Carboxyl group involvement in the meta I and meta II stages in rhodopsin bleaching. A Fourier transform infrared spectroscopic study

Abstract

Structural changes due to photoreceptor membrane bleaching can be studied by Fourier transform infrared difference spectroscopy [1,2]. In this paper we focus on the differences between rhodopsin and metarhodopsin I or II. Peaks in the 1700–1770 cm −1 region are observed, which may be produced by carbonyl groups in either carboxyl (COOH) or ester carbonyl (COOC) groups, the latter being found exclusively in membrane lipids. In order to distinguish between these two types of carbonyl groups, we have studied reconstituted membranes of rhodopsin in a synthetic phosphatidylcholine that lacks ester carbonyl groups. On this basis, we conclude that the major changes in this region are due to rhodopsin carboxyls which undergo either a change in local environment or a protonation/deprotonation reaction. Additional small changes in this region may reflect a direct involvement of phospholipids in the metarhodopsin I-to-II transition. One or more groups responsible for peaks near 1727 and 1702 cm −1 are inaccessible to the outside medium according to hydrogen/deuterium exchange. In contrast, carboxyl group(s) producing peaks near 1710, 1745 and 1768 cm −1 exchange freely with the outside medium and are therefore likely to be located near the membrane surface. Removal of a portion of the C-terminal tail region using proteinase K demonstrates that the carboxyl groups in the C-terminal sequence 248–348 are not involved directly in the rhodopsin to metarhodopsin II transition. At the meta I stage, only carboxyl peaks associated with buried groups appear, suggesting that the initial bleaching events, leading to the formation of this intermediate, produce structural rearrangements in the interior region of rhodopsin. These changes then spread to the peripheral surface regions during the metarhodopsin I-to-II transition.