Comparative Electrochemical Study of N‐, C‐terminal and Integral Centrin on Adsorption and Metal‐Binding Properties

Abstract

The adsorption behavior of apo‐ciliate Euplotes octocarinatus centrin (EoCen) and N(C)‐terminal domain of EoCen (N(C)‐EoCen) at a glassy carbon (GC) electrode is studied by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). Interestingly, the adsorption isotherms of C‐EoCen, N‐EoCen and EoCen at a GC surface differ from one another. It is considered to be associated with the different aggregation properties of three proteins. Furthermore, we analyze the metal‐binding properties of centrin and the followed changes in protein structure upon metal‐binding. Corresponding to the four binding sites of EoCen, it shows four no‐equiv signals of CV or EIS change. It indicates that the four different binding sites of EoCen can be discriminated by the EIS titration curves, which is in contrast with conventional use of spectral method. According to the electrostatic potential at the molecular surface of proteins, the favored orientation of N‐EoCen and C‐EoCen on the GC surface is modeled. These models can well explain the results of titration curves of Eu3+ to N(C)‐EoCen. This work has established the electrochemical methodology which can be used to measure metal‐binding sites involved and reveal the contribution of each metal site to the whole protein conformational change.