Acid-induced conformational remodeling of arachin bidirectionally modulates aflatoxin B1 binding: a multi-spectroscopic and molecular simulation study

Arachin (Ara), the major storage protein in peanuts, is considered a critical bottleneck limiting the accurate detection and effective detoxification of aflatoxin B1 (AFB1) due to its strong binding affinity for this mycotoxin. However, the structure-activity relationship governing Ara-AFB1 interactions and its response to environmental factors remain poorly understood. In this study, we systematically investigated how acid treatment (pH 3.0 or 5.0) followed by neutralization to pH 8.0 alters the conformation of Ara and consequently affects its binding behavior toward AFB1. Multi-spectroscopy and molecular simulations revealed that pH 5.0 treatment followed by neutralization induced partial unfolding, increased α-helix content relative to native Ara, and exposed additional hydrophobic sites, resulting in enhanced AFB1 binding affinity. In contrast, pH 3.0 treatment caused dissociation that was not reversed upon neutralization, accompanied by extensive α-helix loss, a marked increase in random coil content, and hydrophobic core disruption, leading to decreased AFB1 binding capacity. Notably, AFB1 may be physically entrapped within acid-induced protein aggregates, which may contribute to the increased difficulty of detection and detoxification. Our findings reveal, for the first time, a bidirectional regulatory mechanism by which pH-driven conformational remodeling of Ara modulates AFB1 binding in opposite directions depending on acid intensity. This work provides mechanistic insights into matrix-associated mycotoxin behavior and offers theoretical support for AFB1 risk management in food processing environments.