Parallel Chemoselective Profiling for Mapping Protein Structure

Abstract

Highlights d Deep mutational scanning data are used to guide the generation of protein variants d Parallel chemoselective labeling of variants allows mapping of solvent accessibility d Differences in quantified labeling rates are used to infer structure and dynamics

Synthesis of Guaianolide Analogues with a Tunable α-Methylene−γ-lactam Electrophile and Correlating Bioactivity with Thiol Reactivity

“…Moreover, our new approach, ALARM MSPS, provides a method for evaluation of compounds that may function as reversible covalent inhibitors. Recently, a similar assay with isotopic iodoacetamide (IAD) was reported to study protein dynamics, which supports our approach …”

“…The stereochemistry of the carbon adjacent to the lactam carbonyl was not assigned. 1 (3aR*,9bS*)-3,6-Dimethyl-9-phenyl-1,3a,4,5,7,9b-hexahydro-2H-azuleno [4,5-b]pyrrole-2,8(3H)-dione (59). 59 was prepared according to General Procedure I. Allene-yne 57 (17 mg, 0.061 mmol), rhodium dicarbonyl chloride dimer (3 mg, 0.0061 mmol), and toluene (6 mL) yielded lactam 59 (14 mg, 77% yield) as a white solid after purification by column chromatography (gradient elution with 50−100% ethyl acetate in hexanes).…”

“…Recently, a similar assay with isotopic iodoacetamide (IAD) was reported to study protein dynamics, which supports our approach. 59 The experimental workflow of the ALARM MSPS assay involves the following steps (Figure 6, panel A): (1) incubation of the La antigen, containing the reactive cysteine C245, with test compounds 51 or S17; (2) addition of IAD to carbamidomethylate cysteine residues that were not adducted;…”

Triazolinedione protein modification: from an overlooked off-target effect to a tryptophan-based bioconjugation strategy

“…21 In 2010, Barbas and co-workers reported a click like reaction for the more abundant tyrosine (Tyr, 3.3% abundance 7 ) using triazolinedione chemistry, 22 after which many applications and refinements for protein conjugation followed. 23–29 Interestingly, when exploring this powerful Tyr click reaction on Trp containing peptides, we observed a high degree of off-target labelling on Trp residues, even in aqueous buffers. In the initial paper reporting the development of the TAD tyrosine click like reaction, Barbas and co-workers reported that the TAD reaction is amino acid selective for tyrosine in aqueous buffers.…”

Profiling of the drug resistance of thousands of Src tyrosine kinase mutants uncovers a regulatory network that couples autoinhibition to catalytic domain dynamics

“…HDX-MS of Src was performed as describe in (Potter et al, 2020). Briefly, purified Src 3D (WT or W285T) or Src CD (WT and W285T) was diluted to 0.2 mg/mL in protein dilution buffer (50 mM HEPES, pH 7.8, 150 mM NaCl, 1 mM DTT, 5% glycerol).…”