Abstract
The redox potential of a protein disulphide bond is one of the most important factors for determining the role of a disulphide bond. Disulphide bonds can have a stabilizing role for the structure of a protein or they can play a functional role which can regulate protein bioactivity. Determining the redox potential of disulphides can help distinguish the functional from the structural disulphide bonds. In this chapter, two methods for determining the redox potential of a protein disulphide bond are described. The first method uses maleimide-biotin labeling of free cysteine thiols and western blot densitometry to determine the fraction of reduced disulphide bond under various redox-buffering conditions. The second method uses differential cysteine labeling and tandem mass spectrometry to determine the redox potential.
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Schmidt B, Ho L, Hogg PJ (2006) Allosteric disulfide bonds. Biochemistry 45:7429–7433
Gilbert HF (1990) Molecular and cellular aspects of thiol-disulfide exchange. Adv Enzymol Relat Areas Mol Biol 63:69–172
Wunderlich M, Glockshuber R (1993) Redox properties of protein disulfide isomerase (dsba) from Escherichia coli. Protein Sci 2:717–726
Wouters MA, Fan SW, Haworth NL (2010) Disulfides as redox switches: From molecular mechanisms to functional significance. Antioxid Redox Sign 12:53–91
Cook KM, Hogg PJ (2013) Post-translational control of protein function by disulfide bond cleavage. Antioxid Redox Sign 18:1987–2015
Butera D, Cook KM, Chiu J, Wong JW, Hogg PJ (2014) Control of blood proteins by functional disulfide bonds. Blood 123:2000–2007
Cook KM, McNeil HP, Hogg PJ (2013) Allosteric control of beta 11-tryptase by a redox active disulfide bond. J Biol Chem 288:34920–34929
Zhou A, Carrell RW, Murphy MP, Wei Z, Yan Y, Stanley PL, Stein PE, Broughton Pipkin F, Read RJ (2010) A redox switch in angiotensinogen modulates angiotensin release. Nature 468:108–111
Butera D, Passam F, Ju L, Cook KM, Woon H, Aponte-Santamaria C, Gardiner E, Davis AK, Murphy DA, Bronowska A, Luken BM, Baldauf C, Jackson S, Andrews R, Grater F, Hogg PJ (2018) Autoregulation of von Willebrand factor function by a disulfide bond switch. Sci Adv 4:eaaq1477
Ioannou Y, Zhang JY, Passam FH, Rahgozar S, Qi JC, Giannakopoulos B, Qi M, Yu P, Yu DM, Hogg PJ, Krilis SA (2010) Naturally occurring free thiols within beta 2-glycoprotein I in vivo: Nitrosylation, redox modification by endothelial cells, and regulation of oxidative stress-induced cell injury. Blood 116:1961–1970
Giannakopoulos B, Gao L, Qi M, Wong JW, Yu DM, Vlachoyiannopoulos PG, Moutsopoulos HM, Atsumi T, Koike T, Hogg P, Qi JC, Krilis SA (2012) Factor XI is a substrate for oxidoreductases: enhanced activation of reduced FXI and its role in antiphospholipid syndrome thrombosis. J Autoimmun 39:121–129
Kaiser BK, Yim D, Chow IT, Gonzalez S, Dai Z, Mann HH, Strong RK, Groh V, Spies T (2007) Disulphide-isomerase-enabled shedding of tumour-associated nkg2d ligands. Nature 447:482–486
Butera D, Wind T, Lay AJ, Beck J, Castellino FJ, Hogg PJ (2014) Characterization of a reduced form of plasma plasminogen as the precursor for angiostatin formation. J Biol Chem 289:2992–3000
Pijning AE, Chiu J, Yeo RX, Wong JWH, Hogg PJ (2018) Identification of allosteric disulfides from labile bonds in x-ray structures. R Soc Open Sci 5:171058
Li W, Baldus IB, Grater F (2015) Redox potentials of protein disulfide bonds from free-energy calculations. J Phys Chem B 119:5386–5391
Liang Hai Po H, Brophy Teresa M, Hogg Philip J (2011) Redox properties of the tissue factor cys186–cys209 disulfide bond. Biochem J 437:455–460
