Structural Bioinformatics Studies of Integral Transmembrane Enzymes pMMO Complex, C560, CYB, and DHSD and their AlphaFold3-Predicted Water-Soluble QTY Variants

The QTY (glutamine, threonine, tyrosine) code is a simple protein engineering and design tool that systematically replaces the hydrophobic amino acids leucine (L), isoleucine (I), valine (V), and phenylalanine (F) into the hydrophilic amino acids glutamine (Q), threonine (T), and tyrosine (Y), respectively, to enable the water-solubility of membrane proteins including membrane enzymes. In this study, we present the structural bioinformatics study of six membrane protein enzymes with experimentally determined CryoEM structures including the methane monooxygenase pMMO complex (pMOA, pMOB, pMOC), CYB, C560, DHSD, and their AlphaFold3-predicted water-soluble QTY variants. We applied the QTY code only to the transmembrane alpha-helices of the proteins. We then superposed structures of CryoEM-determined native proteins with their water-soluble QTY variants. The QTY code engineered water-soluble QTY variants demonstrate remarkable structural similarity with their native structures, with RMSD (Root Mean Square Deviation) values between 0.506Å and 0.887Å despite significant (33.33–86.61%) changes to the protein sequence of the transmembrane domains. To verify the effectiveness of our study, we show the changes in hydrophobicity surfaces between the native proteins and their QTY variants, and explain the rationale behind such change. Our structural bioinformatics studies provide key insight into differences between the hydrophobic alpha-helices and alpha-hydrophilic helices. The QTY code will likely further aid the future applications of methane oxidation enzymes. The water-soluble QTY variants could aid in studying and utilization of the enzymes in aqueous environments without detergent solubilization and stabilization. These QTY variant membrane enzymes may be particularly useful for future industrial-scale production in aqueous chassis such as filamentous algae.