predict symptom burden or ward experience, except for amitriptyline's inverse relationship with process perception. Discussion: Regarding process perception and therapy outcome, amitriptyline might have unfavorable effects at dose reductions, whereas COX-inhibition could be beneficial at higher dosages. Similar findings have already been described with regard to COX-inhibition in depression and schizophrenia.

Energetic dependencies dictate folding mechanism in a complex protein.

Large proteins with multiple domains are thought to fold cotranslationally to minimize interdomain misfolding. Once folded, domains interact with each other through the formation of extensive interfaces that are important for protein stability and function. However, multidomain protein folding and the energetics of domain interactions remain poorly understood. In elongation factor G (EF-G), a highly conserved protein composed of 5 domains, the 2 N-terminal domains form a stably structured unit cotranslationally. Using single-molecule optical tweezers, we have defined the steps leading to fully folded EF-G. We find that the central domain III of EF-G is highly dynamic and does not fold upon emerging from the ribosome. Surprisingly, a large interface with the N-terminal domains does not contribute to the stability of domain III. Instead, it requires interactions with its folded C-terminal neighbors to be stably structured. Because of the directionality of protein synthesis, this energetic dependency of domain III on its C-terminal neighbors disrupts cotranslational folding and imposes a posttranslational mechanism on the folding of the C-terminal part of EF-G. As a consequence, unfolded domains accumulate during synthesis, leading to the extensive population of misfolded species that interfere with productive folding. Domain III flexibility enables large-scale conformational transitions that are part of the EF-G functional cycle during ribosome translocation. Our results suggest that energetic tuning of domain stabilities, which is likely crucial for EF-G function, complicates the folding of this large multidomain protein.

Facile tethering of stable and unstable proteins for optical tweezers experiments.

Single-molecule force spectroscopy with optical tweezers has emerged as a powerful tool for dissecting protein folding. The requirement to stably attach "molecular handles" to specific points in the protein of interest by preparative biochemical techniques is a limiting factor in applying this methodology, especially for large or unstable proteins that are difficult to produce and isolate. Here, we present a streamlined approach for creating stable and specific attachments using autocatalytic covalent tethering. The high specificity of coupling allowed us to tether ribosome-nascent chain complexes, demonstrating its suitability for investigating complex macromolecular assemblies. We combined this approach with cell-free protein synthesis, providing a facile means of preparing samples for single-molecule force spectroscopy. The workflow eliminates the need for biochemical protein purification during sample preparation for single-molecule measurements, making structurally unstable proteins amenable to investigation by this powerful single-molecule technique. We demonstrate the capabilities of this approach by carrying out pulling experiments with an unstructured domain of elongation factor G that had previously been refractory to analysis. Our approach expands the pool of proteins amenable to folding studies, which should help to reduce existing biases in the currently available set of protein folding models.

The human habenula is responsive to changes in luminance and circadian rhythm.

The habenula is a pivotal structure in the neural network that implements various forms of cognitive and motivational functions and behaviors. Moreover, it has been suggested to be part of the brain's circadian system, not at least because habenular neurons are responsive to retinal illumination and exhibit circadian modulations of their firing patterns in animal research. However, no study has directly investigated the human habenula in this regard. We developed a paradigm in which alternating phases of high and low luminance are used to study human habenular functioning. In two experiments with independent samples, fMRI data of 24 healthy participants were acquired at a field strength of 7T, and of 21 healthy participants at 3T. Region of interest analyses revealed that the human habenula is responsive to light as well, resulting in a decrease in activation when a change in luminance occurs. Although this pattern is not predicted by animal research, we were able to replicate this finding in a second independent data set. Furthermore, we demonstrate that the strength of decrease in activation is modulated in a circadian fashion, being more strongly deactivated in morning than in afternoon sessions. Taken together, these findings provide strong evidence that changes in illumination elicit changes in habenular activation and that these changes appear to be more pronounced in the morning than in the afternoon.

Single-Molecule Chemo-Mechanical Spectroscopy Provides Structural Identity of Folding Intermediates.

Single-molecule force spectroscopy has emerged as a powerful tool for studying the folding of biological macromolecules. Mechanical manipulation has revealed a wealth of mechanistic information on transient and intermediate states. To date, the majority of state assignment of intermediates has relied on empirical demarcation. However, performing such experiments in the presence of different osmolytes provides an alternative approach that reports on the structural properties of intermediates. Here, we analyze the folding and unfolding of T4 lysozyme with optical tweezers under a chemo-mechanical perturbation by adding osmolytes. We find that two unrelated protective osmolytes, sorbitol and trimethylamine-n-oxide, function by marginally decelerating unfolding rates and specifically modulating early events in the folding process, stabilizing formation of an on-pathway intermediate. The chemo-mechanical perturbation provides access to two independent metrics of the relevant states during folding trajectories, the contour length, and the solvent-accessible surface area. We demonstrate that the dependence of the population of the intermediate in different osmolytes, in conjunction with its measured contour length, provides the ability to discriminate between potential structural models of intermediate states. Our study represents a general strategy that may be employed in the structural modeling of equilibrium intermediate states observed in single-molecule experiments.

