Monitoring Oxidative Folding of a Single Protein Catalyzed by the Disulfide Oxidoreductase DsbA

Kahn, Thomas B.; Fernández, Julio M.; Pérez-Jiménez, Raúl

doi:10.1074/jbc.m115.646000

Cited by 28 publications

(35 citation statements)

References 44 publications

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“…Taken together, these data suggest that PDI re-introduces the disulfide bond after the first step of protein folding, collapse of the polypeptide backbone to a high entropy molten globule state. This confirms the conclusions, that protein folding drives disulfide formation, inferred from previous single molecule data that lacked resolution to resolve the individual folding steps (Kahn et al, 2015;Kosuri et al, 2012). Furthermore the shift in folding probability to higher forces suggests a bona fide chaperone activity of the PDI domain with extended polypeptides, indicating a pathway for disulfide bond introduction into titin even under the resting forces it experiences in muscle.…”

Section: Protein Disulfide Isomerase (Pdi) Mediates Disulfide Reformasupporting

confidence: 87%

The Mechanical Power of Protein Folding

Eckels

Haldar

Tapia‐Rojo³

et al. 2018

Preprint

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The delivery of mechanical power, a crucial component of animal motion, is constrained by the universal compromise between force and velocity of its constituent molecular systems. Here we demonstrate a switchable power amplifier in an Ig domain of the massive muscle protein titin.Titin is composed of many tandem repeats of individually foldable Ig domains, which unfold and extend during muscle stretch and readily refold when the force on titin is quenched during a contraction. Cryptic cysteine residues are common in elastic proteins like titin where they can oxidize to form intra-domain disulfide bonds, limiting the extensibility of an unfolding domain.However, the functional significance of disulfide-bonds in titin Ig domains remains unknown and may be fundamental to muscle mechanics. Here we use ultra-stable magnetic tweezers force spectroscopy to study the elasticity of a disulfide bonded modular titin protein operating in the physiological range, with the ability to control the oxidation state of the protein in real time using both organic reagents and oxidoreductase enzymes. We show that presence of an oxidized disulfide bond allows the parent Ig domain to fold at much higher forces, shifting the midpoint folding probability from 4.0 pN to 12.8 pN after formation. The presence of disulfide bonds in titin regulates the power output of protein folding in an all-or-none manner, providing for example at 6.0 pN, a boost from 0 to 6,000 zeptowatts upon oxidation. At this same force, single molecular motors such as myosin are typically stalled and perform little to no work. We further demonstrate that protein disulfide isomerase (PDI) readily reintroduces disulfide bonds into unfolded titin Ig domains, an important mechanism for titin which operates under a resting force of several pN in vivo. Our results demonstrate, for the first time, the functional significance of disulfide bonds as potent power amplifiers in titin and provide evidence that protein folding can generate substantial amounts of power to supplement the myosin motors during a contraction.

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Section: Protein Disulfide Isomerase (Pdi) Mediates Disulfide Reformasupporting

confidence: 87%

The Mechanical Power of Protein Folding

Eckels

Haldar

Tapia‐Rojo³

et al. 2018

Preprint

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“…In this sense, -SOH emerges as a key molecular player able to rescue the protein from misfolding by forming a protective disulfide bond that allows the protein to successfully refold. This kinetic scheme seems to differ from the enzyme-mediated oxidative folding findings catalysed by PDI40 and DsbA61, whereby the same I27 protein mutant was able to refold on its own while keeping the catalytic enzyme attached through a mixed disulfide conformation before the disulfide bond was successfully reformed at a later stage, the overall kinetics being largely controlled by the off-rate dynamics of the catalytic enzyme.…”

Section: Discussionmentioning

confidence: 84%

Protein S-sulfenylation is a fleeting molecular switch that regulates non-enzymatic oxidative folding

2016

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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.

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“…Here we have provided an empiric description and measurement of a chaperone-like behavior of E. coli DsbA, which acts as a mechanical chaperone to accelerate refolding to the native state and to shift the folding probability under force. Previous studies on a disulfide containing protein using AFM based force spectroscopy demonstrated that DsbAcatalyzed oxidative folding occurs ~3-fold faster than folding of the reduced protein [26] . This acceleration in the folding rate with DsbA versus reduced substrate is likely an indirect indication of the chaperone activity of the enzyme, however these studies by AFM lacked sensitivity required to resolve folding events at low force, which we can now directly observe in the magnetic tweezers.…”

Section: Discussionmentioning

confidence: 99%

“…Protein L notably lacks any cysteine residues and thus allows us to probe the mechanism of DsbA chaperone activity independent from its oxidoreductase activity. We previously studied DsbA-catalyzed oxidative folding on a disulfidecontaining protein using AFM-based force spectroscopy (42). In those studies, it was discovered that DsbA-catalyzed oxidative folding occurs ~3-fold faster than folding of the reduced protein.…”

Section: Discussionmentioning

confidence: 99%

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DsbA is a redox-switchable mechanical chaperone

Eckels¹,

Chaudhuri²,

Chakraborty³

et al. 2018

Preprint

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Force is a crucial protein denaturant in cells, occurring during processes including translation, degradation, and translocation. In bacteria, many toxins and virulence factors pass through a translocon pore as unfolded polypeptides en route to periplasmic and extracellular compartments.DsbA is a ubiquitous bacterial oxidoreductase that associates with substrates during and after translocation, yet its involvement in protein folding and translocation remains an open question.Here, using magnetic tweezers-based single molecule force spectroscopy, we characterize a mechanical chaperone activity of DsbA on a cysteine-free domain from the protein L superantigen. Interaction of DsbA with unfolded protein L substrates prominently increases the fraction of time that protein L spends in its native folded state. This chaperone activity is tuned by the oxidation state of DsbA; oxidized DsbA is a strong promoter of folding, but the effect is weakened by reduction of the catalytic CXXC motif. We further localize the chaperone binding site of DsbA using a seven residue peptide which effectively blocks the chaperone activity. We calculated that DsbA assisted folding of proteins in the periplasm generates enough mechanical work to decrease the ATP consumption needed for periplasmic translocation by up to 33%. In turn, pharmacologic inhibition of this chaperone activity may open up a new class of antivirulence agents.

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Monitoring Oxidative Folding of a Single Protein Catalyzed by the Disulfide Oxidoreductase DsbA

Cited by 28 publications

References 44 publications

The Mechanical Power of Protein Folding

The Mechanical Power of Protein Folding

Protein S-sulfenylation is a fleeting molecular switch that regulates non-enzymatic oxidative folding

DsbA is a redox-switchable mechanical chaperone

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