Force-dependent chemical kinetics of disulfide bond reduction observed with single-molecule techniques - PubMed (original) (raw)
Force-dependent chemical kinetics of disulfide bond reduction observed with single-molecule techniques
Arun P Wiita et al. Proc Natl Acad Sci U S A. 2006.
Abstract
The mechanism by which mechanical force regulates the kinetics of a chemical reaction is unknown. Here, we use single-molecule force-clamp spectroscopy and protein engineering to study the effect of force on the kinetics of thiol/disulfide exchange. Reduction of disulfide bonds through the thiol/disulfide exchange chemical reaction is crucial in regulating protein function and is known to occur in mechanically stressed proteins. We apply a constant stretching force to single engineered disulfide bonds and measure their rate of reduction by DTT. Although the reduction rate is linearly dependent on the concentration of DTT, it is exponentially dependent on the applied force, increasing 10-fold over a 300-pN range. This result predicts that the disulfide bond lengthens by 0.34 A at the transition state of the thiol/disulfide exchange reaction. Our work at the single bond level directly demonstrates that thiol/disulfide exchange in proteins is a force-dependent chemical reaction. Our findings suggest that mechanical force plays a role in disulfide reduction in vivo, a property that has never been explored by traditional biochemistry. Furthermore, our work also indicates that the kinetics of any chemical reaction that results in bond lengthening will be force-dependent.
Conflict of interest statement
Conflict of interest statement: No conflicts declared.
Figures
Fig. 1.
Force–clamp spectroscopy identifies single thiol/disulfide exchange events under a stretching force. (A) An engineered disulfide bond was introduced between the 32nd and the 75th residue of the 27th Ig-like domain of cardiac titin (I27G32C–A75C). In the ribbon diagram of I27G32C–A75C, mutated residues 32 and 75 are yellow spheres, residues 1–31 and 76–89 are pictured in red (unsequestered residues), and 33–74, behind the disulfide bond, are in blue (trapped residues). The cartoons on the left depict the three sequential events that take place when we apply a mechanical force to the I27G32C–A75C protein. Applying a mechanical force first triggers the unfolding and extension of the protein, up to the position of the disulfide bond. We call this initial elongation unsequestered unfolding. If DTT is present in the bathing solution, disulfide bond reduction can occur, allowing for the extension of the trapped residues. (B) Force–clamp experiment showing the stepwise elongation (red trace) of an (I27G32C–A75C)8 polyprotein pulled at a constant force of 130 pN (black trace) in the absence of DTT. Seven steps of equal size, ≈10.6 nm, mark the sequential unfolding of the unsequestered region of seven I27G32C–A75C modules in the polyprotein. The brief downward deflections in the force (black trace) are due to lag in the feedback electronics after each unfolding event. (C) When the same experiment is repeated in the presence of 50 mM DTT, we again observe a series of ≈10.8 nm steps corresponding to the unsequestered unfolding events, followed by several ≈13.8 nm steps (blue stars), which mark thiol/disulfide exchange events and the subsequent extension of the trapped residues. In this trace, we also observe an event with an amplitude of ≈24.0 nm (green arrow), which corresponds to the unfolding of a single fully reduced I27 module.
Fig. 2.
A double-pulse protocol separates the unsequestered unfolding from disulfide reduction events. (A) Typical double-pulse force–clamp experiment pulling the (I27G32C–A75C)8 protein first at 130 pN for 1 s and then stepping to a force of 200 pN for 7 s (black trace). In this experiment, the first pulse to 130 pN causes a series of seven unsequestered unfolding events (10.6-nm steps). Upon increasing the force to 200 pN (green arrow), we observed an elastic step elongation of the protein. In the absence of DTT, no further steps are observed. (B) Repeating the same experiment in the presence of 12.5 mM DTT, we again observed six unsequestered unfolding events at 130 pN. Upon stepping to 200 pN and after the elastic elongation of the protein (green arrow), we then observed a series of five steps of ≈14.2 nm corresponding to the disulfide reduction events. Notice the rapid exponential time course followed by the unsequestered unfolding events at 130 pN and the much slower reduction events observed during the second pulse at 200 pN.
Fig. 3.
