Biophysical chemistry最新文献

Protein aggregation is a major hurdle in developing biopharmaceuticals, in particular protein formulation area, but plays a pivotal role in food products. Co-solvents are used to suppress protein aggregation in pharmaceutical proteins. On the contrary, aggregation is encouraged in the process of food product making. Thus, it is expected that co-solvents play a contrasting role in biopharmaceutical formulation and food products. Here, we show several examples that utilize co-solvents, e.g., salting-out salts, sugars, polyols and divalent cations in promoting protein-protein interactions. The mechanisms of co-solvent effects on protein aggregation and solubility have been studied on aqueous protein solution and applied to develop pharmaceutical formulation based on the acquired scientific knowledge. On the contrary, co-solvents have been used in food industries based on empirical basis. Here, we will review the mechanisms of co-solvent effects on protein-protein interactions that can be applied to both pharmaceutical and food industries and hope to convey knowledge acquired through research on co-solvent interactions in aqueous protein solution and formulation to those involved in food science and provide those involved in protein solution research with the observations on aggregation behavior of food proteins.

RNA polymerase I (Pol I) is responsible for synthesizing ribosomal RNA, which is the rate limiting step in ribosome biogenesis. We have reported wide variability in the magnitude of the rate constants defining the rate limiting step in sequential nucleotide additions catalyzed by Pol I. in this study we sought to determine if base identity impacts the rate limiting step of nucleotide addition catalyzed by Pol I. To this end, we report a transient state kinetic interrogation of AMP, CMP, GMP, and UMP incorporations catalyzed by Pol I. We found that Pol I uses one kinetic mechanism to incorporate all nucleotides. However, we found that UMP incorporation is faster than AMP, CMP, and GMP additions. Further, we found that endonucleolytic removal of a dimer from the 3′ end was fastest when the 3′ terminal base is a UMP. It has been previously shown that both downstream and upstream template sequence identity impacts the kinetics of nucleotide addition. The results reported here show that the incoming base identity also impacts the magnitude of the observed rate limiting step.

Bacillus anthracis, the causative agent of anthrax, poses a substantial threat to public health and national security, and is recognized as a potential bioweapon due to its capacity to form resilient spores with enduring viability. Inhalation or ingestion of even minute quantities of aerosolized spores can lead to widespread illness and fatalities, underscoring the formidable lethality of the bacterium. With an untreated mortality rate of 100%, Bacillus anthracis is a disconcerting candidate for bioterrorism. In response to this critical scenario, we employed state-of-the-art computational tools to conceive and characterize flexible RNA aptamer therapeutics tailored for anthrax. The foundational structure of the flexible RNA aptamers was designed by removing the C2’-C3’ in each nucleotide unit. Leveraging the crystal structure of Bacillus anthracis ribosomal protein S8 complexed with an RNA aptamer, we explored the structural, dynamic, and energetic aspects of the modified RNA aptamer – S8 protein complexes through extensive all-atom explicit-solvent molecular dynamics simulations (400 ns, 3 replicas each), followed by drawing comparisons to the control system. Our findings demonstrate the enhanced binding competencies of the flexible RNA aptamers to the S8 protein via better shape complementarity and improved H-bond network compared to the control RNA aptamer. This research offers valuable insights into the development of RNA aptamer therapeutics targeting Bacillus anthracis, paving the way for innovative strategies to mitigate the impact of this formidable pathogen.

In the presented work, a study on the solubility and intermolecular interactions of l-serine and L-cysteine was carried out in binary mixtures of H₂O + dimethylformamide (DMF), H₂O + dimethylsulfoxide (DMSO), and H₂O + acetonitrile (ACN) in the temperature range of T = 288.15 K to 308.15 K. l-serine exhibited the highest solubility in water, while L-cysteine was more soluble in water-DMF. The solvation process was assessed through standard Gibbs energy calculations, indicating the solvation stability order: water-ACN > water-DMSO > water-DMF for l-serine, and water-DMF > water-DMSO > water-ACN for L-cysteine. This study also explored the influence of these amino acids on solvent–solvent interactions, revealing changes in chemical entropies and self-association patterns within the binary solvent mixtures.

The thermal effect of the formation of the “burst-phase” folding intermediate has been studied using a titration calorimeter. It is shown that, unlike the total thermal effect of native structure formation, it can be both positive and negative depending on the temperature. The reasons for this paradoxical behavior are analyzed. A conclusion is drawn about the leading role of dehydration of non-polar groups in the first stage of folding.

Hydrogen peroxide, produced by Dual Oxidase (Duox), is essential for thyroid hormone synthesis. Duox activation involves Ca²⁺ binding to its EF-hand Domain (EFD), which contains two EF-hands (EFs). In this study, we characterized a truncated EFD using spectrometry, calorimetry, electrophoretic mobility, and gel filtration to obtain its Ca²⁺ binding thermodynamic and kinetics, as well as to assess the associated conformational changes. Our results revealed that its 2nd EF-hand (EF2) exhibits a strong exothermic Ca²⁺ binding (K_a = 10⁷ M⁻¹) while EF1 shows a weaker binding (K_a = 10⁵ M⁻¹), resulting in the burial of its negatively charged residues. The Ca²⁺ binding to EFD results in a stable structure with a melting temperature shifting from 67 to 99 °C and induces a structural transition from a dimeric to monomeric form. EF2 appears to play a role in dimer formation in its apo form, while the hydrophobic exposure of Ca²⁺-bound-EF1 is crucial for dimer formation in its holo form. The result is consistent with structures obtained from Cryo-EM, indicating that a stable structure of EFD with hydrophobic patches upon Ca²⁺ binding is vital for its Duox's domain-domain interaction for electron transfer.