Navigating the complexities of multi-domain protein folding.

Proteome complexity has expanded tremendously over evolutionary time, enabling biological diversification. Much of this complexity is achieved by combining a limited set of structural units into long polypeptides. This widely used evolutionary strategy poses challenges for folding of the resulting multi-domain proteins. As a consequence, their folding differs from that of small single-domain proteins, which generally fold quickly and reversibly. Co-translational processes and chaperone interactions are important aspects of multi-domain protein folding. In this review, we discuss some of the recent experimental progress toward understanding these processes.

Stick-slip unfolding favors self-association of expanded HTT mRNA.

In Huntington's Disease (HD) and related disorders, expansion of CAG trinucleotide repeats produces a toxic gain of function in affected neurons. Expanded huntingtin (expHTT) mRNA forms aggregates that sequester essential RNA binding proteins, dysregulating mRNA processing and translation. The physical basis of RNA aggregation has been difficult to disentangle owing to the heterogeneous structure of the CAG repeats. Here, we probe the folding and unfolding pathways of expHTT mRNA using single-molecule force spectroscopy. Whereas normal HTT mRNAs unfold reversibly and cooperatively, expHTT mRNAs with 20 or 40 CAG repeats slip and unravel non-cooperatively at low tension. Slippage of CAG base pairs is punctuated by concerted rearrangement of adjacent CCG trinucleotides, trapping partially folded structures that readily base pair with another RNA strand. We suggest that the conformational entropy of the CAG repeats, combined with stable CCG base pairs, creates a stick-slip behavior that explains the aggregation propensity of expHTT mRNA.

Anomalous signal from S atoms in protein crystallographic data from an X-ray free-electron laser.

X-ray free-electron lasers (FELs) enable crystallographic data collection using extremely bright femtosecond pulses from microscopic crystals beyond the limitations of conventional radiation damage. This diffraction-before-destruction approach requires a new crystal for each FEL shot and, since the crystals cannot be rotated during the X-ray pulse, data collection requires averaging over many different crystals and a Monte Carlo integration of the diffraction intensities, making the accurate determination of structure factors challenging. To investigate whether sufficient accuracy can be attained for the measurement of anomalous signal, a large data set was collected from lysozyme microcrystals at the newly established `multi-purpose spectroscopy/imaging instrument' of the SPring-8 Ångstrom Compact Free-Electron Laser (SACLA) at RIKEN Harima. Anomalous difference density maps calculated from these data demonstrate that serial femtosecond crystallography using a free-electron laser is sufficiently accurate to measure even the very weak anomalous signal of naturally occurring S atoms in a protein at a photon energy of 7.3 keV.

AP profiling resolves co-translational folding pathway and chaperone interactions in vivo.

Natural proteins have evolved to fold robustly along specific pathways. Folding begins during synthesis, guided by interactions of the nascent protein with the ribosome and molecular chaperones. However, the timing and progression of co-translational folding remain largely elusive, in part because the process is difficult to measure in the natural environment of the cytosol. We developed a high-throughput method to quantify co-translational folding in live cells that we term Arrest Peptide profiling (AP profiling). We employed AP profiling to delineate co-translational folding for a set of GTPase domains with very similar structures, defining how topology shapes folding pathways. Genetic ablation of major nascent chain-binding chaperones resulted in localized folding changes that suggest how functional redundancies among chaperones are achieved by distinct interactions with the nascent protein. Collectively, our studies provide a window into cellular folding pathways of complex proteins and pave the way for systematic studies on nascent protein folding at unprecedented resolution and throughput.

3D-2D Distance Maps Conversion Enhances Classification of Craniosynostosis.