Ensemble measurements of the kinetics of thiol/disulfide exchange. (A) Three recordings are shown of single (I27G32C–A75C)8 polyproteins that were extended with the same double-pulse protocol shown in Fig. 2_B_: 12.5 mM DTT, F = 130 pN for 1 s, and then F = 200 pN. The stochastic nature of both the unsequestered unfolding events as well as of the thiol/disulfide exchange events becomes apparent when comparing these recordings. (B Upper) A four-trace average (red trace) of the double-pulse experiments shown in A and Fig. 2_B_ demonstrates the methods used to build up an ensemble of recordings under a set of conditions. Similar four-trace averages are shown for data obtained under two other conditions: 12.5 mM DTT, F = 130 pN for 1 s then F = 300 pN (green trace); and 0 mM DTT, F = 130 pN for 1 s then F = 200 pN (blue trace). Notice that the time course of unsequestered unfolding during the first pulse is similar under all conditions. (B Lower) The averaged force traces are shown.
Fig. 4.
The thiol/disulfide exchange chemical reaction is force and [DTT] dependent. Multiple-trace averages (n > 20 in each trace) of thiol/disulfide exchange events measured by using the double-pulse protocol as a function of force and DTT concentration are shown. Only the second pulse averages are shown; time = 0 s denotes the start of the second pulse. (A) A set of trace averages measured at a constant concentration of DTT (12.5 mM), while varying the force of the second pulse between 100 and 400 pN. Single-exponential fits (continuous lines) measure the time constant, τ_r_, of thiol/disulfide exchange. (B) Plot of the rate of thiol/disulfide exchange, r = 1/τ_r_, as a function of the pulling force at [DTT] = 12.5 mM. The solid line is an exponential fit to the data. (C) Trace averages measured at a constant pulling force (F = 200 pN) and at various DTT concentrations. (D) Plot of r as a function of [DTT]. The solid line is a linear fit to the data. The linear (first-order) dependence on [DTT] demonstrates that the thiol/disulfide exchange reaction in our system is bimolecular. This reaction can be described empirically from B and D by the simple rate equation r = k(F)[DTT], where k(F) is exponentially dependent on the pulling force and k(200 pN) = 27.6 M−1·s−1 from the linear fit in D.
Fig. 5.
Force sensitivity and bond lengthening in the thiol/disulfide exchange chemical reaction. (A) Semilogarithmic plot of the rate of thiol/disulfide exchange, r (filled circles) and of the unsequestered unfolding rate, τ_u_ (open circles) as a function of the pulling force. The solid line is a fit of the equation r = A(exp((F_Δ_x r_−_E_a)/k_B_T))[DTT]. The fit gave values of Δ_x_r = 0.34 Å and E_a = 65 kJ/mol (with A ≈ 1012 s−1·M−1) for thiol/disulfide exchange. The dashed line fits the unsequestered unfolding rate with α_u(F) = α_u(0)exp(F_Δx_u/k_B_T) obtaining Δ_x_ u = 1.75 Å. (B) A simple illustration of the energy landscape of the thiol/disulfide exchange reaction under force. Our Bell-like model predicts that the transition state is located at 0.34 Å along the linear reaction coordinate, explaining the relative insensitivity of this reaction as compared with the unsequestered unfolding of the I27 protein. We calculate that applying 400 pN of force reduces the activation energy barrier for thiol/disulfide exchange by 8.2 kJ/mol. (C) Illustration of the thiol/disulfide exchange reaction between a DTT molecule and a disulfide bond under a stretching force. Only the two participating cysteine residues are shown for simplicity. The sulfur atoms are in green. The disulfide bond length increases from 2.05 Å initially (29) (Left) up to 2.39 Å at the transition state of an SN2 reaction between the DTT molecule and the disulfide bond (Right).
Comment in
- Covalent chemistry on distended proteins.
Discher DE, Bhasin N, Johnson CP. Discher DE, et al. Proc Natl Acad Sci U S A. 2006 May 16;103(20):7533-4. doi: 10.1073/pnas.0602388103. Epub 2006 May 8. Proc Natl Acad Sci U S A. 2006. PMID: 16682625 Free PMC article. No abstract available.
Similar articles
- Single-molecule force spectroscopy measurements of bond elongation during a bimolecular reaction.
