Tailoring protein nanomechanics with chemical reactivity

Abstract

The nanomechanical properties of elastomeric proteins determine the elasticity of a variety of tissues. A widespread natural tactic to regulate protein extensibility lies in the presence of covalent disulfide bonds, which significantly enhance protein stiffness. The prevalent in vivo strategy to form disulfide bonds requires the presence of dedicated enzymes. Here we propose an alternative chemical route to promote non-enzymatic oxidative protein folding via disulfide isomerization based on naturally occurring small molecules. Using single-molecule force-clamp spectroscopy, supported by DFT calculations and mass spectrometry measurements, we demonstrate that subtle changes in the chemical structure of a transient mixed-disulfide intermediate adduct between a protein cysteine and an attacking low molecular-weight thiol have a dramatic effect on the protein's mechanical stability. This approach provides a general tool to rationalize the dynamics of S-thiolation and its role in modulating protein nanomechanics, offering molecular insights on how chemical reactivity regulates protein elasticity.

The force-dependent mechanism of DnaK-mediated mechanical folding

“…Similarly, binding of vinculin to α-catenin also requires mechanical unfolding, making the binding site accessible for binding ( 41 ). From the mechanochemistry perspective, force-exposure of structurally buried disulfide bonds or reactive side chains triggers a variety of posttranslational modifications that regulate protein folding and elasticity ( 42 , 43 ). Here, we add chaperone binding to mechanically exposed cryptic sequences as a key regulator of mechanical folding.…”

Single‐Molecule Observation of Intermediates in Bioorthogonal 2‐Cyanobenzothiazole Chemistry

“…Cys8 . [a] Data for pK a values are taken from references [25,[40][41][42][43].Different pK a values for the same compound from different studies are included.…”

Forcing the reversibility of a mechanochemical reaction

“…In general, in-vivo reformation of disulfide bonds is typically catalyzed by dedicated enzymes 50 , 51 , often employing the low p K a value of the catalytic cysteine as a general mechanism to foster reactivity 52 . However, other non-enzymatic oxidative pathways are slowly coming to light, involving sulfenic acid 40 , 53 or small thiols 54 , 55 , such as the ones studied here. In this vein, our experiments revealed that reducing the disulfide bonds by poor nucleophiles leads to unstable and reactive mixed disulfide products that can readily condense with the neighboring protein thiolate to form a stiff disulfide bond, yielding a properly refolded and mechanically rigid protein form.…”

“…Alternatively, the solvent-exposed mixed disulfide bond can react with free thiols in solution (Supplementary Fig. 25 ), yielding a fully reduced protein that can refold into a native conformation that exhibits intermediate mechanical properties between the compliant, misfolded protein and the stiff, fully oxidized form 54 . The proportion of refolded yet reduced protein experimentally measured in the presence of each distinct thiol nucleophile follows surprisingly well thermodynamic considerations based on DFT calculations of the mixed disulfide and the thiol/cysteine homodimers (Methods, and Supplementary Fig.…”