Installation of electrophiles onto the C-terminus of recombinant ubiquitin and ubiquitin-like proteins

Abstract

Ubiquitin and related Ubiquitin-like proteins (Ubls) influence a variety of cellular pathways including protein degradation and response to viral infections. The chemical interrogation of these complex enzymatic cascades relies on...

Semisynthetic LC3 Probes for Autophagy Pathways Reveal a Noncanonical LC3 Interacting Region Motif Crucial for the Enzymatic Activity of Human ATG3

“…We recently reported access to Ubl proteins bearing thiol-reactive electrophiles at their C-terminus by chemoselective acylation of recombinant protein-hydrazides with carboxylic acid anhydrides . The lack of ABPs for autophagy inspired us to prepare GABARAP ABPs using this protocol.…”

“…We recently reported access to Ubl proteins bearing thiol-reactive electrophiles at their C-terminus by chemoselective acylation of recombinant protein-hydrazides with carboxylic acid anhydrides. 22 The lack of ABPs for autophagy inspired us to prepare GABARAP ABPs using this protocol. GABARAP was expressed as an Mxe GyrA intein fusion to Cterminal glycine deletion (ΔG116) to conserve the atomic register at the C-terminus after the introduction of electrophilic groups.…”

“…Herein, we report the facile preparation of GABARAP and LC3A activity-based probes (ABPs) using a recently established hydrazide acylation protocol. , Access to these ABPs was critical for the identification of an unknown noncanonical LIR motif embedded in an unusual β-sheet conformation in human ATG3. Further investigation of this motif with macrocyclic peptide binders, X-ray crystallography, and CRISPR-enabled in cellulo studies revealed that this LIR motif is crucial for the enzymatic function of ATG3.…”

Enzymatic Fluoromethylation as a Tool for ATP‐Independent Ligation

“…F‐methylation of GFP‐tag‐1 in the presence of a hydroxylamine‐derivatized dye (4‐nitrobenzylhydroxylamine, 20 mM), hydrazine (20 mM) or a hydroxylamine derivatized biotin (20 mM) produced more than 70 % of the corresponding conjugates ( 20 – 22 , Figure 4, Figure S32–S44). Conjugate 23 was obtained by incubation of the GFP‐hydrazide 21 with chloroacetic anhydride (1–50 mM) at pH 3.0, in adaptation of a recently published protocol for the installation of C‐terminal chloride electrophiles (Figure S44) [22] . In the absence of an efficient external nucleophile, F‐methylation of GFP‐tag‐1 resulted in transient accumulation of the F‐methyl ester ( 18 ), which, after 19 hrs, completely transformed into a species that is 18 Da lighter than unmodified GFP‐tag‐1 (GFP‐tag‐1‐H 2 O: calcd: m/z 30477.4, found: 30478, Figure S36), suggesting that the F‐methylation protein has the capacity to slowly undergo intramolecular condensation.…”

Enzymatic Fluoromethylation as a Tool for ATP‐Independent Ligation

“…Conjugate 23 was obtained by incubation of the GFPhydrazide 21 with chloroacetic anhydride (1-50 mM) at pH 3.0, in adaptation of a recently published protocol for the installation of C-terminal chloride electrophiles (Figure S44). [22] In the absence of an efficient external nucleophile, F-methylation of GFP-tag-1 resulted in transient accumulation of the F-methyl ester (18), which, after 19 hrs, completely transformed into a species that is 18 Da lighter than unmodified GFP-tag-1 (GFP-tag-1-H 2 O: calcd: m/z 30477.4, found: 30478, Figure S36), suggesting that the F- methylation protein has the capacity to slowly undergo intramolecular condensation. The same species (GFP-tag-1-H 2 O) also forms in some of the conjugation reactions described above as a minor side product.…”