Structural and Biophysical Characterisation of Seed Storage Proteins

The growing global population and the need to reduce greenhouse gas emissions necessitate the adoption of sustainable, plant-based dietary proteins. Rapeseed (Brassica napus) presents a significant opportunity in this context. This major global oilseed crop, provides a protein-rich meal as a co-product, which is currently underutilized as animal feed. The seed storage proteins (SSPs) of rapeseed have high contents of essential amino acids, making them nutritionally valuable. However, significant hurdles prevent their widespread application human food, including the presence of compounds causing bitterness and astringency, anti-nutritional factors, and concerns regarding the allergenicity of the SSPs. This thesis focuses on molecular descriptions of the main SSPs of rapeseed, cruciferin and napin, in an effort to advance our knowledge of their properties, structures, and interactions. By investigating and presenting basic properties of rapeseed SSPs, we aim to accelerate their use in the food industry.

I present a cryo-EM structure of hexameric, plant-derived cruciferin determined at 1.83 Å resolution. This structure reveals evidence of compositional heterogeneity, arising from the coexistence of multiple isoforms of the monomeric subunit. Grouping of the particles at monomer-level by iterative 3D classifications, suggests that these isoforms can be separated into three groups, and structures of three distinct isoforms can be refined. When taking into account the error-rates of the classifier, the apparent isoform composition aligns perfectly with an independent estimate based on quantitative LC-MS, thus supporting a quantitative interpretation of the monomer classification approach. Comparing the prevalence of different hexameric assemblies formed from the three distinguishable groups of isoforms, reveals that heterohexameric complexes form in a nearly random manner, with only subtle preferences between interacting isoforms.

Another aim of this thesis has been the characterisation of recombinantly produced rapeseed SSPs in efforts towards establishing these as effective model systems for studies of the real, natural proteins. Such systems could provide a foundational framework for future studies aimed at elucidating the structure-function relationships of rapeseed SSPs. Structural characterisation by a combination of cryo-EM and X-ray crystallography of recombinantly produced rapeseed procruciferin reveals multiple surprises regarding the assembly-behaviour of this pre-cursor protein. While usually considered a trimer, procruciferin is found to form a ’misaligned hexamer’ from asymmetric association between two trimers in the absence of salt. This assembly thus distinguishes itself from the hexamer formed from mature, plant-derived cruciferin, which possesses a six-fold symmetry. Further, in the presence of salt (500 mM NaCl), the same procruciferin forms a dodecamer with tetrahedral symmetry, by the association of four trimers.

Finally, molecular dynamics simulations are applied in an investigation of the interaction between napin and a hydrophobic interface, modelled by a biphasic system of water and octane. The adsorption of two isoforms of napin to the interface are compared across multiple simulations, revealing signs of a specific adsorption orientation of the 2SSI_BRANA isoform.