Understanding the mechanism of proton pumping requires a detailed description of the energetics and sequence of events associated with the proton transfers, and of how proton transfer couples to conformational rearrangements of the protein. Here, we discuss our recent advances in using computer simulations to understand how bacteriorhodopsin pumps protons. We emphasize the importance of accurately describing the retinal geometry and the location of water molecules.

Dihedral conformational transitions are analyzed systematically in a model globular protein, cytochrome P450cam, to examine their structural and chemical dependences through combined conventional molecular dynamics (cMD), accelerated molecular dynamics (aMD) and adaptive biasing force (ABF) simulations. The aMD simulations are performed at two acceleration levels, using dihedral and dual boost, respectively. In comparison with cMD, aMD samples protein dihedral transitions approximately two times faster on average using dihedral boost, and ∼ 3.5 times faster using dual boost. In the protein backbone, significantly higher dihedral transition rates are observed in the bend, coil, and turn flexible regions, followed by the β bridge and β sheet, and then the helices. Moreover, protein side chains of greater length exhibit higher transition rates on average in the aMD-enhanced sampling. Side chains of the same length (particularly Nχ = 2) exhibit decreasing transition rates with residues when going from hydrophobic to polar, then charged and aromatic chemical types. The reduction of dihedral transition rates is found to be correlated with increasing energy barriers as identified through ABF free energy calculations. These general trends of dihedral conformational transitions provide important insights into the hierarchical dynamics and complex free energy landscapes of functional proteins.

Kisspeptin controls reproduction by stimulating gonadotrophin-releasing hormone neurones via its receptor Kiss1r. Kiss1r is also expressed other brain areas and in peripheral tissues, suggesting additional nonreproductive roles. We recently determined that Kiss1r knockout (KO) mice develop an obese and diabetic phenotype. In the present study, we investigated whether Kiss1r KOs develop this metabolic phenotype as a result of alterations in the expression of metabolic genes involved in the appetite regulating system of the hypothalamus, including neuropeptide Y (Npy) and pro-opiomelanocortin (Pomc), as well as leptin receptor (Lepr), ghrelin receptor (Ghsr), and melanocortin receptors 3 and 4 (Mc3r, Mc4r). Body weights, leptin levels and hypothalamic gene expression were measured in both gonad-intact and gonadectomised (GNX) mice at 8 and 20 weeks of age that had received either normal chow or a high-fat diet. We detected significant increases in Pomc expression in gonad-intact Kiss1r KO mice at 8 and 20 weeks, although there were no alterations in the other metabolic-related genes. However, the Pomc increases appeared to reflect genotype differences in circulating sex steroids, because GNX wild-type and Kiss1r KO mice exhibited similar Pomc levels, along with similar Npy levels. The altered Pomc gene expression in gonad-intact Kiss1r KO mice is consistent with previous reports of reduced food intake in these mice and may serve to increase the anorexigenic drive, perhaps compensating for the obese state. However, the surprising overall lack of changes in any of the hypothalamic metabolic genes in GNX KO mice suggests that the aetiology of obesity in the absence of kisspeptin signalling may reflect peripheral rather than central metabolic impairments.

The insertion of nascent polypeptide chains into lipid bilayer membranes and the stability of membrane proteins crucially depend on the equilibrium partitioning of polypeptides. For this, the transfer of full sequences of amino-acid residues into the bilayer, rather than individual amino acids, must be understood. Earlier studies have revealed that the most likely reference state for partitioning very hydrophobic sequences is the membrane interface. We have used μs-scale simulations to calculate the interface-to-transmembrane partitioning free energies ΔG_S→TM for two hydrophobic carrier sequences in order to estimate the insertion free energy for all 20 amino acid residues when bonded to the center of a partitioning hydrophobic peptide. Our results show that prior single-residue scales likely overestimate the partitioning free energies of polypeptides. The correlation of ΔG_S→TM with experimental full-peptide translocon insertion data is high, suggesting an important role for the membrane interface in translocon-based insertion. The choice of carrier sequence greatly modulates the contribution of each single-residue mutation to the overall partitioning free energy. Our results demonstrate the importance of quantifying the observed full-peptide partitioning equilibrium, which is between membrane interface and transmembrane inserted, rather than combining individual water-to-membrane amino acid transfer free energies.

