Detection of a Yb3+ binding site in regenerated bacteriorhodopsin that is coordinated with the protein and phospholipid head groups

Abstract

Near infrared Yb3+ vibronic sideband spectroscopy was used to characterize specific lanthanide binding sites in bacteriorhodopsin (bR) and retinal free bacteriorhodopsin (bO). The VSB spectra for deionized bO regenerated with a ratio of 1:1 and 2:1 ion to bO are identical. Application of a two-dimensional anti-correlation technique suggests that only a single Yb3+ site is observed. The Yb3+ binding site in bO is observed to consist of PO2- groups and carboxylic acid groups, both of which are bound in a bidentate manner. An additional contribution most likely arising from a phenolic group is also observed. This implies that the ligands for the observed single binding site are the lipid head groups and amino acid residues. The vibronic sidebands of Yb3+ in deionized bR regenerated at a ratio of 2:1 ion to bR are essentially identical to those in bO. The other high-affinity binding site is thus either not evident or its fluorescence is quenched. A discussion is given on the difference in binding of Ca2+ (or Mg2+) and lanthanides in phospholipid membrane proteins.

Figure 1

Near infrared Yb³⁺ VSB spectra of bO samples containing one Yb³⁺ (Upper) and two Yb³⁺ (Lower) atoms per bO. (A) Low frequency spectra: 878 nm, 450 mW, 500 scans. (B) High frequency spectra: 965 nm excitation, 450 mW, 10,000 scans.

Figure 2

Near infrared Yb³⁺ VSB spectra Yb³⁺ regenerated bR and bO. (A) Zero phonon lines for samples of bR containing two Yb³⁺ (Bottom; 870 nm excitation, 20 mW, 100 scans), bO containing one Yb³⁺ (Middle: 878 nm excitation, 650 mW, 1000 scans), and bO containing two Yb³⁺ atoms (Top: 884 nm excitation, 500 mW, 1000 scans). (B) VSB spectra of bR in the 700–2000 cm⁻¹ VSB shift region, excited at 965 nm (Lower: 250 mW, 2000 scans) and 962 nm (Upper: 250 mW, 2000 scans). Obvious Raman bands from the retinal chromophore are designated R. (C) VSB bands arising from the phosphate symmetric P–O stretching mode of the PGP ( Lower: 886 nm, 180 mW, 200 scans) and P₂O₇²⁻ (Upper: 876 nm, 450 mW, 100 scans) Yb³⁺ complexes.

Figure 3

(A) Near infrared Yb³⁺ VSB spectrum, excited at 965 nm, of bR containing two Yb³⁺ atoms per bR in the 1100–1300 cm⁻¹ VSB shift region (Top: 250 mW, 2000 scans) and the Fourier transform Raman spectrum of the same bR sample excited at 1064 nm (Middle: 280 mW, 2000 scans); 1064 nm excitation results in the observation of essentially only the Raman contributions of the retinal chromophore. Subtracting the retinal Raman contributions (middle spectrum) from the top spectrum results in a difference spectrum (Bottom) revealing VSB bands previously hidden by the Raman contributions. (B) VSB spectrum of the same bR sample excited at 945 nm (Upper: 450 mW, 2000 scans). At this wavelength of excitation, the retinal Raman contributions are not present in the 1400–2000 cm⁻¹ VSB shift spectral region. The Fourier transformed Raman spectrum of the same bR sample is shown for comparison (Lower: excitation 1064 nm, 280 mW, 2000 scans).

Figure 4

VSB spectrum of bO containing one and two Yb³⁺ atoms per bO reconstructed from the two-dimensional correlation analysis. (Inset) Schematic diagram showing the possible configuration of the ligands of Yb³⁺ suggested by the results of this work.