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We present a generically applicable approach to determine an extensive set of size-dependent critical quality attributes inside nanoparticulate pharmaceutical products. By coupling asymmetrical-flow field-flow fractionation (AF4) measurements directly in-line with solution small angle X-ray scattering (SAXS), vital information such as (i) quantitative, absolute size distribution profiles, (ii) drug loading, (iii) size-dependent internal structures, and (iv) quantitative information on free drug is obtained. Here the validity of the method was demonstrated by characterizing complex mRNA-based lipid nanoparticle products. The approach is particularly applicable to particles in the size range of 100 nm and below, which is highly relevant for pharmaceutical products—both biologics and nanoparticles. The method can be applied as well in other fields, including structural biology and environmental sciences.

Photon-by-photon analysis tools for diffusion-based single-molecule Förster resonance energy transfer (smFRET) experiments often describe protein dynamics with Markov models. However, FRET efficiencies are only projections of the conformational space such that the measured dynamics can appear non-Markovian. Model-free methods to quantify FRET efficiency fluctuations would be desirable in this case. Here, we present such an approach. We determine FRET efficiency correlation functions free of artifacts from the finite length of photon trajectories or the diffusion of molecules through the confocal volume. We show that these functions capture the dynamics of proteins from nano- to milliseconds both in simulation and experiment, which provides a rigorous validation of current model-based analysis approaches. The authors show how to compute the autocorrelation function of FRET efficiencies and obtain dynamic information from as little as a few thousand molecules.

The vast majority of protein structures are determined at cryogenic temperatures, which are far from physiological conditions. Nevertheless, it is well established that temperature is an essential thermodynamic parameter for understanding the conformational dynamics and functionality of proteins in their native environments. Time-resolved crystallography is a technique that aims to elucidate protein function by examining structural alterations during processes such as ligand binding, catalysis, or allostery. However, this approach is typically conducted under ambient conditions, which may obscure crucial conformational states, that are only visible at physiological temperatures. In this study, we directly address the interplay between protein structure and activity via a method that enables multi-temperature, time-resolved serial crystallography experiments in a temperature window from below 10 °C to above 70 °C. Via this 5D-SSX, time-resolved experiments can now be carried out at physiological temperatures and with long time delays, providing insights into protein function and enzyme catalysis. Our findings demonstrate the temperature-dependent modulation of turnover kinetics for the mesophilic β -lactamase CTX-M-14 and the thermophilic enzyme xylose isomerase, within the full protein structure Here, the authors present a method that enables time-resolved serial protein crystallography over a wide temperature range (10–70 °C). This 5D crystallography approach can be implemented to illuminate protein function at physiologically relevant conditions.

Biological small‐angle X‐ray scattering (SAXS) is a versatile and powerful technique for investigating the structural and biophysical properties of biologically and pharmaceutically relevant macromolecules and nanoparticles. SAXS offers detailed insights into macromolecular composition, size, shape and internal structure, while addressing key aspects such as oligomeric state, stability, molecular interactions, and conformational flexibility. Recently, asymmetrical‐flow field‐flow fractionation (AF4) was successfully coupled to SAXS, enabling online size‐based fractionation and analysis of polydisperse samples. This approach allows precise, size‐dependent characterization, offering significant advancements in the study of polydisperse systems. We have integrated an AF4 device at the P12 beamline at the European Molecular Biology Laboratory and implemented technical adaptations allowing full automation to make the system suitable for routine user access. We provide streamlined workflows and troubleshooting resources for both novice and advanced SAXS users thereby equipping them with clear guidance on performing AF4–SAXS measurements. The general principles of our set‐up are easily adaptable to other beamlines which have integrated (or are planning to integrate) a similar system. By coupling asymmetrical‐flow field‐flow fractionation to small‐angle X‐ray scattering (AF4–SAXS), we enable precise, size‐resolved analysis of polydisperse samples. Our automated AF4–SAXS system at the EMBL P12 beamline streamlines workflows, making advanced characterization accessible to both novice and experienced users, with principles adaptable to other facilities.

The predominant resistance mechanism observed in Gram-negative bacteria involves the production of β -lactamases, which catalyse the hydrolysis of β -lactam antibiotics, thereby rendering them ineffective. Although Isoxazolyl Penicillins have been available since the 1970s, there are currently no structures in complex with class-A β -lactamases available. Here we have analysed the structure of the clinically relevant β -lactamase CTX-M-14 from Klebsiella pneumoniae near physiological temperatures, via serial synchrotron crystallography. We demonstrate the acyl-enzyme intermediates of the catalytically impaired CTX-M-14 mutant E166A in complex with three Isoxazolyl-Penicillins: Oxacillin, Cloxacillin and Dicloxacillin. Structural comparisons of CTX-M-Penicillin complexes show that, while conserved active-site interactions are maintained, each Isoxazolyl-Penicillin adopts a distinct conformation. While the three derivatives differ only by one and two chlorine atoms, respectively, their conformational heterogeneity appears to be increased by chlorination of the phenyl ring. Class-A β -lactamases can render antibiotics ineffective through hydrolysis, but the associated enzyme-antibiotic complexes remain largely underexplored at room temperatures. Here, the authors use RT serial synchrotron crystallography to report acyl-enzyme intermediates of the catalytically impaired β -lactamase CTX-M-14 mutant (E166A) from Klebsiella pneumoniae, and show that different isoxazolyl-penicillins adopt different conformations at room temperature.

