Structural effects of charge destabilization and amino acid substitutions in amyloid fragments of CsgA

[Display omitted] •Amino acid substitutions change the aggregation propensity and morphology of peptides designed based on R4 fragments of CsgA.•Swapping the charge states within the polypeptide chain influences the preferential formation of amyloid fibrils.•Substituting glycine with lysine in CsgA R4 peptides from S. enterica affects fibril morphology, leading to the generation of spherical fibrils.•Substituting leucine with alanine in the same peptide accelerates the aggregation process and results in the formation of long rigid fibrils.•Electrostatic interactions play a role in the process by which peptides and proteins self-assemble into fibrils. The most studied functional amyloid is the CsgA, major curli subunit protein, which is produced by numerous strains of Enterobacteriaceae. Although CsgA sequences are highly conserved, they exhibit species diversity, which reflects the specific evolutionary and functional adaptability of the major curli subunit. Herein, we performed bioinformatics analyses to uncover the differences in the amyloidogenic properties of the R4 fragments in Escherichia coli and Salmonella enterica and proposed four mutants for more detailed studies: M1, M2, M3, and M4. The mutated sequences were characterized by various experimental techniques, such as circular dichroism, ATR-FTIR, FT-Raman, thioflavin T, transmission electron microscopy and confocal microscopy. Additionally, molecular dynamics simulations were performed to determine the role of buffer ions in the aggregation process. Our results demonstrated that the aggregation kinetics, fibril morphology, and overall structure of the peptide were significantly affected by the positions of charged amino acids within the repeat sequences of CsgA. Notably, substituting glycine with lysine resulted in the formation of distinctive spherically packed globular aggregates. The differences in morphology observed are attributed to the influence of phosphate ions, which disrupt the local electrostatic interaction network of the polypeptide chains. This study provides knowledge on the preferential formation of amyloid fibrils based on charge states within the polypeptide chain.