title: Macromolecular crowding modulates the kinetics and morphology of amyloid self-assembly by β-lactoglobulin.
abstract: The formation of amyloid fibrils by β-lactoglobulin in the presence of GndHCl has been monitored by using thioflavin T (ThT) fluorescence, Congo Red and transmission electron microscopy (TEM). Large quantities of aggregated protein are formed by incubating β-lactoglobulin in 2M GndHCl at room temperature and pH 7.0 for about 20 days. The kinetics of fibrillation process can be described by the lag time for formation of stable nuclei (nucleation) and the apparent rate constant for the growth of fibrils (elongation). Moreover, the effects of macromolecular crowding agents, Dextran 70 and polyethylene glycols (PEG), on the amyloid formation of β-lactoglobulin at pH 7.0 are studied. The results show that the increase in macromolecular crowding agent concentrations results in shorter lag time and faster growth of fibrils. It proves that macromolecular crowding can effectively accelerate the fibril formation of β-lactoglobulin at neutral pH. At the same time, it can be observed that the amplitude of the ThT fluorescence intensity decreases as the Dextran 70 concentration is increased. The observation suggests that the yield of amyloid fibrils is significantly reduced by the addition of macromolecular crowding agents. The conclusion is further confirmed by the transmission electron microscopy. In addition, the results of transmission electron microscopy also indicate that macromolecular crowding can alter the fibril morphology of β-lactoglobulin. In brief, our findings demonstrate that the effects of macromolecular crowding are essential to the understanding of protein amyloid self-assembly occurring in vivo.
authors.affiliation: Department of Physics, Science of College, Nanjing Agricultural University, Nanjing 210095, China. mabaoliang@njau.edu.cn
authors.forename: Baoliang
authors.initials: B
authors.lastname: Ma
authors.forename: Jinbing
authors.initials: J
authors.lastname: Xie
authors.forename: Liangshu
authors.initials: L
authors.lastname: Wei
authors.forename: Wei
authors.initials: W
authors.lastname: Li
mesh_terms: D000682:Amyloid / Q000737:chemistry* / Q000648:ultrastructure; D000818:Animals; D052160:Benzothiazoles; D002417:Cattle; D003224:Congo Red; D005456:Fluorescent Dyes / Q000737:chemistry; D019791:Guanidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007782:Lactoglobulins / Q000737:chemistry* / Q000648:ultrastructure; D046529:Microscopy, Electron, Transmission; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D013844:Thiazoles / Q000737:chemistry
chemical_list: D000682:Amyloid; D052160:Benzothiazoles; D005456:Fluorescent Dyes; D007782:Lactoglobulins; D013844:Thiazoles; C009462:thioflavin T; D003224:Congo Red; D019791:Guanidine