We propose a detailed computational beta cell model that emphasizes the role of anaplerotic metabolism under glucose and glucose-glutamine stimulation. This model goes beyond the traditional focus on mitochondrial oxidative phosphorylation and ATP-sensitive K⁺ channels, highlighting the predominant generation of ATP from phosphoenolpyruvate in the vicinity of K_ATP channels. It also underlines the modulatory role of H₂O₂ as a signaling molecule in the first phase of glucose-stimulated insulin secretion. In the second phase, the model emphasizes the critical role of anaplerotic pathways, activated by glucose stimulation via pyruvate carboxylase and by glutamine via glutamate dehydrogenase. It particularly focuses on the production of NADPH and glutamate as key enhancers of insulin secretion. The predictions of the model are consistent with empirical data, highlighting the complex interplay of metabolic pathways and emphasizing the primary role of glucose and the facilitating role of glutamine in insulin secretion. By delineating these crucial metabolic pathways, the model provides valuable insights into potential therapeutic targets for diabetes.

Reverse micelles (RMs) are spontaneously organizing nanobubbles composed of an organic solvent, surfactants, and an aqueous phase that can encapsulate biological macromolecules for various biophysical studies. Unlike other RM systems, the 1-decanoyl-rac-glycerol (10MAG) and lauryldimethylamine-N-oxide (LDAO) surfactant system has proven to house proteins with higher stability than other RM mixtures with little sensitivity to the water loading (W₀, defined by the ratio of water to surfactant). We investigated this unique property by encapsulating three model proteins – cytochrome c, myoglobin, and flavodoxin – in 10MAG/LDAO RMs and applying a variety of experimental methods to characterize this system's behavior. We found that this surfactant system differs greatly from the traditional, spherical, monodisperse RM population model. 10MAG/LDAO RMs were discovered to be oblate ellipsoids at all conditions, and as W₀ was increased, surfactants redistributed to form a greater number of increasingly spherical ellipsoidal particles with pools of more bulk-like water. Proteins distinctively influence the thermodynamics of the mixture, encapsulating at their optimal RM size and driving protein-free RM sizes to scale accordingly. These findings inform the future development of similarly malleable encapsulation systems and build a foundation for application of 10MAG/LDAO RMs to analyze biological and chemical processes under nanoscale confinement.

The main cysteine protease (M^pro) of coronavirus SARS-CoV-2 has become a promising target for computational development in anti-COVID-19 treatments. Here, we benchmarked the performance of six biomolecular molecular dynamics (MD) force fields (OPLS-AA, CHARMM27, CHARMM36, AMBER03, AMBER14SB and GROMOS G54A7) and three water models (TIP3P, TIP4P and SPC) for reproducing the native fold and the enzymatic activity of M^pro as monomeric and dimeric units. The MD sampling up to 1 μs suggested that the proper choice of the force fields and water models plays an essential role in reproducing the tertiary structure and the inter-residue distance between the catalytic dyad His41-Cys145. We found that while most benchmarked all-atom force fields reproduce well the native fold of M^pro, the CHARMM27/TIP3P and OPLS-AA/TIP4P setups revealed a good performance in reproducing the structure of the catalytic domain. In addition, these FF setups were also well-adopted for MD sampling of M^pro at the physiologic conditions by mimicking the presence of 100 mM NaCl and the elevated temperature of 310 K. Finally, both FFs were also performed well in reproducing the native fold of M^pro in a dimeric form. Therefore, comparing the preservation of the native fold of M^pro and the stability of its catalytic site architecture, our MD benchmarking suggests that the OPLS-AA/TIP4P and CHARMM27/TIP3P MD setups at the physiologic conditions may be well-suited for rapid in silico screening and developing broad-spectrum anti-coronaviral therapeutic agents.

The DNA and RNA aptamers D4 and R4, respectively, emerged from the modification of PC-3 cell-binding aptamer A4. Our objective was to characterize the aptamers in silico and in vitro and begin to identify their target molecules. We represented their structures using computational algorithms; evaluated their binding to several prostate cell lines and their effects on the viability and migration of these cells; and determined their dissociation constant by flow cytometry. We analyzed circulating prostate tumor cells from patients using D4, R4, anti-CD133 and anti-CD44. Finally, the target proteins of both aptamers were precipitated and identified by mass spectrometry to simulate their in silico docking. The aptamers presented similar structures and bound to prostate tumor cells without modifying the cellular parameters studied, but with different affinities. The ligand cells for both aptamers were CD44⁺, indicating that they could identify cells in the mesenchymal stage of the metastatic process. The possible target proteins NXPE1, ADAM30, and MUC6 need to be further studied to better understand their interaction with the aptamers. These results support the development of new assays to determine the clinical applications of D4 and R4 aptamers in prostate cancer.