Improved understanding of properties that mediate protein solubility and resistance to aggregation are important for developing biopharmaceuticals, and more generally in biotechnology and synthetic biology. Recent acquisition of large datasets for antibody biophysical properties enables the search for predictive models. In this report, machine learning methods are used to derive models for 12 biophysical properties. A physicochemical perspective is maintained in analysing the models, leading to the observation that models cluster largely according to charge (cross-interaction measurements) and hydrophobicity (self-interaction methods). These two properties also overlap in some cases, for example in a new interpretation of variation in hydrophobic interaction chromatography. Since the models are developed from differences of antibody variable loops, the next stage is to extend models to more diverse protein sets. Availability The web application for the sequence-based algorithms are available on the protein-sol webserver, at https://protein-sol.manchester.ac.uk/abpred, with models and virtualisation software available at https://protein-sol.manchester.ac.uk/software.
Increased protein solubility is known to correlate with an increase in the proportion of lysine over arginine residues. Previous work has shown that the aggregation propensity of a single-chain variable fragment (scFv) does not correlate with its conformational stability or native-state protein−protein interactions. Here, we test the hypothesis that aggregation is driven by the colloidal stability of partially unfolded states, studying the behavior of scFv mutants harboring single or multiple site-specific arginine to lysine mutations in denaturing buffers. In 6 M guanidine hydrochloride (GdmCl) or 8 M urea, repulsive protein−protein interactions were measured for the wild-type and lysineenriched (4RK) scFvs reflecting weakened short-range attractions and increased excluded volume. In contrast to the arginine-enriched mutant (7KR) scFv exhibited strong reversible association. In 3 M GdmCl, the minimum concentration at which the scFvs were unfolded, the hydrodynamic radius of 4RK remained constant but increased for the wild type and especially for 7KR. Studies of single-point arginine to lysine scFv mutants indicated that the observed aggregation propensity of arginine under denaturing conditions was nonspecific. Interestingly, one such swap generated a scFv with especially low aggregation rates under low/high ionic strengths and denaturing buffers; molecular modeling identified hydrogen bonding between the arginine side chain and main chain peptide groups, stabilizing the structure. The arginine/lysine ratio is not routinely considered in biopharmaceutical scaffold design or current amyloid prediction methods. This work therefore suggests a simple method for increasing the stability of a biopharmaceutical protein against aggregation.
Motivation Evolution couples differences in ambient pH to biological function through protonatable groups, in particular those that switch from buried to exposed and alter protonation state in doing so. We present a tool focusing on structure-based discovery and display of these groups. Results Since prediction of buried group pKas is computationally intensive, solvent accessibility of ionizable groups is displayed, from which the user can iteratively select pKa calculation centers. Results are color-coded, with emphasis on buried groups. Utility is demonstrated with benchmarking against known pH sensing sites in influenza virus hemagglutinin and in variants of murine hepatitis virus, a coronavirus. A pair of histidine residues that are conserved in coronavirus spike proteins are predicted to be electrostatically frustrated at acidic pH in both pre- and post-fusion conformations. We suggest that an intermediate expanded conformation at endosomal pH could relax the frustration, allowing histidine protonation, and facilitating conformational conversion of coronavirus spike protein. Availability This tool is available at http://www.protein-sol.manchester.ac.uk/pka/
Understanding the intricate interplay of interactions between proteins, excipients, ions and water is important to achieve the effective purification and stable formulation of protein therapeutics. The free energy of lysozyme interacting with two kinds of polyanionic excipients, citrate and tripolyphosphate, together with sodium chloride and TRIS-buffer, are analysed in multiple-walker metadynamics simulations to understand why tripolyphosphate causes lysozyme to precipitate but citrate does not. The resulting multiscale decomposition of energy and entropy components for water, sodium chloride, excipients and lysozyme reveals that lysozyme is more stabilised by the interaction of tripolyphosphate with basic residues. This is accompanied by more sodium ions being released into solution from tripolyphosphate than for citrate, whilst the latter instead has more water molecules released into solution. Even though lysozyme aggregation is not directly probed in this study, these different mechanisms are suspected to drive the cross-linking between lysozyme molecules with vacant basic residues, ultimately leading to precipitation.
The vacuolar H+-ATPase (V-ATPase) is an enzymatic complex that functions in an ATP-dependent manner to pump protons across membranes and acidify organelles, thereby creating the proton/pH gradient required for membrane trafficking by several different types of transporters. We describe heterozygous point variants in ATP6V0C, encoding the c-subunit in the membrane bound integral domain of the V-ATPase, in 27 patients with neurodevelopmental abnormalities with or without epilepsy. Corpus callosum hypoplasia and cardiac abnormalities were also present in some patients. In silico modeling suggested that the patient variants interfere with the interactions between the ATP6V0C and ATP6V0A subunits during ATP hydrolysis. Consistent with decreased V-ATPase activity, functional analyses conducted in Saccharomyces cerevisiae revealed reduced LysoSensor fluorescence and reduced growth in media containing varying concentrations of CaCl2. Knockdown of ATP6V0C in Drosophila resulted in increased duration of seizure-like behavior, and the expression of selected patient variants in Caenorhabditis elegans led to reduced growth, motor dysfunction, and reduced lifespan. In summary, this study establishes ATP6V0C as an important disease gene, describes the clinical features of the associated neurodevelopmental disorder, and provides insight into disease mechanisms.
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