This report describes screening of different detergents for preparing the visual GPCR, rhodopsin, and its complex with mini-Go. Biochemical methods characterizing the quality of the complex at different stages during purification are demonstrated. This protocol can be generalized to other membrane protein complexes for their future structural studies.
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.
Determining crystal structures of membrane proteins and their complexes has always been challenging due to difficulties in obtaining well-diffracting crystals. In contrast to soluble proteins, integral membrane proteins comprise a hydrophobic core that spans the cell membrane. To remove membrane proteins from the cell membrane into aqueous buffer, detergents have to be used to form a detergent-protein micelle, thus replacing the lipids around the hydrophobic core of membrane proteins. Stability, activity and integrity of membrane proteins are directly dependent on the chemical and structural properties of the detergent1, and the detergent's properties also determine the size of the micelle. A large detergent micelle may occlude the hydrophilic surfaces of a small membrane protein, thus preventing crystallization due to the lack of crystal contacts when using vapor diffusion method. A small detergent micelle is advantageous for crystallography, but short chain detergents are usually harsher and therefore lead to destabilization and aggregation of the membrane protein. Therefore, before crystallization, an additional detergent screening procedure is indispensable, typically targeting shorter detergents that still maintain protein stability.
G protein-coupled receptors (GPCRs) are integral membrane proteins containing seven transmembrane a-helices. GPCRs exist in two main states, either an inactive state stabilized by inverse agonists or antagonists, or an active state bound to an agonist and stabilized by a G protein, although it is likely that a multitude of sub-states exist between these two extremes. Structure determination of GPCRs initially focused on inactive states bound to inverse agonists and antagonists due to their higher stability than active states2. When GPCRs are activated upon agonist binding, the receptors are highly dynamic, and a cleft forms transiently on the cytoplasmic face of the receptor for G protein coupling. It is thought that this dynamism is why agonist-bound GPCRs are often more unstable than the inactive state. Therefore, it becomes essential to screen for detergents appropriate for the conformational state of the receptor under study, because it is likely that milder detergents will be required for studying an active state compared to an inactive state.
In this report, we use the visual GPCR, bovine rhodopsin3, and its complex with mini-Go protein4,5 for the detergent screening experiments, representing the inactive state and active state, respectively. The detergent screening focused on the classical alkyl maltoside and glucoside detergents and the neopentyl glycol (NPG) detergents. In this context, a classical detergent is built from a sugar head group and an alkyl chain, while the NPG type detergents contains two identical classical detergents that are fused by a quaternary carbon at the interface between the sugars and the alkyl chains6,7,8.
An experimental workflow was designed starting from the purification of rhodopsin in different detergents, followed by formation of the rhodopsin–mini-Go complex and ending with the characterization of the complex using several methods (Figure 1). For the inactive state of rhodopsin, reconstitution of the light sensitive ligand 9-cis retinal was monitored by ultraviolet-visible (UV-VIS) spectroscopy. The spectrum reveals the physicochemical state of the retinal and is indicative of its environment in the retinal binding pocket of rhodopsin. Size exclusion chromatography (SEC) was employed to assess monodispersity of purified rhodopsin as well as formation of the rhodopsin–mini-Go complex. As SEC differentiates protein molecules by their size and shape, aggregated protein population can be identified as they elute in the void volume. To confirm complex formation, fractions from SEC were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to confirm the presence of both rhodopsin and mini-Go.
Another factor that needs to be considered is post-translational modifications (PTM) on the membrane proteins. PTM such as N-glycosylation are often observed on eukaryotic membrane proteins produced in mammalian and insect cell expression systems. A limited N-glycosylation strain of the human embryonic kidney 293 (HEK293) cells was developed by deletion of the gene encoding N-acetylglucosaminyltransferase I (GnTI), resulting in homogenous N-glycosylation by GlcNAc2Man5 at the consensus site Asn-X-Ser/Thr. Although N-glycosylation can be prevented by mutating an amino acid residue in the consensus site, this may also alter the function of the protein or the efficiency of folding. In bovine rhodopsin, mutation of the N-glycosylated residue Asn15 leads to incorrect folding and reduced G protein activation9,10. The rhodopsin used in this report was expressed in HEK 293 GnTI-deficient cell line. However, SDS-PAGE showed the presence of two species of rhodopsin. This heterogeneity could prevent crystal formation and therefore deglycosylation using peptide-N-glycosidase F (PNGase F) and endoglycosidase F1 (Endo F1) was tested. The deglycosylated product was characterized by SDS-PAGE and liquid chromatography-mass spectrometry (LC-MS) to identify the level of glycosylation and its homogeneity.
