Biophysical Studies of LLPS and Aggregation of TDP-43 LCD

Abstract

Growing evidence indicates that liquid–liquid phase separation (LLPS), a phenomenon whereby transient, weak interactions can facilitate self-assembly of proteins into liquid-like droplets and can contribute to the formation of amyloid fibrils. Such an observation has posited that LLPS and the associated formation of membrane-less organelles in the cell can contribute to protein aggregation in neurodegenerative disease. In this chapter, we describe methods for performing biophysical studies on the transactive response DNA-binding protein of 43 kDa (TDP-43), a protein that forms aggregates in amyotrophic lateral sclerosis and frontotemporal lobar degeneration. We describe purification of the disordered low-complexity domain (LCD) of TDP-43 and provide a methodology for studying the protein’s behavior using site-directed spin labeling coupled with electron paramagnetic resonance. We additionally discuss visualization of TDP-43 LCD liquid droplets and methods for quantifying LLPS and aggregation into amyloid fibrils.