Photoactivation of Rhodopsin Causes an Increased Hydrogen-Deuterium Exchange of Buried Peptide Groups

Abstract

A key step in visual transduction is the light-induced conformational changes of rhodopsin that lead to binding and activation of the G-protein transducin. In order to explore the nature of these conformational changes, time-resolved Fourier transform infrared spectroscopy was used to measure the kinetics of hydrogen/deuterium exchange in rhodopsin upon photoexcitation. The extent of hydrogen/deuterium exchange of backbone peptide groups can be monitored by measuring the integrated intensity of the amide II and amide II′ bands. When rhodopsin films are exposed to D 2O in the dark for long periods, the amide II band retains at least 60% of its integrated intensity, reflecting a core of backbone peptide groups that are resistant to H/D exchange. Upon photoactivation, rhodopsin in the presence of D 2O exhibits a new phase of H/D exchange which at 10°C consists of fast (time constant ∼30 min) and slow (∼11 h) components. These results indicate that photoactivation causes buried portions of the rhodopsin backbone structure to become more accessible.