3, 4-Dihydroxyphenylanine (Dopa)-containing proteins are key to wet adhesion in mussels and possibly other sessile organisms also. However, Dopa-mediated adhesive bonding is a hard act to follow in that, at least in mussels, bonding depends on Dopa in both reduced and oxidized forms, for adhesion and cohesion, respectively. Given the vulnerability of Dopa to spontaneous oxidation, the most significant challenge to using it in practical adhesion is controlling Dopa redox in a temporally- and spatially defined manner. Mussels appear to achieve such control in their byssal attachment plaques, and factors involved in redox control can be measured with precision using redox probes such as the diphenylpicryl hydrazyl (DPPH) free radical. Understanding the specifics of natural redox control may provide fundamentally important insights for adhesive polymer engineering and antifouling strategies.

Common seafood pollutants inhibit a crucial cellular defense protein.

The free radical method using 1,1-diphenyl-2-picryl-hydrazyl (DPPH) is a well established assay for the in vitro determination of antioxidant activity in food and biological extracts. The standard DPPH assay uses methanol or ethanol as solvents, or buffered alcoholic solutions in a ratio of 40%/60% (buffer/alcohol, v/v) to keep the hydrophobic hydrazyl radical and phenolic test compounds soluble while offering sufficient buffering capacity at different pHs tested. Following this protocol, we were unable to keep proteinaceous antioxidants soluble at different pHs to test for their antioxidant activity. Thus, the assay protocol was modified as follows to improve its utility:•Non-ionic detergents were added to keep the DPPH radical soluble and to provide a mild and non-denaturing environment for the antioxidant protein.•Maximal concentration of DPPH was limited to 100 μM to stay within the sensitivity range of the detector at the given wavelength (515 nm) and to increase the dynamic range of the assay.•0.1 M citrate phosphate buffer was introduced to prevent experimental artifacts due to changing buffer compositions at different pHs.

BetP is an Na+‐coupled betaine‐specific transporter of the betaine–choline–carnitine (BCC) transporter family involved in the response to hyperosmotic stress. The crystal structure of BetP revealed an overall fold of two inverted structurally related repeats (LeuT‐fold) that BetP shares with other sequence‐unrelated Na+‐coupled symporters. Numerous structures of LeuT‐fold transporters in distinct conformational states have contributed substantially to our understanding of the alternating access mechanism of transport. Nevertheless, coupling of substrate and co‐transported ion fluxes has not been structurally corroborated to the same extent. We converted BetP by a single‐point mutation—glycine to aspartate—into an H+‐coupled choline‐specific transporter and solved the crystal structure of this mutant in complex with choline. The structure of BetP‐G153D demonstrates a new inward‐facing open conformation for BetP. Choline binding to a location close to the second, low‐affinity sodium‐binding site (Na2) of LeuT‐fold transporters is facilitated by the introduced aspartate. Our data confirm the importance of a cation‐binding site in BetP, playing a key role in a proposed molecular mechanism of Na+ and H+ coupling in BCC transporters.