Abstract
The key to determining crystal structures of membrane protein complexes is the quality of the sample prior to crystallization. In particular, the choice of detergent is critical, because it affects both the stability and monodispersity of the complex. We recently determined the crystal structure of an active state of bovine rhodopsin coupled to an engineered G protein, mini-Go, at 3.1 Å resolution. Here, we detail the procedure for optimizing the preparation of the rhodopsin–mini-Go complex. Dark-state rhodopsin was prepared in classical and neopentyl glycol (NPG) detergents, followed by complex formation with mini-Go under light exposure. The stability of the rhodopsin was assessed by ultraviolet-visible (UV-VIS) spectroscopy, which monitors the reconstitution into rhodopsin of the light-sensitive ligand, 9-cis retinal. Automated size-exclusion chromatography (SEC) was used to characterize the monodispersity of rhodopsin and the rhodopsin–mini-Go complex. SDS-polyacrylamide electrophoresis (SDS-PAGE) confirmed the formation of the complex by identifying a 1:1 molar ratio between rhodopsin and mini-Go after staining the gel with Coomassie blue. After cross-validating all this analytical data, we eliminated unsuitable detergents and continued with the best candidate detergent for large-scale preparation and crystallization. An additional problem arose from the heterogeneity of N-glycosylation. Heterologously-expressed rhodopsin was observed on SDS-PAGE to have two different N-glycosylated populations, which would probably have hindered crystallogenesis. Therefore, different deglycosylation enzymes were tested, and endoglycosidase F1 (EndoF1) produced rhodopsin with a single species of N-glycosylation. With this strategic pipeline for characterizing protein quality, preparation of the rhodopsin–mini-Go complex was optimized to deliver the crystal structure. This was only the third crystal structure of a GPCR–G protein signaling complex. This approach can also be generalized for other membrane proteins and their complexes to facilitate sample preparation and structure determination.
Highlights
Determining crystal structures of membrane proteins and their complexes has always been challenging due to difficulties in obtaining welldiffracting crystals
UV-VIS spectroscopy revealed rhodopsin stability Stability of the retinal-reconstituted rhodopsin was assessed by its optical absorbance (Figure 2)
The samples purified in the neopentyl glycol (NPG) detergents (LMNG, decyl maltose neopentyl glycol (DMNG), Cymal-6NG, Cymal-5NG) show optical profiles suggesting a suboptimal binding environment for retinal except for the octyl glucose neopentyl glycol (OGNG) sample, which gave the same optical profile as the dodecyl maltoside (DDM) sample
Summary
Determining crystal structures of membrane proteins and their complexes has always been challenging due to difficulties in obtaining welldiffracting crystals. Activity and integrity of membrane proteins are directly dependent on the chemical and structural properties of the detergent[1], and the detergent's properties determine the size of the micelle. We use the visual GPCR, bovine rhodopsin[3], and its complex with mini-Go protein[4,5] for the detergent screening experiments, representing the inactive state and active state, respectively. NOTE: For 10% detergent stock solution, dissolve 1 g of detergent powder in ultrapure water with gentle rocking, and adjust the final volume to 10 mL. The 1D4 immunoaffinity agarose works as affinity purification material to capture proteins that contain a C-terminal 1D4 sequence This purification material can be prepared[9,11] or purchased. Prepare 1D4 peptide (sequence TETSQVAPA): 800 μM, dissolved in water
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