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Web-based display of protein surface and pH-dependent properties for assessing the developability of biotherapeutics.

ABSTRACT: Protein instability leads to reversible self-association and irreversible aggregation which is a major concern for developing new biopharmaceutical leads. Protein solution behaviour is dictated by the physicochemical properties of the protein and the solution. Optimising protein solutions through experimental screens and targeted protein engineering can be a difficult and time consuming process. Here, we describe development of the protein-sol web server, which was previously restricted to protein solubility prediction from amino acid sequence. Tools are presented for calculating and mapping patches of hydrophobicity and charge on the protein surface. In addition, predictions of folded state stability and net charge are displayed as a heatmap for a range of pH and ionic strength conditions. Tools are evaluated in the context of antibodies, their fragments and interactions. Surprisingly, antibody-antigen interfaces are, on average, at least as polar as Fab surfaces. This benchmarking process provides the user with thresholds with which to assess non-polar surface patches, and possible solubility implications, in proteins of interest. Stability heatmaps compare favourably with experimental data for CH2 and CH3 domains. Display and quantification of surface polarity and pH/ionic strength dependence will be useful generally for investigation of protein biophysics.

SUBMITTER: Hebditch M

PROVIDER: S-EPMC6374528 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Web-based display of protein surface and pH-dependent properties for assessing the developability of biotherapeutics.

Hebditch Max M Warwicker Jim J

Scientific reports 20190213 1

Protein instability leads to reversible self-association and irreversible aggregation which is a major concern for developing new biopharmaceutical leads. Protein solution behaviour is dictated by the physicochemical properties of the protein and the solution. Optimising protein solutions through experimental screens and targeted protein engineering can be a difficult and time consuming process. Here, we describe development of the protein-sol web server, which was previously restricted to prote ...[more]

PMID: 30760735

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Project description:A recently engineered mutant of cyan fluorescent protein (WasCFP) that exhibits pH-dependent absorption suggests that its tryptophan-based chromophore switches between neutral (protonated) and charged (deprotonated) states depending on external pH. At pH 8.1, the latter gives rise to green fluorescence as opposed to the cyan color of emission that is characteristic for the neutral form at low pH. Given the high energy cost of deprotonating the tryptophan at the indole nitrogen, this behavior is puzzling, even if the stabilizing effect of the V61K mutation in proximity to the protonation/deprotonation site is considered. Because of its potential to open new avenues for the development of optical sensors and photoconvertible fluorescent proteins, a mechanistic understanding of how the charged state in WasCFP can possibly be stabilized is thus important. Attributed to the dynamic nature of proteins, such understanding often requires knowledge of the various conformations adopted, including transiently populated conformational states. Transient conformational states triggered by pH are of emerging interest and have been shown to be important whenever ionizable groups interact with hydrophobic environments. Using a combination of the weighted-ensemble sampling method and explicit-solvent constant pH molecular dynamics (CPHMD(MS?D)) simulations, we have identified a solvated transient state, characterized by a partially open ?-barrel where the chromophore pKa of 6.8 is shifted by over 20 units from that of the closed form (6.8 and 31.7, respectively). This state contributes a small population at low pH (12% at pH 6.1) but becomes dominant at mildly basic conditions, contributing as much as 53% at pH 8.1. This pH-dependent population shift between neutral (at pH 6.1) and charged (at pH 8.1) forms is thus responsible for the observed absorption behavior of WasCFP. Our findings demonstrate the conditions necessary to stabilize the charged state of the WasCFP chromophore (namely, local solvation at the deprotonation site and a partial flexibility of the protein ?-barrel structure) and provide the first evidence that transient conformational states can control optical properties of fluorescent proteins.

Dataset Information

Web-based display of protein surface and pH-dependent properties for assessing the developability of biotherapeutics.

Publications

Web-based display of protein surface and pH-dependent properties for assessing the developability of biotherapeutics.

Similar Datasets

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