Self-catalyzed growth of S layers via an amorphous-to-crystalline transition limited by folding kinetics

Abstract

The importance of nonclassical, multistage crystallization pathways is increasingly evident from theoretical studies on colloidal systems and experimental investigations of proteins and biomineral phases. Although theoretical predictions suggest that proteins follow these pathways as a result of fluctuations that create unstable dense-liquid states, microscopic studies indicate these states are long-lived. Using in situ atomic force microscopy to follow 2D assembly of S-layer proteins on supported lipid bilayers, we have obtained a molecular-scale picture of multistage protein crystallization that reveals the importance of conformational transformations in directing the pathway of assembly. We find that monomers with an extended conformation first form a mobile adsorbed phase, from which they condense into amorphous clusters. These clusters undergo a phase transition through S-layer folding into crystalline clusters composed of compact tetramers. Growth then proceeds by formation of new tetramers exclusively at cluster edges, implying tetramer formation is autocatalytic. Analysis of the growth kinetics leads to a quantitative model in which tetramer creation is rate limiting. However, the estimated barrier is much smaller than expected for folding of isolated S-layer proteins, suggesting an energetic rationale for this multistage pathway.in situ atomic force microscopy imaging | protein crystal growth | two-step crystallization | amorphous precursors | assembly kinetics S elf-assembled protein architectures exhibit a range of structural motifs (1) including particles (2), fibers (3), ribbons (4), and sheets (5). Their functions include selective transport (5), structural scaffolding (6), mineral templating (4, 7), and propagation of or protection from pathogenesis (3,8). Although the molecular structures of the isolated proteins dictate their governing interactions, these functions emerge from the nanoscale organization that arises out of self-assembly. Recent theoretical investigations have predicted that assembly pathways during crystallization of proteins can deviate from the classical picture in which order arises concomitantly with condensation (9). Instead, dense-liquid droplets with little long-range order, which arise through transient fluctuations, have been found to lie along the path of least steep ascent over the free energy barrier to nucleation of the ordered phase. After formation of these denseliquid droplets, relaxation to the lower energy-ordered state occurs. Experimental studies of bulk protein crystallization (10, 11) have led to similar conclusions and provide convincing evidence for a dense-liquid phase that precedes order. But due to experimental limitations associated with viewing assembly in three dimensions with molecular resolution, a molecular-scale picture of multistage pathways has not been obtained. Moreover, in many protein systems, folding events and conformational transformations to oligomeric forms are an inherent part of assembly, but their role in defining the assembly pathw...

Do hierarchical structures assemble best via hierarchical pathways?

“…1 tein clusters seen in AFM images of SbpA self-assembly 37 , and allows formation of macroscopic liquid phases implicated in protein self-assembly generally 1,30,38,39 . In general, the nonspecific interaction mediates transitions from gas to liquid to solid, while specific interactions determine whether those phases consist of monomers, dimers, or tetramers.…”

“…We can begin to answer this question through the intensive study of computer models inspired by real systems. A simple example of a natural hierarchical material is provided by the SbpA surface-layer (S-layer) protein, which forms a membrane on the outsides of the bacterium Lysinibacillus sphaericus and on surfaces in vitro [21][22][23][24] . This membrane, which is robust and controllably porous, is a square crystal lattice whose repeat unit is a tetramer, and so is a member of perhaps the simplest class of hierarchical materials, a one-component structure possessing order on two length scales (that of the monomer and that of the tetramer).…”

Direct observation of kinetic traps associated with structural transformations leading to multiple pathways of S-layer assembly

“…S3). As shown by the height profile, the average height of the tall domains is 8-9 nm, which corresponds to a single layer of S-layer tetramers (17), and the average height difference between the short and tall domains is consistently approximately 2-3 nm. A native protein gel analysis confirms that the purified protein has a homogeneous native state in solution (SI Appendix, Fig.…”

The energetics of prenucleation clusters in lattice solutions

“…8 Many phases also appear to precipitate by twostep processes, whereby liquid-like droplets form first and crystals subsequently form within these droplets. [9][10][11][12] Finally, the structural properties of very small clusters have been called into question, with several studies suggesting that the smallest clusters may be polymeric or liquid-like in structure, rather than being compact crystal-like objects. 12,13 Taken together, these behaviors suggest that the energy landscape for cluster formation can be significantly more complex than imagined in the early formulations of CNT, especially when clusters are very small.…”