Hydrazide
Mimics for Protein Lysine Acylation To Assess
Nucleosome Dynamics and Deubiquitinase Action
- Publication date
- Publisher
Abstract
A range of acyl-lysine (acyl-Lys)
modifications on histones and
other proteins have been mapped over the past decade but for most,
their functional and structural significance remains poorly characterized.
One limitation in the study of acyl-Lys containing proteins is the
challenge of producing them or their mimics in site-specifically modified
forms. We describe a cysteine alkylation-based method to install hydrazide
mimics of acyl-Lys post-translational modifications (PTMs) on proteins.
We have applied this method to install mimics of acetyl-Lys, 2-hydroxyisobutyryl-Lys,
and ubiquityl-Lys that could be recognized selectively by relevant
acyl-Lys modification antibodies. The acyl-Lys modified histone H3
proteins were reconstituted into nucleosomes to study nucleosome dynamics
and stability as a function of modification type and site. We also
installed a ubiquityl-Lys mimic in histone H2B and generated a diubiquitin
analog, both of which could be cleaved by deubiquitinating enzymes.
Nucleosomes containing the H2B ubiquityl-Lys mimic were used to study
the SAGA deubiquitinating module’s molecular recognition. These
results suggest that acyl-Lys mimics offer a relatively simple and
promising strategy to study the role of acyl-Lys modifications in
the function, structure, and regulation of proteins and protein complexes