Molecular dynamics simulations and structural bioinformatics of bacterial integral alpha-helical membrane enzymes and their AlphaFold2-predicted water-soluble QTY analogues

Abstract

The study of integral membrane proteins has long been challenging because of their poor solubility in aqueous environments. We previously used QTY code to enhance the hydrophilicity in alpha-helices, beta-barrels, and monoclonal antibodies by systematically pairwise replacing the hydrophobic amino acids L (leucine) to Q (glutamine), V(valine)/I(isoleucine) to T (threonine), and F (phenylalanine) to Y (tyrosine). The superposed AlphaFold2-predicted structures of alpha-helical transmembrane enzyme variants with >41% amino acid substitutions displayed remarkable similarity to native structures (RMSD 0.3Å-0.7 Å). We conducted molecular dynamics (MD) simulations, which revealed that, even in the absence of a lipid bilayer, the QTY-modified enzymes retained stable dynamics comparable to their membrane-bound forms. Root mean square fluctuation (RMSF) values remained below 2 Å across the transmembrane and core regions, and residue-wise root mean square deviation (RMSD) values were minimal (<3 Å), indicating that the structural integrity of the protein core was largely preserved. These results suggest that the QTY variants, designed for soluble environments, effectively mimic the stability and conformational rigidity of natural membrane-bound enzymes. Our findings show that the QTY code is a simple method for designing water-soluble membrane protein enzymes in different biological scenarios, and it may encourage further experiments to validate our structural bioinformatics research.