Antibiotic resistance is a growing global health threat that risks the lives of millions. Among the resistance mechanisms, that mediated by metallo-β-lactamases is of particular concern as these bacterial enzymes dismantle most β-lactam antibiotics, which are our widest applied and cheapest to produce antibiotic agents. So far, no clinically applicable metallo-β-lactamase inhibitors are available. Aiming to adapt to structural variations, we introduce the inhibitor concept: dynamically chiral phosphonic acids. We demonstrate that they are straightforward to synthesize, penetrate bacterial membranes, inhibit the metallo-β-lactamase enzymes NDM-1, VIM-2 and GIM-1, and are non-toxic to human cells. Mimicking the transition state of β-lactam hydrolysis, they target the Zn ions of the metallo-β-lactamase active site. As a unique feature, both of their stereoisomers bind metallo-β-lactamases, which provides them unparalleled adaptability to the structural diversity of these enzymes, and may allow them to hamper bacteria’s ability for resistance development. Metallo-β-lactamases dismantle most β-lactam antibiotics and cause antibiotic resistance, however, no metallo-β-lactamase inhibitors are clinically available. Here, the authors report stereodynamically chiral phosphonic acids as potential metallo-β-lactamase inhibitors, showing unparalleled adaptability to the structural diversity of metallo-β-lactamases.

Protein stability in detergent or membrane-like environments is the bottleneck for structural studies on integral membrane proteins (IMP). Irrespective of the method to study the structure of an IMP, detergent solubilization from the membrane is usually the first step in the workflow. Here, we establish a simple, high-throughput screening method to identify optimal detergent conditions for membrane protein stabilization. We apply differential scanning fluorimetry in combination with scattering upon thermal denaturation to study the unfolding of integral membrane proteins. Nine different prokaryotic and eukaryotic membrane proteins were used as test cases to benchmark our detergent screening method. Our results show that it is possible to measure the stability and solubility of IMPs by diluting them from their initial solubilization condition into different detergents. We were able to identify groups of detergents with characteristic stabilization and destabilization effects for selected targets. We further show that fos-choline and PEG family detergents may lead to membrane protein destabilization and unfolding. Finally, we determined thenmodynamic parameters that are important indicators of IMP stability. The described protocol allows the identification of conditions that are suitable for downstream handling of membrane proteins during purification.

Background Spinal Muscular Atrophy (SMA) is characterized by progressive and predominantly proximal and axial muscle atrophy and weakness. Respiratory muscle weakness results in impaired cough with recurrent respiratory tract infections, nocturnal hypoventilation, and may ultimately lead to fatal respiratory failure in the most severely affected patients. Treatment strategies to either slow down the decline or improve respiratory muscle function are wanting. Objective The aim of this study is to assess the feasibility and efficacy of respiratory muscle training (RMT) in patients with SMA and respiratory muscle weakness. Methods The effect of RMT in patients with SMA, aged ≥ 8 years with respiratory muscle weakness (maximum inspiratory mouth pressure [PImax] ≤ 80 Centimeters of Water Column [cmH2O]), will be investigated with a single blinded randomized sham-controlled trial consisting of a 4-month training period followed by an 8-month open label extension phase. Intervention The RMT program will consist of a home-based, individualized training program involving 30-breathing cycles through an inspiratory and expiratory muscle training device. Patients will be instructed to perform 10 training sessions over 5–7 days per week. In the active training group, the inspiratory and expiratory threshold will be adjusted to perceived exertion (measured on a Borg scale). The sham-control group will initially receive RMT at the same frequency but against a constant, non-therapeutic resistance. After four months the sham-control group will undergo the same intervention as the active training group (i.e., delayed intervention). Individual adherence to the RMT protocol will be reviewed every two weeks by telephone/video call with a physiotherapist. Main study parameters/endpoints We hypothesize that the RMT program will be feasible (good adherence and good acceptability) and improve inspiratory muscle strength (primary outcome measure) and expiratory muscle strength (key secondary outcome measure) as well as lung function, patient reported breathing difficulties, respiratory infections, and health related quality of life (additional secondary outcome measures, respectively) in patients with SMA. Discussion RMT is expected to have positive effects on respiratory muscle strength in patients with SMA. Integrating RMT with recently introduced genetic therapies for SMA may improve respiratory muscle strength in this patient population. Trial registration Retrospectively registered at clinicaltrial.gov: NCT05632666.

Antibiotic resistance is a growing global health threat that risks the lives of millions. Among the resistance mechanisms, that mediated by metallo-beta-lactamases is of particular concern as these bacterial enzymes dismantle most beta-lactam antibiotics, which are our widest applied and cheapest to produce antibiotic agents. So far, no clinically applicable metallo-beta-lactamase inhibitors are available. Aiming to adapt to structural variations, we introduce the inhibitor concept: dynamically chiral phosphonic acids. We demonstrate that they are straightforward to synthesize, penetrate bacterial membranes, inhibit the metallo-beta-lactamase enzymes NDM-1, VIM-2 and GIM-1, and are non-toxic to human cells. Mimicking the transition state of beta-lactam hydrolysis, they target the Zn ions of the metallo-beta-lactamase active site. As a unique feature, both of their stereoisomers bind metallo-beta-lactamases, which provides them unparalleled adaptability to the structural diversity of these enzymes, and may allow them to hamper bacteria's ability for resistance development.