Graduate Student University of British Columbia, Canada
There is a growing interest in developing functional amyloid as a novel biomaterial (e.g., for use in encapsulation, gellation and bioscaffolding) owing to the unique physiochemical properties of amyloid fibrils. Legume seed storage proteins, mainly 7S and 11S globulins, could be a sustainable and cost-effective input for plant-based functional amyloid. Crude extracts of legume proteins can be induced to fibrillate by heating at acidic pH, although mechanistic details are currently lacking. In this work, we sought to identify which proteins and hydrolyzed peptide fragments form the amyloid fibrils, and then link the fibril core regions with fibrillation kinetics and morphology. Pea and soy proteins were selected as models. Crude proteins were fractionated by differential precipitation and anion exchange chromatography into 7S- and 11S-enriched fractions. 7S, 11S globulins and crude extracts were incubated at pH 2, 80°C. The fibrillation kinetics were determined using ThT fluorescence; protein hydrolysis during fibrillation was monitored by SDS-PAGE; fibril morphologies were characterized by TEM, and fibril core composition was determined using LC-MS/MS. Partially purified 7S and 11S globulins displayed faster fibrillation kinetics than crude proteins. Pea 7S globulin displayed no decipherable lag phase and the fastest growth kinetics. Fibrils formed by pea proteins were mainly straight and long, while soy fibrils were more worm-like. Mass spectrometry identified over 150 unique peptides in each sample, with pea 7S exhibiting the most abundant fibril-forming regions representing half its sequence. 7S globulins from both pea and soy possess a higher fibril-forming capacity than 11S. Soy and pea protein-enriched fractions possess copious fibril-forming peptides compared to previously studied β-lactoglobulin and lysozyme, making them suitable alternatives to animal proteins for creating functional amyloid.