Evidence for protein conformational change at a Au(110)/protein interface

Abstract

Evidence is presented that reflection anisotropy spectroscopy (RAS) can provide real-time measurements of conformational change in proteins induced by electron transfer reactions. A bacterial electron transferring flavoprotein (ETF) has been modified so as to adsorb on an Au(110) electrode and enable reversible electron transfer to the protein cofactor in the absence of mediators. Reversible changes are observed in the RAS of this protein that are interpreted as arising from conformational changes accompanying the transfer of electrons.

Fig. 1

Molecular graphics ribbon diagram for the structure of M. methyltrophus ETF showing the mutation sites where a cysteine residue was introduced by directed mutagenesis, replacing either the asparate-16 or the serine-193 residues on the surface of the protein to produce, respectively, the D16C and S193C mutants. The sites of modifications are shown as spheres. The α-subunit of the enzyme is shown in dark grey, the β-subunit in light grey and the FAD in black.

Fig. 2

Spectral changes accompanying titration of the engineered S193C ETF during potentiometric measurements. Black arrows indicate the direction of absorbance changes at 2.6 eV (470 nm) and at 3.3 eV (370 nm) as the potential is reduced. Inset: plot of absorbance change at 470 nm (•) and at 370 nm (○) against the recorded potentials for the engineered S193C ETF protein. E′₁ =+64mV, E′₂ ⪡ −250mV. The ETF_2e is inaccessible on reducing the engineered protein chemically as observed for a wild-type protein [9]. However, the reduction of ETF to the 2-electron reduced species is achievable by electrochemical methods [19]. Protein (63 μM) was contained in 50 mM potassium phosphate buffer, pH 7.0 at 25 °C and maintained under anaerobic conditions.

Fig. 3

RA spectra of the engineered ETF protein assembled on the Au(110) electrode surface in 0.1 M NaH₂PO₄/K₂HPO₄. (a) RA spectrum of the Au(110) electrode (solid line); RA spectrum of the Au(110) electrode assembled with the engineered S193C ETF protein (dashed line); RA spectrum of the S193C ETF after subtracting the Au(110) spectrum (○), all obtained at +244mV, vs. NHE. (b) RA spectra of the engineered S193C ETF protein assembled on the Au(110) electrode surface at +300mV (solid line), −100mV (dashed line) and at −356mV (○) representing the RA spectra of the oxidised, one-electron reduced and two-electron reduced species of the S193C ETF protein, respectively. In each case the RA spectrum of the Au(110) obtained at the corresponding potential has been subtracted from that of the adsorbed protein.

Fig. 4

Time dependence of the spectral intensity of the RAS signal at 2.5 eV following a stepped voltage change recorded for the engineered ETF proteins adsorbing onto the Au(110) electrode. Proteins were contained in 0.1 M NaH₂PO₄/K₂HPO₄. (a) For the S193C ETF protein, the voltage is stepped from +300mV to −100mV (solid line), from +300mV to −356mV (×) and from −356mV to −100mV (ο) for 10 cycles each of 60 s. The spectra are offset for clarity and the scale, 1 × 10⁻³, is marked on the y-axis. (b) The voltage is stepped from +300mV to −100mV to −356mV and then back to −100mV and finally to +300mV. The trace for the S193C ETF protein is the solid line and for the D16C ETF protein is shown by circles with each voltage maintained for 60 s. The spectra are offset for clarity and the scale, 1 × 10⁻³, is marked on the y-axis.