ABSTRACT
Recombinant proteins play pivotal roles in numerous applications including industrial biocatalysts or therapeutics. Despite the recent progress in computational protein structure prediction, protein solubility and reduced aggregation propensity remain challenging attributes to design. Identification of aggregation-prone regions is essential for understanding misfolding diseases or designing efficient protein-based technologies, and as such has a great socio-economic impact. Here, we introduce AggreProt, a user-friendly webserver that automatically exploits an ensemble of deep neural networks to predict aggregation-prone regions (APRs) in protein sequences. Trained on experimentally evaluated hexapeptides, AggreProt compares to or outperforms state-of-the-art algorithms on two independent benchmark datasets. The server provides per-residue aggregation profiles along with information on solvent accessibility and transmembrane propensity within an intuitive interface with interactive sequence and structure viewers for comprehensive analysis. We demonstrate AggreProt efficacy in predicting differential aggregation behaviours in proteins on several use cases, which emphasize its potential for guiding protein engineering strategies towards decreased aggregation propensity and improved solubility. The webserver is freely available and accessible at https://loschmidt.chemi.muni.cz/aggreprot/.
Subject(s)
Internet , Protein Aggregates , Software , Protein Engineering/methods , Algorithms , Proteins/chemistry , Proteins/genetics , Neural Networks, Computer , Protein Folding , Solubility , Protein ConformationABSTRACT
Thermostability is an essential requirement for the use of enzymes in the bioindustry. Here, we compare different protein stabilization strategies using a challenging target, a stable haloalkane dehalogenase DhaA115. We observe better performance of automated stabilization platforms FireProt and PROSS in designing multiple-point mutations over the introduction of disulfide bonds and strengthening the intra- and the inter-domain contacts by in silico saturation mutagenesis. We reveal that the performance of automated stabilization platforms was still compromised due to the introduction of some destabilizing mutations. Notably, we show that their prediction accuracy can be improved by applying manual curation or machine learning for the removal of potentially destabilizing mutations, yielding highly stable haloalkane dehalogenases with enhanced catalytic properties. A comparison of crystallographic structures revealed that current stabilization rounds were not accompanied by large backbone re-arrangements previously observed during the engineering stability of DhaA115. Stabilization was achieved by improving local contacts including protein-water interactions. Our study provides guidance for further improvement of automated structure-based computational tools for protein stabilization.
