Studying the formation and properties of β-lactoglobulin (βlg) and β-casein (βcn) nanofibrils using atomic force microscopy

Using two atomic force microscopy (AFM) modes, Quantitative Nanomechanical Mapping (QNM) and force spectroscopy, we investigated the structure, process of formation, and mechanical properties of βlg and βcn fibers. βlg required 1 day of heating at 80 C, pH 2 to form fibrils in solution, while βcn required 3 days of heating to form fibrils in similar conditions. From AFM images, the proteins' change from native to fibril form was observed. Using QNM, an average modulus of 4.3 GPa was measured for βlg fibers and 3.1 GPa for βcn fibers. A persistence length (Lp) of 920 nm was determined by measuring the end to end distance and contour length of βlg fibers; for βcn fibrils an Lp of 2200 nm was determined. The average rupture length from performing forced unfolding of βlg fibrils was found to correspond to the unfolded length of native βlg monomers, but not in the case of βcn fibrils. From our data, hypotheses are made about the relation between native and fibrillar structures of the protein. This work shows that AFM can probe the structure and properties of amyloid fibrils. Understanding the formation and properties of these fibrils is important to unraveling their biological role and also for applications in nanotechnology and material science.