Spectroscopic Validation of Crystallographic Structures of a Protein Active Site by Chiroptical Spectroscopy

Abstract

Out-of-plane distortions of a cofactor molecule in a protein active site are functionally important, and in photoreceptors, it has been proposed that they are crucial for spectral tuning and energy storage in photocycle intermediates. However, these subtle structural features are often beyond the grasp of structural biology. This issue is strikingly exemplified by photoactive yellow protein: its 14 independently determined crystal structures exhibit considerable differences in the dihedral angles defining the chromophore geometry, even though most of these are at excellent resolution. Here we developed a strategy to verify cofactor distortions in crystal structures by using quantum chemical calculations and chiroptical spectroscopy, particularly Raman optical activity and electronic circular dichroism spectroscopies. Based on this approach, we identify seven crystal structures with the chromophore geometries inconsistent with the experimentally observed data. The strategy implemented here promises to be widely applicable to uncovering cofactor distortions at active sites and to studies of reaction intermediates.

Near-Infrared Excited Raman Optical Activity as a Tool to Uncover Active Sites of Photoreceptor Proteins

“…Although the ROA spectral features of GR and NaR were well explained from their crystal structures (cf. Figure ), we recently used the ROA spectrum to show that some crystal structures of PYP did not accurately represent the correct conformation of the p CA chromophore in solution . The crystal structures of sensory rhodopsin II were likewise found to exhibit a twist in the chromophore that contradicted with the ROA spectra in solution .…”

“…The observed spectra are dominated by bands due to the p CA chromophore, while some protein bands, such as amide I (∼1667 cm –1 ), amide III (1250∼1350 cm –1 ), and phenylalanine F12 mode (1004 cm –1 ), are discerned . The assignment of the chromophore bands was confirmed by extensive isotope editing experiments. ,, Using DFT calculations (traces c and d) based on a simplified active site model, the ROA spectrum was shown to originate from the distorted C4C7C8C9O moiety, and the conformation was determined.…”

“…Experimental Raman and ROA spectra of PYP in comparison with the calculated spectra (at the B3LYP/6-31+G** level) . (a and b) Experimental Raman and ROA spectra, respectively.…”

“…As depicted in Figure , PYP exhibits two intense ROA bands ascribed to the ethylenic hydrogen out-of-plane (HOOP) modes, γ 2 (984 cm –1 ) and γ 8 (825 cm –1 ). ,, We performed systematic DFT calculations to explore what structural factors affect the sign and intensity of the HOOP bands . We focused on three dihedral angles τ­(C3–C4– C7–C8), τ­(C4–C7–C8–C9), and τ­(C7–C8–C9 = O).…”

“…From this perspective, we present general principles for ROA and briefly describe ROA instruments. We will then discuss several research topics where ROA spectroscopy has been instrumental in uncovering the active site structures of photoreceptor proteins. ,− …”