Nucleation of protein mesocrystals via oriented attachment

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.

A sweet protein monellin as a non-antibody scaffold for synthetic binding proteins

Synthetic binding proteins that have the ability to bind with molecules can be generated using various protein domains as non-antibody scaffolds. These designer proteins have been used widely in research studies, as their properties overcome the disadvantages of using antibodies. Here, we describe the first application of a phage display to generate synthetic binding proteins using a sweet protein, monellin, as a non-antibody scaffold. Single-chain monellin (scMonellin), in which two polypeptide chains of natural monellin are connected by a short linker, has two loops on one side of the molecule. We constructed phage display libraries of scMonellin, in which the amino acid sequence of the two loops is diversified. To validate the performance of these libraries, we sorted them against the folding mutant of the green fluorescent protein variant (GFPuv) and yeast small ubiquitin-related modifier. We successfully obtained scMonellin variants exhibiting moderate but significant affinities for these target proteins. Crystal structures of one of the GFPuv-binding variants in complex with GFPuv revealed that the two diversified loops were involved in target recognition. scMonellin, therefore, represents a promising non-antibody scaffold in the design and generation of synthetic binding proteins. We termed the scMonellin-derived synthetic binding proteins “SWEEPins.”

Nonclassical nucleation of protein mesocrystals via oriented attachment

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 which in turn become the building blocks of a next-level assembly process driven by oriented attachment (OA). As it stands, OA has not been observed for proteins. Here we employ 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. The OA mechanism yields crystal morphologies that are not attainable through conventional crystallization routes. Based on these insights we revisit a system of protein crystallization that has long been classified as non-classical, but our data is in direct conflict with that conclusion supporting a classical mechanism that implicates OA. These observations raise further questions about past conclusions for other proteins and illustrate the importance of maturation stages after primary nucleation has taken place.

Expanding the space of protein geometries by computational design of de novo fold families

Naturally occurring proteins use a limited set of fold topologies, but vary the precise geometries of structural elements to create distinct shapes optimal for function. Here we present a computational design method termed LUCS that mimics nature’s ability to create families of proteins with the same overall fold but precisely tunable geometries. Through near-exhaustive sampling of loop-helix-loop elements, LUCS generates highly diverse geometries encompassing those found in nature but also surpassing known structure space. Biophysical characterization shows that 17 (38%) out of 45 tested LUCS designs were well folded, including 16 with designed non-native geometries. Four experimentally solved structures closely match the designs. LUCS greatly expands the designable structure space and provides a new paradigm for designing proteins with tunable geometries customizable for novel functions.One Sentence SummaryA computational method to systematically sample loop-helix-loop geometries expands the structure space of designer proteins.