Determination of Proteasomal Unfolding Ability

Abstract

We use an in vitro degradation assay with a model substrate to assess proteasomal unfolding ability. Our substrate has an unstructured region that is the site of ubiquitination, followed by an easy-to-unfold domain and a difficult-to-unfold domain. Degradation proceeds through the unstructured and easy-to-unfold domains, but the difficult-to-unfold domain can be degraded completely or, if the proteasome stalls, can be released as a partially degraded fragment. The ratio between these two possible outcomes allows us to quantify the unfolding ability and determine how processively the proteasome degrades its substrates.

Keywords: ATP-dependent protease; Proteasome; Protein Degradation; Protein Unfolding; Ubiquitin-proteasome system.