Abstract The resistance–nodulation–division efflux machinery confers antimicrobial resistance to Gram-negative bacteria by actively pumping antibiotics out of the cell. The protein complex is powered by proton motive force; however, the proton transfer mechanism itself and indeed even its stoichiometry is still unclear. Here we review computational studies from the last decade that focus on elucidating the number of protons transferred per conformational cycle of the pump. Given the difficulties in studying proton movement using even state-of-the-art structural biology methods, the contributions from computational studies have been invaluable from a mechanistic perspective.

Abstract Respiratory disease and breathing abnormalities worsen with dehydration of the upper airways. We find that humidification of inhaled air occurs by evaporation of water over mucus lining the upper airways in such a way as to deliver an osmotic force on mucus, displacing it towards the epithelium. This displacement thins the periciliary layer of water beneath mucus while thickening topical water that is partially condensed from humid air on exhalation. With the rapid mouth breathing of dry air, this condensation layer, not previously reported while common to transpiring hydrogels in nature, can deliver an osmotic compressive force of up to around 100 cm H2O on underlying cilia, promoting adenosine triphosphate secretion and activating neural pathways. We derive expressions for the evolution of the thickness of the condensation layer, and its impact on cough frequency, inflammatory marker secretion, cilia beat frequency and respiratory droplet generation. We compare our predictions with human clinical data from multiple published sources and highlight the damaging impact of mouth breathing, dry, dirty air and high minute volume on upper airway function. We predict the hypertonic (or hypotonic) saline mass required to reduce (or amplify) dysfunction by restoration (or deterioration) of the structure of ciliated and condensation water layers in the upper airways and compare these predictions with published human clinical data. Preserving water balance in the upper airways appears critical in light of contemporary respiratory health challenges posed by the breathing of dirty and dry air.

Abstract While RNA folding was originally seen as a simple problem to solve, it has been shown that the promiscuous interactions of the nucleobases result in structural polymorphism, with several competing structures generally observed for non-coding RNA. This inherent complexity limits our understanding of these molecules from experiments alone, and computational methods are commonly used to study RNA. Here, we discuss three advanced sampling schemes, namely Hamiltonian-replica exchange molecular dynamics (MD), ratchet-and-pawl MD and discrete path sampling, as well as the HiRE-RNA coarse-graining scheme, and highlight how these approaches are complementary with reference to recent case studies. While all computational methods have their shortcomings, the plurality of simulation methods leads to a better understanding of experimental findings and can inform and guide experimental work on RNA polymorphism.

AbstractChaperones protect other proteins against misfolding and aggregation, a key requirement for maintaining biological function. Experimental observations of changes in solubility of amyloid proteins in the presence of certain chaperones are discussed here in terms of thermodynamic driving forces. We outline how chaperones can enhance amyloid solubility through the formation of heteromolecular aggregates (co-aggregates) based on the second law of thermodynamics and the flux towards equal chemical potential of each compound in all phases of the system. Higher effective solubility of an amyloid peptide in the presence of chaperone implies that the chemical potential of the peptide is higher in the aggregates formed under these conditions compared to peptide-only aggregates. This must be compensated by a larger reduction in chemical potential of the chaperone in the presence of peptide compared to chaperone alone. The driving force thus relies on the chaperone being very unhappy on its own (high chemical potential), thus gaining more free energy than the amyloid peptide loses upon forming the co-aggregate. The formation of heteromolecular aggregates also involves the kinetic suppression of the formation of homomolecular aggregates. The unhappiness of the chaperone can explain the ability of chaperones to favour an increased population of monomeric client protein even in the absence of external energy input, and with broad client specificity. This perspective opens for a new direction of chaperone research and outlines a set of outstanding questions that aim to provide additional cues for therapeutic development in this area.

AbstractInfrared (IR) spectroscopy is increasingly being used to probe the secondary structure of proteins, especially for high-concentration samples and biopharmaceuticals in complex formulation vehicles. However, the small path lengths required for aqueous protein transmission experiments, due to high water absorbance in the amide I region of the spectrum, means that the path length is not accurately known, so only the shape of the band is ever considered. This throws away a dimension of information. Attenuated total reflectance (ATR) IR spectroscopy is much easier to implement than transmission IR spectroscopy and, for a given instrument and sample, gives reproducible spectra. However, the ATR-absorbance spectrum varies with sample concentration and instrument configuration, and its wavenumber dependence differs significantly from that observed in transmission spectroscopy. In this paper, we determine, for the first time, how to transform water and aqueous protein ATR spectra into the corresponding transmission spectra with appropriate spectral shapes and intensities. The approach is illustrated by application to water, concanavalin A, haemoglobin and lysozyme. The transformation is only as good as the available water refractive index data. A hybrid of literature data provides the best results. The transformation also allows the angle of incidence of an ATR crystal to be determined. This opens the way to using both spectral shape and spectra intensity for protein structure fitting.