Force-dependent chemical kinetics of disulfide bond reduction observed with single-molecule techniques

Wiita, Arun P.; Ainavarapu, Sri Rama Koti; Huang, Hongtai; Fernández, Julio M.

doi:10.1073/pnas.0511035103

Cited by 328 publications

(390 citation statements)

References 47 publications

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“…Protein Expression and Purification-Preparation of (I27 SS ) 8 polyprotein was prepared as described previously (18,24) with a few differences. Two residues were mutated in the I27 module using a QuikChange site-directed mutagenesis kit (Stratagene) to engineer a buried intracellular disulfide bond.…”

Section: Methodsmentioning

confidence: 99%

Force-Clamp Spectroscopy Detects Residue Co-evolution in Enzyme Catalysis

Pérez-Jiménez

Wiita

Rodríguez-Larrea

et al. 2008

Journal of Biological Chemistry

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Understanding how the catalytic mechanisms of enzymes are optimized through evolution remains a major challenge in molecular biology. The concept of co-evolution implicates that compensatory mutations occur to preserve the structure and function of proteins. We have combined statistical analysis of protein sequences with the sensitivity of single molecule forceclamp spectroscopy to probe how catalysis is affected by structurally distant correlated mutations in Escherichia coli thioredoxin. Our findings show that evolutionary anti-correlated mutations have an inhibitory effect on enzyme catalysis, whereas positively correlated mutations rescue the catalytic activity. We interpret these results in terms of an evolutionary tuning of both the enzyme-substrate binding process and the chemistry of the active site. Our results constitute a direct observation of distant residue co-evolution in enzyme catalysis.The acquisition of adequate activity by an enzyme is the result of natural selection through mutagenesis (1). Mutations that represent a functional advantage may prevail, reflecting not only the optimization of function but also the emergence of a new enzymatic mechanism (1-4). Disadvantageous mutations can be compensated by mutations in other positions (5-8), as proposed by the concept of co-evolution. This hypothesis suggests that pairs of positions in the sequence evolve together in such a way that mutations in one of the residues induce a coherent response in the other to compensate for a certain functional or structural effect (5, 7). Over the last decades, numerous statistical methods have been applied to detect residue co-evolution in protein sequences (9, 10). Analysis of co-evolving residues has been used to explore functional coupling in processes like protein-protein interaction (11), gating of potassium channels (12), and allosteric communications in proteins (13). Experimental validation has been provided for the identification of networks of energetically coupled residues mediating allostery (14 -16). However, in the case of enzyme catalysis, only theoretical studies have been reported (17). The limitation of the available techniques in providing insights into the mechanism of catalysis has made it difficult to experimentally validate co-evolution as a natural means of catalysis optimization. Here, we have combined statistical sequence analysis and single molecule force-clamp spectroscopy to probe the effect of evolutionary correlated mutations in the Escherichia coli Trx catalytic mechanism, a ubiquitous disulfide bond reductase.In a previous study, we developed single molecule forceclamp spectroscopy as a novel sensitive tool to study the mechanism of the catalytic reaction of E. coli Trx (18). We showed that Trx reduces disulfide bonds using two distinct force-dependent catalytic mechanisms. The first mechanism is favored at low forces (up to 200 pN), 3 whereas the second mechanism is predominant at high forces (over 200 pN). These two chemical reduction mechanisms are likely to have evolved to al...

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Section: Methodsmentioning

confidence: 99%

Force-Clamp Spectroscopy Detects Residue Co-evolution in Enzyme Catalysis

Pérez-Jiménez

Wiita

Rodríguez-Larrea

et al. 2008

Journal of Biological Chemistry

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“…At the same time, the Trx catalysis proceeds by the S N 2 reaction mechanism, 11,12 which requires that the disulfide bond and the sulfur atom of the enzyme align in the same direction forming a straight line. 15,25,29,30 Therefore, in order for the S N 2 reaction to proceed, the system must overcome a deformation energy barrier associated with the rotation of the disulfide bond with respect to the Trx grove. Simulations show 30 that the bond rotation causes a contraction of the I27 domain, and that the contraction is opposed by the pulling force.…”

Section: Deformation Model Of Enzyme Catalysis In the Presence Of Forcementioning

confidence: 99%

“…Recently, Fernandez and co-workers published a series of articles 15,25,29,30 reporting novel and unexpected results regarding the enzymatic dissociation of disulfide bonds subjected to external force. A number of different enzymes were investigated.…”

Section: Introductionmentioning

confidence: 99%

Deformation Model for Thioredoxin Catalysis of Disulfide Bond Dissociation by Force

Pereverzev

Prezhdo

2009

Cel. Mol. Bioeng.

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Abstract-We develop a theoretical model describing the effect of applied force on the rate of enzymatic dissociation of disulfide bonds (Wiita, A. P. et al., Nature 450:124-127, 2007). The timescale characterizing the extension of the 8-domain protein chain, in which every domain contains a disulfide bond, exhibits anomalous force dependence: The extension time first increases and then decreases with increasing force. This type of force dependence indicates catch-binding and is under active investigation in a variety of systems. The disulfide system presents the first example of a chemical catch-bond. The key difference between the deformation model developed here and the two-pathway model used in the original publication is the bond-deformation term in the force dependence of the dissociation rate constant. The bond-deformation concept provides a different interpretation of the phenomenon. Rather than invoking a new dissociation pathway, which is difficult to rationalize for a simple S-S bond, catch-binding is explained by a force-induced deformation in the protein system disfavoring bond dissociation by thioredoxin. The analysis of the experimental data is performed within the Michaelis-Menten kinetic mechanism of enzyme catalysis. A simplified version of the MichaelisMenten mechanism containing only four parameters is found to provide a quantitative description of the key features of the experimental data.

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“…These Ig domains are found in many adhesion and matrix proteins, and some cytoskeletal proteins also are reported to have at times an intact S-S bond (7). Wiita et al (5) engineered the well characterized titinIg27 domain to have a similar disulfide. Stretching such proteins by AFM had been done on wild-type Ig-cell adhesion molecules (CAMs) (8, 9) and had suggested that a one-dimensional version of Eq.…”

mentioning

confidence: 99%

“…Moreover, the log-dependence on force also fits the one-dimensional theory of bond extension, giving ⌬x* ϭ 0.34 Å for the engineered titin domain (5). Although Wiita et al (5) point out that it is not yet known how often any disulfide bond in vivo will be exposed to the force levels that they explored, it does seem likely that the sensitivity of any particular thiol͞disulfide exchange reaction to a pulling force will depend very specifically on the environment surrounding the bond. Temperature already has proven to strongly influence protein unfolding and refolding in AFM (11)(12)(13).…”

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confidence: 99%