Amyloid-Like Aggregation in Native Protein and its Suppression in the Bio-Conjugated Counterpart

Abstract

<jats:p>Prevention of protein aggregation and thus stabilization of proteins has large biological and biotechnological implications. Here we introduce Dynamic Light Scattering (DLS) and DLS-based microrheology to show how native bovine serum albumin (nBSA) forms amyloid fibrils in weakly denaturing conditions as function of time, and how stoichiometric conjugation of BSA with polymer-surfactants (PSpBSA) protects the protein form such aggregation. Employing a combination of Thioflavin-T fluorescence, Fourier transform infrared spectroscopy and other methods, we show that nBSA forms filamentous aggregates with amyloid-like structure, while PSpBSA proteins remain fully dispersed with only minor changes in their folding state, even when continuously heated for up to 5 days in denaturation conditions at 65 °C. Time-resolved DLS-based microrheology studies demonstrate that suspensions of the filamentous nBSA aggregates become viscoelastic for concentrations ≥200 <jats:italic>μ</jats:italic>M. Our results indicate that after 6 days in aggregation conditions, the elastic modulus <jats:italic>G</jats:italic>′(<jats:italic>ω</jats:italic>) of nBSA solutions went from zero initially to values of up to 3.6 Pa, indicating that the filaments become long enough to form an entangled, viscoelastic network. Interestingly, heating 200 <jats:italic>μ</jats:italic>M native BSA solutions at 65 °C for 2 days in Eppendorf tubes resulted in self-standing films rather than dispersed filaments. These films exhibited strong ThT-fluorescence intensities and a predominant <jats:italic>β</jats:italic>-sheet secondary structure in FTIR studies, suggesting that the self-standing microstructure of the film resulted from hierarchical self-assembly of the amyloid fibrils.</jats:p>