Infective endocarditis (IE) is a cardiovascular disease often caused by bacteria of the viridans group of streptococci, which includes Streptococcus gordonii and Streptococcus sanguinis. Previous research has found that serine-rich repeat (SRR) proteins on the S. gordonii bacterial surface play a critical role in pathogenesis by facilitating bacterial attachment to sialylated glycans displayed on human platelets. Despite their important role in disease progression, there are currently no anti-adhesive drugs available on the market. Here, we performed structure-based virtual screening using an ensemble docking approach followed by consensus scoring to identify novel small molecule effectors against the sialoglycan binding domain of the SRR adhesin protein Hsa from the S. gordonii strain DL1. The screening successfully predicted nine compounds which were able to displace the native ligand (sialyl-T antigen) in an in vitro assay and bind competitively to Hsa. Furthermore, hierarchical clustering based on the MACCS fingerprints showed that eight of these small molecules do not share a common scaffold with the native ligand. This study indicates that SRR family of adhesin proteins can be inhibited by diverse small molecules and thus prevent the interaction of the protein with the sialoglycans. This opens new avenues for discovering potential drugs against IE.

Kisspeptin and its receptor, Kiss1r, act centrally to stimulate reproduction. Recent evidence indicates that kisspeptin is also important for body weight and metabolism, as whole-body Kiss1r KO mice, developed with gene trap technology, display obesity and reduced metabolism. Kiss1r is expressed in brain and multiple peripheral tissues, but it is unknown which is responsible for the metabolic phenotype. Here, we sought to confirm that 1) the metabolic phenotype of the gene trap Kiss1r KOs is due to disruption of kisspeptin signaling and not off-target effects of viral mutagenesis, and 2) the Kiss1r flox line is suitable for creating conditional KOs to study the metabolic phenotype. We used Cre/lox technology (Zp3-Cre/Kiss1r flox) to develop a new global Kiss1r KO ("Kiss1r gKO") to compare with the original gene trap KO phenotype. We confirmed that deleting exon 2 of Kiss1r from the entire body induces hypogonadism in both sexes. Moreover, global deletion of Kiss1r induced obesity in females, but not males, along with increased adiposity and impaired glucose tolerance, similar to the gene trap Kiss1r KOs. Likewise, Kiss1r gKO females had decreased VO₂ and VCO₂, likely underlying their obesity. These findings support that our previous results in gene trap Kiss1r KOs are due to disrupted kisspeptin signaling, and further highlight a role for Kiss1r signaling in energy expenditure and metabolism besides controlling reproduction. Moreover, given Kiss1r expression in multiple cell-types, our findings indicate that the Kiss1r flox line is viable for future investigations to isolate specific target cells of kisspeptin's metabolic effects.

Carbon dioxide (CO2) is a detrimental greenhouse gas and is the main contributor to global warming. In addressing this environmental challenge, a promising approach emerges through the utilization of deep eutectic solvents (DESs) as an ecofriendly and sustainable medium for effective CO2 capture. Chemically reactive DESs, which form chemical bonds with the CO2, are superior to nonreactive, physically based DESs for CO2 absorption. However, there are no accurate computational models that provide accurate predictions of the CO2 solubility in chemically reactive DESs. Here, we develop machine learning (ML) models to predict the solubility of CO2 in chemically reactive DESs. As training data, we collected 214 data points for the CO2 solubility in 149 different chemically reactive DESs at different temperatures, pressures, and DES molar ratios from published work. The physics-driven input features for the ML models include σ-profile descriptors that quantify the relative probability of a molecular surface segment having a certain screening charge density and were calculated with the first-principle quantum chemical method COSMO-RS. We show here that, although COSMO-RS does not explicitly calculate chemical reaction profiles, the COSMO-RS-derived σ-profile features can be used to predict bond formation. Of the models trained, an artificial neural network (ANN) provides the most accurate CO2 solubility prediction with an average absolute relative deviation of 2.94% on the testing sets. Overall, this work provides ML models that can predict CO2 solubility precisely and thus accelerate the design and application of chemically reactive DESs.