OBJECTIVE: Diagnosis of craniosynostosis using photogrammetric 3D surface scans is a promising radiation-free alternative to traditional computed tomography. We propose a 3D surface scan to 2D distance map conversion enabling the usage of the first convolutional neural networks (CNNs)-based classification of craniosynostosis. Benefits of using 2D images include preserving patient anonymity, enabling data augmentation during training, and a strong under-sampling of the 3D surface with good classification performance. METHODS: The proposed distance maps sample 2D images from 3D surface scans using a coordinate transformation, ray casting, and distance extraction. We introduce a CNN-based classification pipeline and compare our classifier to alternative approaches on a dataset of 496 patients. We investigate into low-resolution sampling, data augmentation, and attribution mapping. RESULTS: Resnet18 outperformed alternative classifiers on our dataset with an F1-score of 0.964 and an accuracy of 98.4%. Data augmentation on 2D distance maps increased performance for all classifiers. Under-sampling allowed 256-fold computation reduction during ray casting while retaining an F1-score of 0.92. Attribution maps showed high amplitudes on the frontal head. CONCLUSION: We demonstrated a versatile mapping approach to extract a 2D distance map from the 3D head geometry increasing classification performance, enabling data augmentation during training on 2D distance maps, and the usage of CNNs. We found that low-resolution images were sufficient for a good classification performance. SIGNIFICANCE: Photogrammetric surface scans are a suitable craniosynostosis diagnosis tool for clinical practice. Domain transfer to computed tomography seems likely and can further contribute to reducing ionizing radiation exposure for infants.

Efficacy of Oral Rabies Vaccine Baits Containing SPBN GASGAS in Domestic Dogs According to International Standards.

(1) Background: The oral vaccination of free-roaming dogs against rabies has been developed as a promising complementary tool for mass dog vaccination. However, no oral rabies vaccine has provided efficacy data in dogs according to international standards. (2) Methods: To test the immunogenicity and efficacy of the third-generation oral rabies virus vaccine strain, SPBN GASGAS, in domestic dogs, dogs were offered an egg-flavoured bait containing 3.0 mL of the vaccine (107.5 FFU/mL) or a placebo egg-flavoured bait. Subsequently, these 25 vaccinated and 10 control animals were challenged approximately 6 months later with a dog rabies virus isolate. Blood samples were collected at different time points postvaccination and examined by ELISA and RFFIT. (3) Results: All but 1 of the 25 vaccinated dogs survived the challenge infection; meanwhile, all 10 control dogs succumbed to rabies. The serology results showed that all 25 vaccinated dogs seroconverted in ELISA (>40% PB); meanwhile, only 13 of the 25 vaccinated dogs tested seropositive ≥ 0.5 IU/mL) in RFFIT. (4) Conclusions: The SPBN GASGAS rabies virus vaccine meets the efficacy requirements for live oral rabies vaccines as laid down by the European Pharmacopoeia and the WOAH Terrestrial Manual. SPBN GASGAS already fulfilled the safety requirements for oral rabies vaccines targeted at dogs. Hence, the egg-flavoured bait containing SPBN GASGAS is the first oral vaccine bait that complies with WOAH recommendations for the intended use of oral vaccination of free-roaming dogs against rabies.

Discovery of a selective and biologically active low-molecular weight antagonist of human interleukin-1ß.

Human interleukin-1ß (hIL-1ß) is a pro-inflammatory cytokine involved in many diseases. While hIL-1ß directed antibodies have shown clinical benefit, an orally available low-molecular weight antagonist is still elusive, limiting the applications of hIL-1ß-directed therapies. Here we describe the discovery of a low-molecular weight hIL-1ß antagonist that blocks the interaction with the IL-1R1 receptor. Starting from a low affinity fragment-based screening hit 1, structure-based optimization resulted in a compound (S)-2 that binds and antagonizes hIL-1ß with single-digit micromolar activity in biophysical, biochemical, and cellular assays. X-ray analysis reveals an allosteric mode of action that involves a hitherto unknown binding site in hIL-1ß encompassing two loops involved in hIL-1R1/hIL-1ß interactions. We show that residues of this binding site are part of a conformationally excited state of the mature cytokine. The compound antagonizes hIL-1ß function in cells, including primary human fibroblasts, demonstrating the relevance of this discovery for future development of hIL-1ß directed therapeutics.

Time-resolving state-specific molecular dissociation with XUV broadband absorption spectroscopy.

The electronic and nuclear dynamics inside molecules are essential for chemical reactions, where different pathways typically unfold on ultrafast timescales. Extreme ultraviolet (XUV) light pulses generated by free-electron lasers (FELs) allow atomic-site and electronic-state selectivity, triggering specific molecular dynamics while providing femtosecond resolution. Yet, time-resolved experiments are either blind to neutral fragments or limited by the spectral bandwidth of FEL pulses. Here, we combine a broadband XUV probe pulse from high-order harmonic generation with an FEL pump pulse to observe dissociation pathways leading to fragments in different quantum states. We temporally resolve the dissociation of a specific O2+ state into two competing channels by measuring the resonances of ionic and neutral fragments. This scheme can be applied to investigate convoluted dynamics in larger molecules relevant to diverse science fields.