S-Layer Self-Assembly on Supported Lipid-Bilayers: The Importance of Amorphous Precursors and Folding Transitions

Abstract

The outermost membranes of many archaea and bacteria are comprised of highly-ordered 2D arrays of surface layer (S-layer) proteins. Their functions include selective transport, structural scaffolding, mineral templating and propagation of or protection from pathogenesis. Although the primary and secondary structures of the isolated proteins determine their governing interactions, their functions emerge from the tertiary and quaternary architecture that stems from S-layer self-assembly, a process that is poorly understood. Here we report results using in situ AFM to follow 2D self-assembly of monomeric SbpA of Lysinibacillus sphaericus on supported lipid bi-layers (SLBs) at the molecular-scale. We show that the assembly process begins with adsorption of unstructured monomers, which form a mobile phase on the SLBs. These then condense into amorphous clusters, which undergo a phase transition to ordered 2D clusters of 2 to 15 folded tetramers. The ordered clusters then enter a growth phase in which new tetramers form from unstructured monomers exclusively at unoccupied lattice sites along the cluster edges, implying that new tetramer formation is auto-catalytic. We show that the analysis of growth dynamics leads to a quantitative model in which the main rate limiting parameter is the probability of tetramer creation. The estimated energy barrier of 51 kJ/mole for this process is much less than expected form scaling laws for folding of isolated proteins. Finally we present preliminary results from dynamic Monte Carlo simulations that show how the combination of non-specific interactions and directional bonds characteristic of many proteins lead to non-classical assembly pathways, such as the one observed here involving formation of amorphous clusters followed by relaxation to the ordered state.