Conformations of a highly expressed Z19 α-zein studied with AlphaFold2 and MD simulations

Abstract

Alpha-zeins are amphiphilic maize proteins with interesting material properties suitable for numerous applications, e.g., in renewable plastics, foods, therapeutics and additive manufacturing (3D-printing). To exploit their full potential, molecular-level insights are essential. Since alpha-zeins have resisted experimental atomic-resolution characterization, molecular models have been central to elucidating their structure, but deep-learning alpha-zein models are largely unexplored. Therefore, this work studies an AlphaFold2 model of a highly expressed zein with molecular dynamics (MD) simulations. The mature protein sequence of the alpha-zein cZ19C2 gave a loosely packed model with 7 alpha-helical segments connected by turns/loops and 68% total alpha-helicity. Compact tertiary structure was limited to a C-terminal bundle of three alpha-helices, each aligning well with the published repeat sequence NPAAYLQQQQLLPFNQLA(V/A)(L/A). The model was subjected to MD simulations in water containing 0, 3, 23, 50, and 100 mol% ethanol for 400 ns, with extension of selected simulations to the us timescale. Although the simulations gave structurally diverse endpoints, several patterns were observed: In water and less than or equal to 2 mol% ethanol, the model rapidly formed compact globular structures, largely preserving the C-terminal bundle. At greater than or equal to 50 mol% ethanol, extended conformations prevailed, consistent with previous SAXS studies. Tertiary structure was partially stabilized in water and in 2 mol% and 23 mol% ethanol, but was disrupted in greater than or equal to 50 mol% ethanol. Averaged results indicated slightly increased helicity with ethanol concentration. Multiple copies of a globular model conformation rapidly formed branched aggregates in aqueous all-atom and coarse-grained MD simulations. Beta-sheet content was typically <1% across all simulations. In aqueous simulations with glycidyl methacrylate (GMA), 55% of GMA was found within 4 Angstrom of the model. Pre-reaction complexes for methacrylation were indicated by GMA epoxide carbons within 2.9 - 3.3 Angstrom of side chain hydroxyl oxygens, suggesting accessibility of reactive sites in compact alpha-zein conformations.