Nucleation of protein mesocrystals via oriented attachment

Abstract

Self-assembly of proteins holds great promise for the bottom-up design and production of synthetic biomaterials. In conventional approaches, designer proteins are pre-programmed with specific recognition sites that drive the association process towards a desired organized state. Although proven effective, this approach poses restrictions on the complexity and material properties of the end-state. An alternative, hierarchical approach that has found wide adoption for inorganic systems, relies on the production of crystalline nanoparticles that become the building blocks of a next-level assembly process driven by oriented attachment (OA). As it stands, OA has not yet been observed for protein systems. Here we employ cryo-transmission electron microscopy (cryoEM) in the high nucleation rate limit of protein crystals and map the self-assembly route at molecular resolution. We observe the initial formation of facetted nanocrystals that merge lattices by means of OA alignment well before contact is made, satisfying non-trivial symmetry rules in the process. As these nanocrystalline assemblies grow larger we witness imperfect docking events leading to oriented aggregation into mesocrystalline assemblies. These observations highlight the underappreciated role of the interaction between crystalline nuclei, and the impact of OA on the crystallization process of proteins.

The Non‐Classical Crystallization Mechanism of a Composite Biogenic Guanine Crystal

“…The crystal lamellae are in perfect structural registry, and the guanine composite thus resembles a mesocrystal. [ 30,47 ] We do not know if the same formation mechanism is employed in other guanine systems. However, it was recently observed that forming guanine crystals in hatchling lizards comprise ≈12 nm crystal plates, [ 21 ] which eventually merge into single crystals.…”

Molecular Mechanism of Organic Crystal Nucleation: A Perspective of Solution Chemistry and Polymorphism

“…However, the nucleation of proteins does not have to be a two-step process. Van Driesch et al observed the direct self-assembly of glucose isomerase protein molecules into polyhedral nanocrystals with surprising smooth surfaces and sharp vertices, indicating a one-step nucleation mechanism [80]. The more detailed theoretical developments of the two-step nucleation mechanism can be found here [81,82].…”

“…Recent studies show citrate altering the nucleation pathway of calcium oxalate hydrates by interacting with pre-nucleation aggregates and amorphous intermediates mainly through water incorporation and local structure ordering [158]. More recently, multiple nucleation pathways are also seen in protein systems [31,80]. Multiple nucleation pathways were previously suggested by De Yoreo [64] in explaining the nucleation behavior of an inorganic system.…”

“…The associates or aggregates as illustrated in the aforementioned systems bear the inevitable solvation effect [127,147], and desolvation of solvent molecules [148][149][150] accompanied by superamolecular reconstruction leads to the formation of a nucleus. Recent studies have revealed the detailed kinetics of orientated attachment and the importance of desolvation on particle aggregation during crystal nucleation [80,151]. The large barrier of this step results in the rare event of crystal nucleation, which may also produce simultaneous nucleation of two crystal structures (polymorphs) [152][153][154], i.e., concomitant crystallization of polymorphs when the nucleation barrier for two polymorphs were comparable.…”

Multistep Crystallization of Pharmaceutical Amorphous Nanoparticles via a Cognate Pathway of Oriented Attachment: Direct Evidence of Nonclassical Crystallization for Organic Molecules

“…However, the classical nucleation theory (CNT) fails to explain the crystallization mechanism of many organic molecules. , The crystallization of organic molecules is more complicated than that of inorganic materials because the conformational degrees of freedom in molecular systems introduce dynamical factors that render them distinct from inorganic systems . Nonclassical crystallization, also known as crystallization by particle attachment, , is now recognized as a predominant crystallization pathway for many organic compounds ranging from small molecules to proteins. − Amorphous precursor phases are formed as metastable intermediates via two-step nucleation before substantial crystallization. − Apart from the conventional monomer-by-monomer crystal growth mechanism, small nanocrystals can grow via a so-called “oriented attachment” pathway, − which refers to the spontaneous self-organization and fusion of adjacent nanocrystals by sharing a common crystallographic orientation . Oriented attachment reduces the surface energy and thus thermodynamically lowers the enthalpy of the system .…”