Koti Ainavarapu SR, Wiita AP, Dougan L, Uggerud E, Fernandez JM. Koti Ainavarapu SR, et al. J Am Chem Soc. 2008 May 21;130(20):6479-87. doi: 10.1021/ja800180u. Epub 2008 Apr 24. J Am Chem Soc. 2008. PMID: 18433129 - Influence of external force on properties and reactivity of disulfide bonds.
Iozzi MF, Helgaker T, Uggerud E. Iozzi MF, et al. J Phys Chem A. 2011 Mar 24;115(11):2308-15. doi: 10.1021/jp109428g. Epub 2011 Mar 2. J Phys Chem A. 2011. PMID: 21366304 - Force-activated reactivity switch in a bimolecular chemical reaction.
Garcia-Manyes S, Liang J, Szoszkiewicz R, Kuo TL, Fernández JM. Garcia-Manyes S, et al. Nat Chem. 2009 Jun;1(3):236-42. doi: 10.1038/nchem.207. Epub 2009 May 10. Nat Chem. 2009. PMID: 21378854 - Kinetics and mechanisms of thiol-disulfide exchange covering direct substitution and thiol oxidation-mediated pathways.
Nagy P. Nagy P. Antioxid Redox Signal. 2013 May 1;18(13):1623-41. doi: 10.1089/ars.2012.4973. Epub 2013 Jan 9. Antioxid Redox Signal. 2013. PMID: 23075118 Free PMC article. Review. - Thiol/disulfide exchange equilibria and disulfide bond stability.
Gilbert HF. Gilbert HF. Methods Enzymol. 1995;251:8-28. doi: 10.1016/0076-6879(95)51107-5. Methods Enzymol. 1995. PMID: 7651233 Review. No abstract available.
Cited by
- Structural analysis of a ligand-triggered intermolecular disulfide switch in a major latex protein from opium poppy.
Carr SC, Facchini PJ, Ng KKS. Carr SC, et al. Acta Crystallogr D Struct Biol. 2024 Sep 1;80(Pt 9):675-685. doi: 10.1107/S2059798324007733. Epub 2024 Aug 29. Acta Crystallogr D Struct Biol. 2024. PMID: 39207895 Free PMC article. - Conformational activation and inhibition of von Willebrand factor by targeting its autoinhibitory module.
Arce NA, Markham-Lee Z, Liang Q, Najmudin S, Legan ER, Dean G, Su AJ, Wilson MS, Sidonio RF, Lollar P, Emsley J, Li R. Arce NA, et al. Blood. 2024 May 9;143(19):1992-2004. doi: 10.1182/blood.2023022038. Blood. 2024. PMID: 38290109 - The role of single protein elasticity in mechanobiology.
Beedle AE, Garcia-Manyes S. Beedle AE, et al. Nat Rev Mater. 2023 Jan;8:10-24. doi: 10.1038/s41578-022-00488-z. Epub 2022 Oct 24. Nat Rev Mater. 2023. PMID: 37469679 Free PMC article. - Engineering Tissue-Scale Properties with Synthetic Cells: Forging One from Many.
Lin AJ, Sihorwala AZ, Belardi B. Lin AJ, et al. ACS Synth Biol. 2023 Jul 21;12(7):1889-1907. doi: 10.1021/acssynbio.3c00061. Epub 2023 Jul 7. ACS Synth Biol. 2023. PMID: 37417657 Free PMC article. Review. - Bond breaking of furan-maleimide adducts via a diradical sequential mechanism under an external mechanical force.
Cardosa-Gutierrez M, De Bo G, Duwez AS, Remacle F. Cardosa-Gutierrez M, et al. Chem Sci. 2023 Jan 4;14(5):1263-1271. doi: 10.1039/d2sc05051j. eCollection 2023 Feb 1. Chem Sci. 2023. PMID: 36756317 Free PMC article.
References
- Beyer M. K., Clausen-Schaumann H. Chem. Rev. 2005;105:2921–2948. - PubMed
- Grandbois M., Beyer M., Rief M., Clausen-Schaumann H., Gaub H. E. Science. 1999;283:1727–1730. - PubMed
- Rubio-Bollinger G., Bahn S. R., Agrait N., Jacobsen K. W., Vieira S. Phys. Rev. Lett. 2001;87:026101.
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources