Abstract
The post-translational modification S-sulfenylation functions as a key sensor of oxidative stress. Yet the dynamics of sulfenic acid in proteins remains largely elusive due to its fleeting nature. Here we use single-molecule force-clamp spectroscopy and mass spectrometry to directly capture the reactivity of an individual sulfenic acid embedded within the core of a single Ig domain of the titin protein. Our results demonstrate that sulfenic acid is a crucial short-lived intermediate that dictates the protein's fate in a conformation-dependent manner. When exposed to the solution, sulfenic acid rapidly undergoes further chemical modification, leading to irreversible protein misfolding; when cryptic in the protein's microenvironment, it readily condenses with a neighbouring thiol to create a protective disulfide bond, which assists the functional folding of the protein. This mechanism for non-enzymatic oxidative folding provides a plausible explanation for redox-modulated stiffness of proteins that are physiologically exposed to mechanical forces, such as cardiac titin.
Highlights
The post-translational modification S-sulfenylation functions as a key sensor of oxidative stress
While reactive oxygen species (ROS) were originally known for oxidizing various cellular compartments and promoting aging and a broad range of pathologies, more recent evidence shows that ROS acts as signalling molecules that regulate basic cellular processes including growth, differentiation and cell migration[26,50]
It is increasingly clear that a major mechanism of redox signalling is the dynamic regulation of protein function by the chemiselective oxidation of cysteine residues
Summary
-SOH triggers disulfide bond formation. -SOH is typically induced by exposing a cysteine residue to high concentrations of hydrogen peroxide (10–107 M À 1 s À 1)[25,26]. This tendency was quantitatively verified, displaying the percentage of disulfide bond reformation as a function of the stretching force. Aldehyde formation has been long reported as a sub-product of the decomposition of -SOH under high alkali conditions[28,47,48] While both the protein conformation and the timescales sampled in the MS and singlemolecule mechanics experimental approaches are certainly different, the MS data provide excellent complementary information that illustrates the most plausible chemical processes responsible for the misfolding events captured in the nanomechanical experiments, greatly affecting the folding and elastic properties of the individual titin polyproteins. The resulting MS/MS fragmentation spectra identified, for both 24 and 55 cysteines, a shift in Dm/z 1⁄4 þ 138 Da (Fig. 5d, Supplementary Fig. 9 and Supplementary Table 2), which confirmed the presence of dimedone
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