Computational Simulations of Biofilm-Associated Proteins Generated by Pathogenic Escherichia coli Identified by...

Five proteins extracted from bacterial biofilms of a pathogenic Escherichia coli strain were identified by MALDI-TOF-TOF mass spectrometry and top-down protein analysis. They are cold-shock proteins CspC and CspE, DNA-binding proteins HU-α and HU-β and CsgA (the major subunit of curli). The fragmentation efficiency of these singly charged protein ions varied considerably. The pattern of fragment ions obtained from tandem mass spectrometry postsource decay of these protein ions was compared to in silico protein structures obtained using AlphaFold3 to better understand the factors that contribute to their gas phase dissociation as well as their likelihood of unfolding during MALDI sample preparation. Molecular dynamic simulations were performed to calculate the root-mean-square fluctuations (RMSF) of Cα atoms and root-mean-square deviation (RMSD) of the protein backbone to assess their propensity to unfold/denature during MALDI sample preparation. A molecular dynamics simulation was performed on a curli multimer composed of one CsgB (the minor curli subunit) and five CsgA which showed rapid disintegration of this short curli complex in hexafluoro-isopropanol (HFIP) consistent with experimental observations. Strong hydrogen bonding of HFIP appears to disrupt the intermolecular hydrogen bonds of this amyloid protein complex but does not unfold CsgA monomer because of 138 intramolecular hydrogen bonds present in its solenoidal β-sheet structure. Finally, electrostatic analysis of the curli complex reveals partitioned regions of positive and negative charge along the fibril axis that may facilitate its assembly.