Heat-induced fibril assembly of vicilin at pH2.0: Reaction kinetics, influence of ionic strength and protein concentration, and molecular mechanism

Although the heat-induced fibril assembly of legume 7S/11S globulins at pH 2.0 has been reported, the kinetics and molecular mechanism of the assembly are still not fully understood. In this paper, the reaction kinetics, and dependence of fibril assembly on protein concentration and ionic strength, as well as the changes in fibril morphology of three selected vicilins (from kidney, mung and red beans; denoted as Vk, Vm and Vr, respectively) were investigated and compared by dynamic laser scattering, Thioflavin T fluorescence, and atomic force microscopy. The polypeptide hydrolysis, zeta-potential, and conformation changes during the assembly were also characterized, with the aim to unravel the molecular mechanism of the assembly. The results indicated that Vk exhibited a much higher ability to form highly ordered fibrils than Vm (or Vr), though among the three vicilins, Vm exhibited the highest potential. The differences were closely associated with the differences in their susceptibility of the polypeptides to acid hydrolysis, and more basically, amino acid composition. The amount of the formed protofibrils or fibrils in all the vicilins progressively increased with increasing protein concentration, but their size seemed to be unaffected. The fibril assembly of Vk could be greatly improved by electrostatic screening, in an ionic strength dependent way, while that of the other two vicilins was only slightly affected. This further confirmed that Vk exhibited a much better ability to assemble into fibrils. The fibril assembly of vicilins involves at least four steps: thermal denaturation and aggregation (to form amorphous aggregates), polypeptide hydrolysis of the aggregates, initiation of fibril assembly (or formation of protofibrils), and fibril elongation (to form highly ordered fibrils) or chain termination. The findings would be of great importance for understanding the molecular mechanism of fibril assembly of oligomeric proteins from plant sources.