Cross-linking of bovine rhodopsin with sulfosuccinimidyl 4-(N maleimidomethyl)cyclohexane-1-carboxylate affects its functionality.

Rhodopsin is the photoreceptor protein involved in visual excitation in retinal rods. The functionality of bovine rhodopsin was determined following treatment with sulfosuccinimidyl 4-(N maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC), a bifunctional reagent capable of forming covalent cross-links between suitable placed lysines and cysteines. Denaturing polyacrylamide gel electrophoresis showed that rhodopsin incubated with sulfo-SMCC generated intermolecular dimers, trimers, and higher oligomers, although most of the sulfo-SMCC-treated protein remained as a monomer. Minor alterations on the absorption spectrum of light-activated sulfo-SMCC-treated rhodopsin were observed. However, only about 2% stimulation of the guanine nucleotide binding activity of transducin was measured in the presence of sulfo-SMCC-cross-linked photolyzed rhodopsin. Moreover, rhodopsin kinase was not able of phosphorylating sulfo-SMCC-cross-linked rhodopsin after illumination. Rhodopsin was purified in the presence of either 0.1% or 1% n-dodecyl β-D-maltoside, to obtain dimeric and monomeric forms of the protein, respectively. Interestingly, no generation of the regular F1 and F2 thermolytic fragments was perceived with sulfo-SMCC-cross-linked rhodopsin either in the dimeric or monomeric state, implying the formation of intramolecular connections in the protein that might thwart the light-induced conformational changes required for interaction with transducin and rhodopsin kinase. Structural analysis of the rhodopsin three-dimensional structure suggested that the following lysine and cysteine pairs: Lys66/Lys67 and Cys316, Cys140 and Lys141, Cys140 and Lys248, Lys311 and Cys316, and/or Cys316 and Lys325 are potential candidates to generate intramolecular cross-links in the protein. Yet, the lack of fragmentation of sulfo-SMCC-treated Rho with thermolysin is consistent with the formation of cross-linking bridges between Lys66/Lys67 and Cys316, and/or Cys140 and Lys248.

.However, an incomplete formation of cross-linked products was generally observed, and plenty of the monomeric form of Rho still remained in the sample.

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Immunostaining using 1D4 monoclonal antibodies confirmed the generation of 256 dimers, trimers and oligomeric forms of Rho, in addition to the Rho monomeric 257 species (data not shown).

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Both Rho and sulfo-SMCC-cross-linked Rho showed the typical absorption 259 spectrum in the dark with a maximum peak at 500 nm (Fig. 2). Following 260 illumination for 7 min, at room temperature, this band was shifted to 380 nm that 261 corresponded to the metarhodopsin II activated state (Fig. 2). Therefore, the 262 ultraviolet/visible absorption spectra of sulfo-SMCC-modified Rho (dark state) and Rho sample (Fig. 2), which appears to indicate that more than two species were in 268 equilibrium after photolysis in the sample modified with the bifunctional reagent.

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With the intention of tracking spectral modifications in the cross-linked sample, the 270 absorption spectrum of sulfo-SMCC-treated Rho was measured at 4 ºC using a 271 spectrophotometer that was refrigerated with an in-line cooling circulating water 272 bath. As shown in Fig. 3, an altered spectrum was obtained for sulfo-SMCC-cross-273 linked Rho*, at 4 ºC, and after 1 min of illumination. Instead of a peak absorbing at 274 380 nm, a photointermediate species absorbing at about 470 nm was detected 275 (Fig. 3), with a concomitant reduction of the dark species that absorbs at 500 nm. 276 Apparently, the incorporation of the covalently bound cross-linking reagent seems 277 Downloaded from https://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200376/883255/bcj-2020-0376.pdf by guest on 09 June 2020 Biochemical Journal. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version is available at https://doi.org/10.1042/BCJ20200376 to hinder the light-induced conformational change in the photoreceptor protein, 278 probably stabilizing an inactive photointermediate.

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Given that sulfo-SMCC is capable of forming bridges between suitable 280 located Cys and Lys residues, purified Rho samples were pre-incubated either with 281 NEM to modify cysteines, or with PITC to modify lysines, or with both compounds, 282 in order to determine whether these site-specific compounds hindered the band, which implied that the higher size was probably due to the modification of 296 individual cysteyl or lysyl residues in the protein (Fig. 1).

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The ability of sulfo-SMCC-cross-linked Rho* to stimulate the guanine 298 nucleotide binding activity of T was assayed as shown in Fig. 4A. [ 3 H] GMPpNp 299 binding by bovine T was measured after light-dependent activation of either 300 untreated or sulfo-SMCC-treated Rho. As anticipated, Rho* was capable of activating T (Fig. 4A). However, only about 2% stimulation of the guanine 302 nucleotide binding activity of T was detected in the presence of sulfo-SMCC-cross-303 linked Rho* (Fig. 4A). These results clearly showed that treatment with sulfo-304 SMCC affected the light-dependent capability of the photoreceptor protein to 305 activate T.

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The C-terminal tail of Rho possesses a cluster of Ser and Thr residues that 307 are exposed following illumination, and can be rapidly phosphorylated by rhodopsin   In an attempt to assess whether intramolecular cross-links can be generated  distance of 12 Å (Fig. 9). In all these cases, the separating distances between the 494 candidate amino acid residues are very close to the cross-linker's length of 11.6 Å, 495 which suggests that sulfo-SMCC can fit and be easily inserted. Interestingly, the 496 thermolysin cleaving site (carboxylic side of Ser 240 ) is also contained within the 497 region that ligates these last pairs of potential targeted residues. Hence, if these 498 residues are covalently connected intramolecularly by sulfo-SMCC-mediated 499 cross-linking bonds, the F1 and F2 fragments will not be separated even if they are 500 generated after hydrolysis with the thermolysin protease. As said before, another