The success in protein crystallization strongly relies on the protein sample, especially membrane proteins and their complexes due to the complication caused by detergents. This report demonstrates detergent screening and evaluation of sample quality for GPCR–mini-G protein signaling complexes. A variety of methods have been widely used to study the biochemical property of membrane proteins, for example, thermostability assay using fluorescent dyes16,17, binding assay to detect complex formation by measuring the change in tryptophan fluorescence signal18 or the resonance energy transfer with biosensors19. However, the chemical environments used in those methods are quite different from those for preparing a crystallization sample, either proteins are at a thousand-fold lower concentration for fluorescence-based measurement, or proteins are embedded in lipid bilayers or in one fixed detergent condition. In this protocol, the used methods are also standardized in large-scale sample preparation before crystallization. Therefore, the optimized parameters can be easily transferred for crystallization-scale preparation without further major screening and optimization.
The aim of this protocol is to optimize the preparation of a stable and homogeneous GPCR–mini-G protein complex for vapor diffusion crystallization and structure determination by X-ray crystallography. The protocol integrates a set of methods to qualitatively evaluate the impact of detergent and deglycosylation during preparation of the rhodopsin–mini-Go complex. Rhodopsin at inactive state and light-activated state bound with and without the transducin peptide has been crystallized when purified in the detergents octyl glucoside (C8G)20,21,22 and C9G23,24. As the rhodopsin–mini-Go complex purified in C8G and C9G did not yield crystals (data not shown), we then explored a wider range of other detergents using the strategy described (Figure 1). By taking the advantage of the light sensitivity of rhodopsin, we could very well follow the reconstitution of retinal at wavelengths other than 280 nm. In both UV-VIS spectroscopy and SEC, we detected retinal at either 380 nm or 488 nm. However, most membrane proteins do not have such a convenient chromophore to follow functionality during purification. Other options would be to make a ligand detectable by adding a light-detectable chromophore or by using radioligand-binding and thermal shift assays25.
Rhodopsin has a molecular weight of 40 kDa. Due to the mass of detergent it binds, its apparent molecular weight on SEC is about 120 kDa. It is thus no surprise that the binding of mini-Go (24 kDa) was not easily detected on SEC, as this would necessitate differentiation of proteins with apparent masses of 120 kDa and 144 kDa. Analysis of SEC fractions by SDS-PAGE was therefore used to confirm sample purity and complex formation. Even if SEC profiles show a clear shift upon complex formation, it is still recommended to perform SDS-PAGE analysis to confirm the complex formation with correct binding partners rather than other co-purified protein contaminants.
Both rhodopsin and mini-Go were purified in milligram quantities, which allowed the use of low sensitivity detection of the complexes, such as UV-VIS absorption during SEC and Commassie Blue staining of SDS-PAGE gels. Where samples are limited, more sensitive detection should be used, such as an LC purifier equipped with a fluorescence detector to trace tryptophan signals from the protein (280 nm excitation, 350 nm emission) and silver-staining for SDS-PAGE gels. Another approach would be to fuse a fluorescent protein, such as green fluorescence protein (GFP) to the protein of interest, which would also allow detection even during protein expression26 but it should be removed before crystallization.
It is essential to ensure that the purified protein is also free of heterogeneity arising from variable PTMs. In the case described here, the two populations of rhodopsin observed on SDS-PAGE gels were characterized as having either one or two N-glycans. Variable modification of a protein would potentially prevent the formation of well-diffracting crystals, so we therefore deglycosylated rhodopsin. The endoglycosidase Endo F1 was the most effect endoglycosidase tested and treatment led to a single species of unglycosylated receptor, while PNGase F only partially removed the glycans on rhodopsin and resulted in a mixture of rhodopsin fully unglycosylated or with one N-glycan remained. Rhodopsin without deglycosylase treatment has been successfully crystallized3,27,28, and the N-glycan on rhodopsin Asn15 is important to form crystal contact in those cases. In the case of rhodopsin–mini-Go, it is necessary to remove N-glycans by Endo F1 to obtain crystals. There is no standardized rule to deglycosylate proteins of interest before crystallization, but removal of heterogenous PTMs should be considered when proteins fail to crystallize after extensive crystallization trials.
