Catalyst-free click PEGylation reveals substantial mitochondrial ATP synthase sub-unit alpha oxidation before and after fertilisation

Publication date: Available online 18 June 2019Source: Redox BiologyAuthor(s): James N. Cobley, Anna Noble, Eduardo Jimenez-Fernandez, Manuel-Thomas Valdivia Moya, Matthew Guille, Holger HusiAbstractUsing non-reducing Western blotting to assess protein thiol redox state is challenging because most reduced and oxidised forms migrate at the same molecular weight and are, therefore, indistinguishable. While copper catalysed Click chemistry can be used to ligate a polyethylene glycol (PEG) moiety termed Click PEGylation to mass shift the reduced or oxidised form as desired, the potential for copper catalysed auto-oxidation is problematic. Here we define a catalyst-free transcyclooctene-methyltetrazine (TCO-Tz) inverse electron demand Diels Alder chemistry approach that affords rapid (k ∼2000 M−1 s−1), selective and bio-orthogonal Click PEGylation. We used TCO-Tz Click PEGylation to investigate how fertilisation impacts reversible mitochondrial ATP synthase F1-Fo sub-unit alpha (ATP-α-F1) oxidation—an established molecular correlate of impaired enzyme activity—in Xenopus laevis. TCO-Tz Click PEGylation studies reveal substantial (∼65%) reversible ATP-α-F1 oxidation at evolutionary conserved cysteine residues (i.e., C244 and C294) before and after fertilisation. A single thiol is, however, preferentially oxidised likely due to greater solvent exposure during the catalytic cycle. Selective reduction experiments show that: S-glutathionylation accounts for ∼50–60% ...

Source: Redox Biology - June 18, 2019 Category: Biology Source Type: research