Investigation of the Conformational State of the 26S Proteasome Using FRET

Abstract

The 26S proteasome is a multiprotein complex in eukaryotic organisms that mechanically degrades misfolded, mutated, and obsolete proteins. As a result, proteasome function, or the lack thereof, has implications for neurodegenerative diseases. As it unfolds and degrades protein substrates, the proteasome undergoes a series of conformational changes that have been seen in CryoEM structures, proceeding from the s1, or substrate‐accepting state, to the s3 and s3‐like substrate processing and translocating states. In this research, the 19S particle of yeast proteasome was tagged with the fluorescent proteins (FPs) mTurquoise and mNeonGreen. The distance‐dependent spectroscopic technique Förster Resonance Energy Transfer (FRET) was employed to track the conformational changes associated with proteasomal function. The non‐hydrolyzable ATP analog ATPγS increased the observed FRET signal relative to ATP, consistent with the known ability of ATPγS to bias the proteasome’s conformation toward the s3 or s3‐like states.Support or Funding InformationJRC is a Beckman Scholar. This material is based upon work supported by the National Science Foundation under Grant No. 1515229 to DAK.