Assessing the Self-Assembly of Native P1 Extensin Glycoproteins on Highly Oriented Pyrolytic Graphite (HOPG) Using Atomic Force Microscopy (AFM)

Extensins (EXTs) are structural glycoproteins found in plant cell walls that contribute to the growth and extension of plant cells. As stated in prior studies, these are self-assembled biopolymers formed by the association of EXT units, resulting in the formation of more complex scaffold structures. The nature of EXT's structural self-assembly as a result of molecular-level interactions remains to be explored. The topography of the structural 3D growth of the EXT network was monitored using atomic force microscopy (AFM). In this work, we investigated the effects of the monomer concentration and incubation time on the self-assembly of the tomato EXT precursor 1 (P1) on a highly ordered pyrolytic graphite (HOPG) substrate, which was monitored using AFM. The self-assembly was assessed after 1 min of incubation for different P1 bulk concentrations prepared on HOPG. We found that the precursor concentration affected the extent and nature of surface coverage. From 5 to 100 μg/mL of P1, the percent surface coverage after a minute increased from 12% to 95%, generating a homogeneous layer of laterally assembled EXTs. At 5 μg/mL P1 concentration, singular or fewer interacting P1 monomers were observed and considered to be at the initial stages of the self-assembly. After 1 min of incubation, the length, height, and width of a single P1 unit were estimated at 47 ± 5 nm, 0.9 ± 0.2, and 13 ± 3 nm, respectively. Changing the incubation time from 1 to 8 min has resulted in the formation of elongated and highly branched structures. At the fourth and eighth minute of incubation, structural formation via stacking and parallel interactions of P1 monomers was observed, providing a detailed illustration of the sequence of P1 self-assembly. The knowledge gathered from the presented AFM topographical analysis provides insight into the initial self-assembly of EXT. Understanding these stages of EXT network formation is crucial for navigating complex molecular-level arrangements within plant cell walls, which can then be integrated into the design of synthetic biopolymers.