Biophysical Society Thematic Meeting| Lima 2019

Revisiting the Central Dogma of Molecular Biology at the Single-Molecule Level

Saturday Speaker Abstracts

DIRECT OBSERVATION OF SUBSTRATE TRANSLOCATION AND CONFORMATIONAL DYNAMICS IN THE 26S PROTEASOME Erik Jonsson 1 ; Jared Bard 2 ; Andreas Martin 1 ; 1 UC Berkeley, Molecular & Cell Biology, Berkeley, CA, USA

2 The University of Chicago, Biochemistry & Molecular Biology, Chicago, IL, USA The 26S proteasome is a molecular machine that selectively destroys proteins targeted for degradation. It does so by recognizing ubiquitin modifications attached to substrate proteins, followed by engagement of the polypeptide and its mechanical unfolding and tanslocation into a proteolytic core. The precise nature of how the proteasome reads the ubiquitin code, generates mechanical force, and processes its substrates, as well as the conformational landscape it explores during degradation, has remained elusive. Here we employ single-molecule FRET to observe the interactions between fluorescently labeled proteasomes and substrates, and also study the intra-molecular conformational dynamics of dual labeled proteasomes. In order to circumvent previous limitations of working with endogenous holoenzymes, we reconstitute proteasomes from subcomplexes that were recombinantly produced with non-standard amino acid reprograming for fluorescent dye labeling. Fluorescently labeled substrates are enzymatically modified with ubiquitin chains, and we can observe their molecular trajectories as they pass through the various stages of proteasomal engagement, translocation, unfolding, and deubiquitination. By monitoring the conformational state of the proteasome using a FRET pair designed to capture the transition between substrate-free and -engaged states, we observe a highly dynamic holoenzyme at various stages of substrate processing. These observations offer important new insights into the operation of the proteasome and its underlying mechanisms for ATP-dependent degradation.

26