Huber-Wunderlich M, Glockshuber R (1998) A single dipeptide sequence modulates the redox properties of a whole enzyme family. Fold Des 3:161–171
Chiu J, Wong JWH, Hogg PJ (2014) Redox regulation of methionine aminopeptidase 2 activity. J Biol Chem 289:15035–15043
Pasquarello C, Sanchez JC, Hochstrasser DF, Corthals GL (2004) N-t-butyliodoacetamide and iodoacetanilide: two new cysteine alkylating reagents for relative quantitation of proteins. Rapid Commun Mass Spectrom 18:117–127
Bekendam RH, Bendapudi PK, Lin L, Nag PP, Pu J, Kennedy DR, Feldenzer A, Chiu J, Cook KM, Furie B, Huang M, Hogg PJ, Flaumenhaft R (2016) A substrate-driven allosteric switch that enhances PDI catalytic activity. Nat Commun 7:12579
Read SA, O’Connor KS, Suppiah V, Ahlenstiel CLE, Obeid S, Cook KM, Cunningham A, Douglas MW, Hogg PJ, Booth D, George J, Ahlenstiel G (2017) Zinc is a potent and specific inhibitor of ifn-λ3 signalling. Nat Commun 8:15245
Rothwarf DM, Scheraga HA (1992) Equilibrium and kinetic constants for the thiol-disulfide interchange reaction between glutathione and dithiothreitol. Proc Natl Acad Sci USA 89:7944–7948
Gatlin CL, Kleemann GR, Hays LG, Link AJ, Yates JR 3rd (1998) Protein identification at the low femtomole level from silver-stained gels using a new fritless electrospray interface for liquid chromatography-microspray and nanospray mass spectrometry. Anal Biochem 263:93–101
He L, Diedrich J, Chu YY, Yates JR 3rd (2015) Extracting accurate precursor information for tandem mass spectra by RawConverter. Anal Chem 87:11361–11367
Frickel EM, Frei P, Bouvier M, Stafford WF, Helenius A, Glockshuber R, Ellgaard L (2004) Erp57 is a multifunctional thiol-disulfide oxidoreductase. J Biol Chem 279:18277–18287
Passam F, Chiu J, Ju L, Pijning A, Jahan Z, Mor-Cohen R, Yeheskel A, Kolsek K, Tharichen L, Aponte-Santamaria C, Grater F, Hogg PJ (2018) Mechano-redox control of integrin de-adhesion. elife 7. https://doi.org/10.7554/eLife.34843
Chambers JE, Tavender TJ, Oka OBV, Warwood S, Knight D, Bulleid NJ (2010) The reduction potential of the active site disulfides of human protein disulfide isomerase limits oxidation of the enzyme by ero1α. J Biol Chem 285:29200–29207
Krause G, Lundstrom J, Barea JL, Pueyo de la Cuesta C, Holmgren A (1991) Mimicking the active site of protein disulfide-isomerase by substitution of proline 34 in escherichia coli thioredoxin. J Biol Chem 266:9494–9500
Cleland WW (1964) Dithiothreitol, a new protective reagent for SH groups*. Biochemistry 3:480–482
Ravilious GE, Nguyen A, Francois JA, Jez JM (2012) Structural basis and evolution of redox regulation in plant adenosine-5′-phosphosulfate kinase. Proc Natl Acad Sci U S A 109:309–314
Matthias LJ, Azimi I, Tabrett CA, Hogg PJ (2010) Reduced monomeric CD4 is the preferred receptor for HIV. J Biol Chem 285:40793–40799
Nishii W, Kukimoto-Niino M, Terada T, Shirouzu M, Muramatsu T, Kojima M, Kihara H, Yokoyama S (2015) A redox switch shapes the Lon protease exit pore to facultatively regulate proteolysis. Nat Chem Biol 11:46–51
Jin X, Stamnaes J, Klock C, DiRaimondo TR, Sollid LM, Khosla C (2011) Activation of extracellular transglutaminase 2 by thioredoxin. J Biol Chem 286:37866–37873
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Cook, K.M. (2019). Determining the Redox Potential of a Protein Disulphide Bond. In: Hogg, P. (eds) Functional Disulphide Bonds. Methods in Molecular Biology, vol 1967. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9187-7_5
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DOI: https://doi.org/10.1007/978-1-4939-9187-7_5
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