Kisspeptin and its receptor, Kiss1r, act centrally to stimulate reproduction. Recent evidence indicates that kisspeptin is also important for body weight and metabolism, as whole-body Kiss1r KO mice, developed with gene trap technology, display obesity and reduced metabolism. Kiss1r is expressed in brain and multiple peripheral tissues, but it is unknown which is responsible for the metabolic phenotype. Here, we sought to confirm that 1) the metabolic phenotype of the gene trap Kiss1r KOs is due to disruption of kisspeptin signaling and not off-target effects of viral mutagenesis, and 2) the Kiss1r flox line is suitable for creating conditional KOs to study the metabolic phenotype. We used Cre/lox technology (Zp3-Cre/Kiss1r flox) to develop a new global Kiss1r KO ("Kiss1r gKO") to compare with the original gene trap KO phenotype. We confirmed that deleting exon 2 of Kiss1r from the entire body induces hypogonadism in both sexes. Moreover, global deletion of Kiss1r induced obesity in females, but not males, along with increased adiposity and impaired glucose tolerance, similar to the gene trap Kiss1r KOs. Likewise, Kiss1r gKO females had decreased VO₂ and VCO₂, likely underlying their obesity. These findings support that our previous results in gene trap Kiss1r KOs are due to disrupted kisspeptin signaling, and further highlight a role for Kiss1r signaling in energy expenditure and metabolism besides controlling reproduction. Moreover, given Kiss1r expression in multiple cell-types, our findings indicate that the Kiss1r flox line is viable for future investigations to isolate specific target cells of kisspeptin's metabolic effects.

Kisspeptin is vital for the regulation of both fertility and metabolism. Kisspeptin receptor (Kiss1r) knockout (KO) mice exhibit increased adiposity and reduced energy expenditure in adulthood. Kiss1r mRNA is expressed in brown adipose tissue (BAT) and Kiss1r KO mice exhibit reduced Ucp1 mRNA in BAT and impaired thermogenesis. We hypothesised that mice with diminished kisspeptin signalling would exhibit reduced core body temperature (Tc) and altered dynamics of circadian and ultradian rhythms of Tc. Tc was recorded every 15-min over 14-days in gonadectomised wild-type (WT), Kiss1r KO, and also Kiss1-Cre (95% reduction in Kiss1 transcription) mice. Female Kiss1r KOs had higher adiposity and lower Ucp1 mRNA in BAT than WTs. No change was detected in Kiss1-Cre mice. Mean Tc during the dark phase was lower in female Kiss1r KOs versus WTs, but not Kiss1-Cre mice. Female Kiss1r KOs had a lower mesor and amplitude of the circadian rhythm of Tc than did WTs. In WT mice, there were more episodic ultradian events (EUEs) of Tc during the dark phase than the light phase, but this measure was similar between dark and light phases in Kiss1r KO and Kiss1-Cre mice. The amplitude of EUEs was higher in the dark phase in female Kiss1r KO and male Kiss1-Cre mice. Given the lack of clear metabolic phenotype in Kiss1-Cre mice, 5% of Kiss1 transcription may be sufficient for proper metabolic control, as was shown for fertility. Moreover, the observed alterations in Tc suggest that kisspeptin has a role in circadian and ultradian rhythm-driven pathways.