Spectroscopic ruler for measuring active-site distortions based on Raman optical activity of a hydrogen out-of-plane vibration

Abstract

Photoactive yellow protein (PYP), from the phototrophic bacterium Halorhodospira halophila, is a small water-soluble photoreceptor protein and contains p-coumaric acid (pCA) as a chromophore. PYP has been an attractive model for studying the physical chemistry of protein active sites. Here, we explore how Raman optical activity (ROA) can be used to extract quantitative information on distortions of the pCA chromophore at the active site in PYP. We use ¹³C8-pCA to assign an intense signal at 826 cm^-1 in the ROA spectrum of PYP to a hydrogen out-of-plane vibration of the ethylenic moiety of the chromophore. Quantum-chemical calculations based on density functional theory demonstrate that the sign of this ROA band reports the direction of the distortion in the dihedral angle about the ethylenic C=C bond, while its amplitude is proportional to the dihedral angle. These results document the ability of ROA to quantify structural deformations of a cofactor molecule embedded in a protein moiety.

Keywords: chromophore; density functional theory; molecular strain; photoreceptor; vibrational spectroscopy.

Fig. 1.

The structures of the pCA chromophore for PYP. (A) The structure and atom numbering of the chromophore. (B and C) The structures of the chromophore in two representative crystal structures (PDB ID codes 1NWZ and 2QJ7). (D and E) Dihedral angles τ(C3–C4–C7–C8), τ(C7–C8–C9–O2), and τ(C4–C7–C8–C9) as a function of crystallographic resolution. The data for 1NWZ and 2QJ7 are displayed as closed circles and squares, respectively.

Fig. 2.

Observed and calculated Raman and ROA spectra of PYP whose chromophore is unlabeled (black) and ¹³C8 labeled (red). (A) The observed Raman spectra. (B) The calculated Raman spectra. (C) The observed and (D) calculated ¹³C minus ¹²C difference Raman spectra. (E) The observed ROA spectra. (F) The calculated ROA spectra. (G) The observed and (H) calculated ¹³C minus ¹²C difference ROA spectra. PYP samples were dissolved in 10 mM Tris⋅HCl, pH 7.4, and the sample concentration was 4−5 mM. The spectra were obtained with 785-nm excitation (∼200 mW). The calculated spectra were based on model 1, and Gaussian band shapes with a 10 cm⁻¹ width were used except a 20 cm⁻¹ width for the highest band at 1,539 cm⁻¹. Raman and ROA intensities for the highest intensity bands were reduced by a factor of 2 (1,319, 1,314, 1,310, 1,268, 1,266, and 1,259 cm⁻¹) or 10 (1,633, 1,628, and 1,539 cm⁻¹) to make the other bands visible in the figure. The ROA spectra are magnified by a factor of 2,000.

Fig. 3.

Simulated Raman and ROA spectra of the active-site models for PYP. (A) Raman (a) and ROA (b–h) spectra are shown. The dihedral angle about the C7=C8 moiety was varied up to ±30° from a planar geometry of τ(C4–C7–C8–C9) = 180°. The ROA spectra are magnified by a factor of 2,000. (B–D) ROA intensities as a function of the dihedral twists for the chromophore model of PYP. K is a constant (SI Appendix). The Inset shows the normal mode γ₈ for model 1.