Identifying the Mechanism for Amyloid Formation Using Single-Molecule Spectroscopy

We are investigating the mechanism for the initial stages of protein self-assembly leading to amyloid growth using single molecule spectroscopy (SMS). $\beta $-lactoglobulin ($\beta $-LG) has been shown to form amyloid under denaturing conditions and has been chosen as a model protein for this study. Initial bulk experiments have been performed utilizing dynamic light scattering along with steady state and time-resolved fluorescence of conformationally sensitive fluorophores, and a preliminary mechanism of amyloid growth has been formulated. However, SMS directly identifies critical intermediates that may only be hypothesized by bulk experiments. A single molecule imaging experiment utilizing incubated samples of mono-labeled TMR-($\beta $-LG) has been designed to count number of precursor monomers per aggregate species by counting the number of photobleaching steps required to extinguish a single particle's fluorescence. The time evolution of the particle number distribution is fit to a kinetic model representing a mechanism of amyloid growth. Results of bulk and single-molecule experiments will be discussed.