Structure and function in rhodopsin: destabilization of rhodopsin by the binding of an antibody at the N-terminal segment provides support for involvement of the latter in an intradiscal tertiary structure

Abstract

A monoclonal anti-rhodopsin antibody (B6-30N), characterized by Hargrave and coworkers [Adamus, G., Zam, Z. S., Arendt, A., Palczewski, K., McDowell, J. M. & Hargrave, P. (1991) Vision Res. 31, 17-31] as recognizing a short peptide sequence at the N terminus, failed to bind to rhodopsin when the latter was solubilized in dodecylmaltoside (DM). Of the detergents tested thus far, DM affords maximum stability to rhodopsin. Solubilization of rhodopsin in cholate allowed binding of the antibody, but the binding caused destabilization as evidenced by the accelerated loss of absorbance at 500 nm. The result provides support for the earlier conclusion that the N-terminal segment is an integral part of a tertiary structure in the intradiscal domain of native rhodopsin coupled to a tertiary structure in the transmembrane domain. Additional comparative studies on the stability of rhodopsin in different detergents were carried out after direct solubilization from rod outer segments and after extensive treatments to remove the endogenous phospholipids. Purification of rhodopsin in DM resulted in essentially quantitative removal of endogenous phospholipids. When rhodopsin thus purified was treated with the above antibody in DM and in cholate, enhanced destabilization (5-fold) was observed in the latter detergent.

Figure 1

The location of the epitopes of monoclonal anti-rhodopsin antibodies 1D4 and B6–30N on a secondary structure model of bovine rhodopsin. The lengths of the transmembrane (TM) helices are as deduced by Schertler and coworkers (21) from electron diffraction studies. The solid bar next to P₁₄₂ shows the end of the N-terminal fragment, noncovalently linked to the remainder of the opsin sequence (10). The N-terminal fragment N₂–M₃₉ is also shown. The dashed line shows the conserved disulfide bond between C₁₁₀ and C₁₈₇.

Figure 2

Kinetics of binding of opsin, N-terminal fragments of rhodopsin, and rhodopsin to the antibody B6–30N in different detergents. (A) Binding of opsin and N-terminal fragments: ³⁵S-labeled M₁-P₁₄₂ (I), ³H-labeled N₂-M₃₉ (II), and opsin (III), respectively_. (B) Binding of rhodopsin: binding of A₅₀₀ (I) and binding of ³⁵S-labeled rhodopsin (II). The detergents used were DM (open circles), cholate (closed circles), and a mixture of DM (0.1%) and cholate (2% vol/vol) (triangles).

Figure 3

Kinetics of decrease of A₅₀₀ in rhodopsin samples solubilized in different detergents. For details, see text.

Figure 4

Kinetics of decrease of A₅₀₀ in rhodopsin samples purified and kept in different detergents. For details, see text.

Figure 5

TLC analysis of residual PLs in rhodopsin after purification and extensive washing with different detergents (details in text). Lane 1, total PLs from HEK293S cells; lanes 2–5, residual PL after purification and extensive washings in the detergents shown. Mobilities of control samples of phosphatidylserine (PS), phosphatidylcholine (PC), and phosphatidylethanolamine (PE) are shown. Quantitation of radioactive PC remaining is shown in Table 2.

Figure 6

Stability of purified rhodopsin in detergents in the absence and presence of mAb B6–30N. For details, see text.