Characterization of the fibrillation of pumpkin seed protein: Kinetics, structure, and fibril-forming regions

Abstract

Protein fibril formed via the self-assembly of ordered cross-β sheets have attracted considerable attention owing to their functional properties. In this study, we evaluate the fibrillation of pumpkin seed protein (PSP) under acidic heating conditions (pH 2.0, 85 °C, 0–72 h). The growth kinetics, conformational and morphological transitions, and rheological properties of the fibril are analyzed using thioflavin T (Th T) fluorescence, Fourier-transform infrared spectroscopy, circular dichroism, atomic force microscopy, and rotational rheometry. Fibril-forming regions were identified by trapped ion mobility spectrometry time-of-flight mass spectrometry. PSP fibrillation is accompanied by protein hydrolysis, yielding peptide fragments (∼10 kDa) within 24 h. A significant lag phase (0–9 h) is followed by an elongation phase (9–48 h) and subsequent plateau phase (48–60 h), with a conversion rate of 52%. Mature fibrils measuring approximately 1 μm have a height of 7 nm and a compact periodic pitch of 33.4 nm. The variation in height along the direction perpendicular to the fibril axis suggests that fibrillation occurs via a secondary nucleation mechanism. Additionally, the ratio of antiparallel to parallel β-sheets decreased with fibril growth. The solution retained its liquid-like behavior despite an increase in the storage modulus with the degree of fibrillation. The PSP contained abundant hydrophobic fibril-forming regions, with 32 unique peptides (32 unique peptides, 61.04% coverage) from 11S, and 12 unique peptides (12 unique peptides, 60.99% coverage) from 2S. This study provides valuable insights into the fibrillation dynamics of PSP and provides a foundation for its future use as a functional biocompatible material.