Introduction
Praja1 (PJA1) is an intrinsically disordered protein (IDP) consisting of 643 amino acid residues. Different from structured proteins, IDPs are characterized by the lack of a fixed or ordered three-dimensional structure in the absence of thier binding partners. PJA1 has been reported to promote ubiquitination and degradation of aggregate-prone proteins in neurons although the details of protein-protein interactions remain unclear [1,2,3]. Whereas most structured proteins on metal surfaces would lose their functionality by disruption of their higher-order structure, PJA1 is anticipated to retain its functionality in terms of protein interactions due to its inherent properties as an IDP. If PJA1 maintains its function on a solid surface, then studying its behavior leads to understanding and exploiting its interactions with aggregated proteins. In this work, we investigated the behaviors of PJA1 adsorbed on gold substrate by using electrochemical methods: changes in the state of PJA1 by the interaction with alpha-synuclein (α-syn), which is also an IDP and known to be one of interaction partners of PJA1, were examined from the electrode reaction of ferricyanide added as a marker.
Method
Human PJA1 protein was put on a gold substrate (a vacuum-deposited gold thin film on silicon wafer) by drop-casting its aqueous solution, corresponding to 3.7 μg/mm2 on the surface, for 1 h at room temperature. To remove non-adsorbed components, the substrate was rinsed with deionized water and dried with N2 blowing. The interaction with α-syn (140 amino acid residues) was investigated by putting its solution on the substrate followed by incubation at room temperature. After non-reacted component was removed by rinsing, the substrate was dried with N2 blow. Cyclic voltammetry and electrochemical impedance spectroscopy were carried out with these gold substrates as a working electrode, a Pt wire counter electrode, and an Ag/AgCl (3 M, NaCl) reference electrode, in the electrolyte of 5 mM [Fe(CN)6]3- in 0.1 M KCl.
Results
In the cyclic voltammograms for the gold electrodes with PJA1 at different concentration of α-syn (0, 0.01, and 0.1 μg/mL), a pair of peaks attributed to the oxidation and reduction of [Fe(CN)6]3-/4- was observed at ~0.24 and ~0.16 V, respectively, for all three conditions. The magnitude of peak current for 0 μg/mL α-syn was much smaller than that for the case without PJA1, while peak currents increased under the presence of α-syn, suggesting that the suppression of [Fe(CN)6]3-/4- electrode reaction by the presence of PJA1 is reduced by the interaction with α-syn. From the electrochemical impedance measurement, the magnitude of charge transfer resistance was suggested to be largest for 0 μg/mL α-syn among the three conditions and decreased under the presence of α-syn. These results may be caused by the exposure of the gold surface as a consequence of the conformational change in PJA1, and thus demonstrated that PJA1 can interact with α-syn even in its adsorbed state on gold substrate. Details will be presented in the poster session.
References
[1] K. Watabe, M. Niida-Kawaguchi, M. Tada, Y. Kato, M. Murata, K. Tanji, K. Wakabayashi, M. Yamada, A. Kakita, and N. Shibata, Neuropathology, 42, 6, 488-504 (2022).
[2] W. Onodera, K. Kawasaki, M. Oishi, S. Aoki, and T. Asahi, J. Mol. Evol., 92, 1, 21-29 (2024).
[3] S. Aoki, K. Kawasaki, K. Imadegawa, M. Oishi, T. Asahi, and W. Onodera, BioRxiv, 2024.06.10.598176 (2024).