The data and methodology described here guided us to choose OGNG as the most preferred detergent for crystallization of the rhodopsin–mini-Go complex due to its small micelle size and its ability to stabilize the complex. We also used Endo F1 to ensure the purified rhodopsin was a homogeneous species. Crystals were subsequently obtained and we determined the crystal structure to ~3.1 Å4, which was only the third crystal structure of a GPCR–G protein signaling complex14,29.
For membrane proteins bound with and without a partner protein, they should be considered as two different proteins. A protein at different functional states has different conformations and is at different energy level. Therefore, it is recommended to optimize the preparation protocol for each functional state as the parameter for the inactive-state may not be fully transferrable to the activated state. Also, not to mention the change in the protein property complicated by binding a partner protein. The protocol uses methods that are standardized for preparing a crystallization sample to prepare inactive membrane protein in different detergents, followed by protein activation and complex formation, and to characterize protein quality. Thus, this protocol can easily be generalized to other membrane proteins and their complexes for structural studies with minor modification.
The authors have nothing to disclose.
We thank Prof. Dr. Gebhard F. X. Schertler for his long-term support in this project, Dr. Roger J.P. Dawson and Hoffmann La Roche for support in cell culture. This work was sponsored by Swiss National Science Foundation (grants 210030_153145 and 310030B_173335 to GFXS), and funding to CGT from the European Research Council (EMPSI, 339995) and the Medical Research Council (MRC U105197215). FP acknowledges ETH Zürich through the National Center of Competence in Research Molecular Ultrafast Science and Technology (NCCR MUST) and the ETH Femtosecond and Attosecond Science and Technology (ETH FAST) programs. FP, JM, AB and CJT acknowledge long-term financial support from the Paul Scherrer Institute.
1D4 peptide | Peptide2.0 | Under request | |
9-cis retinal | Sigma-Aldrich | R5754 | |
Autosampler A-900 | GE Healthcare | Discontinued | |
C9G | Anatrace | N324 | |
cOmplete, EDTA-free protease inhibitor coctail | Roche | 5056489001 | |
Cymal-5 | Anatrace | C325 | |
Cymal-5NG | Anatrace | NG325 | |
Cymal-6 | Anatrace | C326 | |
Cymal-6NG | Anatrace | NG326 | |
DDM | Anatrace | D310 | |
DM | Anatrace | D322 | |
DMNG | Anatrace | NG322 | |
Econo column | Bio-Rad | 7372512 | |
Ettan LC | GE Healthcare | Discontinued | |
FRAC-950 | GE Healthcare | Discontinued | |
HPLC Water 2795 Separation Module | Waters AG | 720000358EN | |
InstantBlue Protein Stain | Expedeon | ISB1L | |
LCT Premier mass spectrometer (ESI-TOF) | Waters AG | – | |
LMNG | Anatrace | NG310 | |
Monitor UV-900 | GE Healthcare | 18110835 | |
Nanodrop 1000 | Witec AG/ThermoFisher | Discontinued | |
NuPAGE 4-12% Bis-Tris gel 1.0 mm, 15 well | ThermoFisher | NP0323BOX | |
NuPAGE MES SDS buffer (20x) | ThermoFisher | NP0002 | |
OGNG | Anatrace | NG311 | |
PAGEr Minigel Chamber | Lonza | 59905 | |
Reprosil 200 C18-AQ column | Morvay Analytik GmbH | #s1503 | |
Superdex 200 Increase GL column | GE Healthcare | 28990944 | |
Tabletop centrifuge 5424R | Eppendorf | 5404000413 | |
Ultracentrifuge Optima XE-100 | Beckmann Coulter | A94516 | |
ULTRA-TURRAX T25 | IKA WERKE | 0003725003 | |
UV-VIS spectrophotometer | Shimadzu | UV-2401PC | |
Waters 2487 Dual λ Absorbance Detector | Waters AG | – |