Improved Protein Semi-Synthesis Enables Biophysical Studies of Thioamide Destabilization of β-Sheet Interactions.
Improved Protein Semi-Synthesis Enables Biophysical Studies of Thioamide Destabilization of β-Sheet Interactions.
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Philadelphia, US · Author affiliation
Department of Chemistry, School of Arts and Sciences, University of Pennsylvania, 231 S. 34th Street, Philadelphia, Pennsylvania19104, United States.Location evidence
Shizuoka, JP · Author affiliation
Department of Engineering, Graduate School of Integrated Science and Technology, Shizuoka University, 3-5-1 Johoku, Hamamatsu, Shizuoka432-8561, Japan.Location evidence
Hamamatsu, JP · Author affiliation
Department of Engineering, Graduate School of Integrated Science and Technology, Shizuoka University, 3-5-1 Johoku, Hamamatsu, Shizuoka432-8561, Japan.Location evidence
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Original abstract
Thioamides are natural post-translational modifications of the peptide backbone and can be introduced synthetically to probe protein folding or functionalize peptides for translational applications. In this work, we demonstrate that thioamide-containing peptides with C-terminal thioesters can be efficiently generated using Knorr pyrazole activation and used in subsequent native chemical ligation reactions to generate thioamide-containing proteins. We compare this method to acyl azide activation and find that both routes provide similar yields. We also investigate ultrasound-mediated desulfurization of the ligation site cysteine for potential advantages over chemical radical initiators. Scaling up our syntheses allows us to study thioamide perturbations to the β-sheet region of the B1 domain of protein G (GB1) as well as β-strand interactions in amyloid fibrils of the Parkinson's disease protein α-synuclein. In both contexts, we observe dramatic destabilization of the β-sheet networks, manifested in decreased GB1 thermal stability and altered folding and slowed aggregation of α-synuclein. These findings illustrate the impact that a single atom substitution can have on cooperative hydrogen-bonding networks and prompt future study of both systems.