X-ray diffraction of heavy-atom labelled two-dimensional crystals of rhodopsin identifies the position of cysteine 140 in helix 3 and cysteine 316 in helix 8

Abstract

We have used site-specific heavy-atom labelling and X-ray diffraction to localize single amino acid residues in the cytoplasmic domain of the integral membrane protein rhodopsin, the dim-light photoreceptor of retinal vertebrate rod cells. Two-dimensional orthorhombic crystals of the space group p22(1)2(1) (a=59.5(+/-1) A and b=82.7(+/-1.5) A) were produced from detergent-solubilized, partially delipidated rhodopsin. To obtain milligram amounts of two-dimensional crystals, which are required for X-ray diffraction, the yield of the crystalline material was significantly increased by reconstitution of rhodopsin in the presence of cholesterol (1:2 to 1:10 mol/mol) and by adding polar organic solvents to the dialysis buffer. The native cysteine residues C140 and C316 were then selectively labelled with mercury using the sulphydryl-specific reagent p-chloromercuribenzoate (1.6-2.1 mol Hg per mol rhodopsin). The labelling did not affect the unit cell dimensions. Optical absorption spectra of labelled and native two-dimensional rhodopsin crystals showed the characteristic 11-cis-retinal peak at 498 nm, which corresponds to the dark state of rhodopsin. The in-plane position of the mercury label was calculated at 9.5 A resolution from the intensity differences in the X-ray diffraction patterns of labelled and native crystals using Fourier difference methods and the phase information from electron crystallography. The label positions were in excellent agreement with the positions of C140 at the cytoplasmic end of helix 3 and of C316 in the cytoplasmic helix 8 recently obtained from three-dimensional rhodopsin crystals. Whereas these high-resolution diffraction studies were performed under cryogenic conditions (100 K), our results were obtained at room temperature with fully hydrated membranes and in the absence of loop-loop crystal contacts. To study the structural changes of the cytoplasmic loops involved in activation and signal transduction, our more physiological conditions offer important advantages. Furthermore, the localization of C316 is the first direct proof that the electron density on top of helix 1 observed by cryo-electron microscopy is a part of the C-terminal loop. Our approach is of particular interest for investigations of other membrane proteins, for which 3D crystals are not available. Structural constraints from heavy-atom labels at strategic sites enable the assignment of a position in the amino acid sequence to features visible in a low-resolution density map and the study of conformational changes associated with different functional states of the membrane protein.