Structural Changes of Cephalopod Rhodopsin and β-Arrestin Measured by FTIR Difference Spectroscopy and Isotope Editing

Abstract

Invertebrate rhodopsin is the primary photoreceptor found in the eyes of cephalopods. Importantly, unlike vertebrate rhodopsin, invertebrate rhodopsins such as sepia rhodopsin (s-Rh) can be activated by light and then rapidly cycled back to the original state with a second red-shifted photon, thereby facilitating a variety of novel biophysical studies. Additionally, invertebrate rhodopsins can bind to the ubiquitous β-Arrestin2 which is used in regulating signal transduction in many GCPRs. In this study, we used static and time-resolved FTIR difference spectroscopy to investigate the photocycle of s-Rh complexed to β-Arrestin2. In the spectrum of s-Rh alone, difference spectra obtained using two colors to cycle between the ground (rho) and acid meta state show an 11-cis to all-trans photocycle as previously described. Several bands between 1750-1700 cm-1 are assigned using a D2O induced shift to an as yet unknown carboxyl groups. Other large bands are seen especially in the amide I and II regions which indicate significant backbone structural changes. Upon addition of β-Arrestin2, the difference spectrum is altered, especially in the amide I and II regions, reflecting additional structural changes occurring in β-Arrestin2 upon photoactivation. A negative band at 1742 cm-1 was shifted higher in the complex indicating the β-Arrestin2 is perturbing at least one carboxyl group in s-Rh. In order to assign these changes, total 15N isotope labeling of β-Arrestin2 was utilized. Comparison of difference spectra from s-Rh complexes containing unlabelled and 15N labeled β-Arrestin2 reveals ∼ 3 cm-1 downshift of a negative/positive feature at 1668/1655 cm-1 indicating that these bands reflect at least partially conformational changes of the β -arrestin involving α-helical structure. These results offer a promising new tool to investigate the molecular mechanism of β-Arrestin interactions with GCPRs to regulate downstream signaling.