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

Abstract

Abstractα-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 α-zeins have resisted experimental atomic-resolution characterization, molecular models have been central to elucidating their structure, but deep-learning α-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 α-zein cZ19C2 gave a loosely packed model with 7 α-helical segments connected by turns/loops and 68% total α-helicity. Compact tertiary structure was limited to a C-terminal bundle of three α-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 μs timescale. Although the simulations gave structurally diverse endpoints, several patterns were observed: In water and ≤ 2 mol% ethanol, the model rapidly formed compact globular structures, largely preserving the C-terminal bundle. At ≥ 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 ≥ 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. β-sheet content was typically <1% across all simulations. In aqueous simulations with glycidyl methacrylate (GMA), 55% of GMA was found within 4 Å of the model. Pre-reaction complexes for methacrylation were indicated by GMA epoxide carbons within 2.9 – 3.3 Å of side chain hydroxyl oxygens, suggesting accessibility of reactive sites in compact α-zein conformations.