Rational identification of aggregation hotspots based on secondary structure and amino acid hydrophobicity

Abstract

Insolubility of proteins expressed in the Escherichia coli expression system hinders the progress of both basic and applied research. Insoluble proteins contain residues that decrease their solubility (aggregation hotspots). Mutating these hotspots to optimal amino acids is expected to improve protein solubility. To date, however, the identification of these hotspots has proven difficult. In this study, using a combination of approaches involving directed evolution and primary sequence analysis, we found two rules to help inductively identify hotspots: the α-helix rule, which focuses on the hydrophobicity of amino acids in the α-helix structure, and the hydropathy contradiction rule, which focuses on the difference in hydrophobicity relative to the corresponding amino acid in the consensus protein. By properly applying these two rules, we succeeded in improving the probability that expressed proteins would be soluble. Our methods should facilitate research on various insoluble proteins that were previously difficult to study due to their low solubility.

Self-assemble peptide biomaterials and their biomedical applications

“…B, Thin hSAFs, specific interactions at all b, c and f sites were replaced with weaker, more-general interactions, to result in smaller, more flexible, bundles of thinner fibres [31]. (Reproduced and adapted with permission from Nature) C, Hypothesis of self-assembly from peptide monomers to supramolecular networks of Schematic model based on the α-helix rule [32]. (Reproduced and adapted with permission from Nature).…”

Ultraefficiently Calming Cytokine Storm Using Ti3C2Tx MXene

“…[ 37,38 ] More importantly, when the secondary structure of IL‐6 is changed, the hydrophobic functional groups which are originally wrapped inside IL‐6 molecules are exposed, which greatly reduces their dispersibility in aqueous solutions. [ 39,40 ] On the contrary, the surfaces of most traditional commercial absorbents (ACs or resins, etc.) are hydrophobic, and therefore there is no chemical adsorption ability for absorbing IL‐6 molecules.…”

Protein engineering of the aldoxime dehydratase from Bacillus sp. OxB-1 based on a rational sequence alignment approach

“…The program INTMSAlign was originally developed by the Asano group for the identification of novel hydroxynitrile lyases based on multi-sequence alignment 35 . In a later work, the program was modified by implementation of a tool for amino acid hydropathy calculation (INTMSAlign_HiSol) 36 . This additional tool is suitable to compare the amino acid hydropathy of each position in the target protein with the conserved hydropathy in the protein family.…”

“…This calculation method has been utilised for identification of aggregation hotspots in proteins. When the hydropathy of amino acid residues located in α-helices was inverted by exchange with highly conserved amino acids, several proteins could be solubilised for recombinant expression in E. coli 36 . We assumed that the hydropathy switch induces structural changes on the protein constitution, which can also influence the catalytic efficiency.…”