Article

Cite This: Langmuir 2019, 35, 4319−4327 pubs.acs.org/Langmuir

A Chemical Strategy for Replacement in Membrane Research † ‡ § ∥ † § ∥ ⊥ § # ¶ † ‡ § ∥ † Dongxiang Xue, , , , Jingjing Wang, , , , Xiyong Song, , Wei Wang, Tao Hu, , , , Lintao Ye, † † † ‡ § ∥ ¶ † ∥ Yang Liu, Qingtong Zhou, Fang Zhou, , , , Zhong-Xing Jiang, Zhi-Jie Liu,*, , † and Houchao Tao*, † iHuman Institute, ShanghaiTech University, Ren Building, 393 Middle Huaxia Road, Shanghai 201210, China ‡ Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, China § University of Chinese Academy of Sciences, No. 19A, Yuquan Road, Beijing 100049, China ∥ School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China ⊥ Institute of and Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China # National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China ¶ Hubei Province Engineering and Technology Research Center for Fluorinated Pharmaceuticals, School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, China

*S Supporting Information

ABSTRACT: Membrane mimics are indispensable tools in the structural and functional understanding of membrane (MPs). Given stringent requirements of integral MP manipulations, amphiphile replacement is often required in sample preparation for various biophysical purposes. Current protocols generally rely on physical methodologies and rarely reach complete replacement. In comparison, we report herein a chemical alternative that facilitates the exhaustive exchange of membrane-mimicking systems for MP reconstitution. This method, named sacrifice-replacement strategy, was enabled by a class of chemically cleavable detergents (CCDs), derived from the disulfide incorporation in the traditional detergent n- dodecyl-β-D-maltopyranoside. The representative CCD behaved well in both solubilizing the diverse α-helical human G protein- coupled receptors and refolding of the β-barrel bacterial outer X, and more importantly, it could also be readily degraded under mild conditions. By this means, the A2A adenosine receptor was successfully reconstituted into a series of

Downloaded via INST OF BIOPHYSICS on May 16, 2019 at 02:52:40 (UTC). commercial detergents for stabilization screening and nanodiscs for electron microscopy analysis. Featured by the simplicity and compatibility, this CCD-mediated strategy would later find more applications when being integrated in other biophysics studies. See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. ■ INTRODUCTION detergents exhibited favorable stabilization properties, their applications suffer from poor solubilization or accessibil- Membrane proteins (MPs) are highly pursued pharmaceutical 3,9−11 targets because of their important biological roles in ity. Similarly, bilayer systems, like liposomes, bicelles, information and material exchange between and within and nanodiscs, are deemed to maintain MPs in a more native- cells.1,2 Nevertheless, the exploration of MPs has been like state, which however are neither amenable to extraction, and thus, MP reconstitution has to commence with a challenging compared to soluble proteins, as indicated in the 3,9−13 protein data bank (PDB) where MPs only contribute to about detergent-solubilized . Therefore, amphiphile 2% of known protein structures.3 One particular challenge is replacement is often required in reconstitution of challenging the reconstitution of MPs in a proper mimicking membrane MPs in an ideal mimic system to facilitate further studies. system that facilitates subsequent biochemical and biophysical Current detergent replacement protocols primarily involve − characterizations.4 7 in a physical methodology by means of dilution, dialysis, or 3,4,12,14 Over the past decades, various membrane mimic cassettes BioBeads adsorption. Nevertheless, these physical have been developed to prompt in vitro MP studies. Detergents are the most commonly used lipid surrogates, Received: December 7, 2018 which usually have unmatched solubilization but suboptimal Revised: February 10, 2019 stabilization to MPs.8 Though some conceptually novel Published: February 19, 2019

© 2019 American Chemical Society 4319 DOI: 10.1021/acs.langmuir.8b04072 Langmuir 2019, 35, 4319−4327 Langmuir Article

Figure 1. In situ sacrifice-replacement strategy for reconstitution of MPs mediated by CCDs. approaches usually fail to achieve a thorough and clean centrifugation at 8000g for 20 min and stored at −80 °C until exchange. For example, dilution and dialysis can only minimize purification. The cell pellet was lysed in the lysis buffer (100 mM NaCl, 0.5 mM diamine tetraacetic acid (EDTA), 20 mM the of the original detergents, and thus, the fi second mimic system will inevitably be contaminated with Tris-HCl, pH 7.4) by sonication. The lysate was then clari ed at 35 residual detergents. As for physical adsorption, BioBeads are 000g for 30 min and loaded onto a nickel nitrilotriacetic acid (NTA) affinity column. The column was washed sequentially with 75 mL of supposedly able to distinguish detergents from lipids, but each following buffer: (i) 100 mM NaCl, 0.5 mM EDTA, 30 mM actually the selectivity is much less desirable (Figure S1). In imidazole, 20 mM Tris-HCl, pH 7.4; (ii) 100 mM NaCl, 0.5 mM addition, when dealing with fragile MPs such as G protein- EDTA, 50 mM imidazole, 20 mM Tris-HCl, pH 7.4; and (iii) 100 coupled receptors (GPCRs), laborious optimization might be mM NaCl, 0.5 mM EDTA, 100 mM imidazole, 20 mM Tris-HCl, pH required in a preliminary stage for MP reconstitution.12,15 7.4. Finally, MSP2N2 was eluted from the resin with buffer (100 mM Tedious sample-handling steps could easily arise protein NaCl, 0.5 mM EDTA, 400 mM imidazole, 20 mM Tris-HCl, pH 7.4) aggregations. and dialyzed against the dialysis buffer (100 mM NaCl, 0.5 mM As an alternative to conventional physical methods, we EDTA, 20 mM Tris-HCl, pH 7.4) overnight at 4 °C. Meanwhile, the present herein a chemical methodologythe sacrifice- N-terminal His-tag was removed using TEV . The cleaved replacement strategyfor facile and exhaustive detergent MSP2N2 was separated from the His-tag by an additional Ni-NTA purification step. The MSP2N2 protein was further filtered through a replacement during MP reconstitution. This strategy is enabled 0.22 μm syringe filter and stored at −80 °C until use. by a novel series of chemically cleavable detergents (CCDs). In Δ G Protein-Coupled Receptors. The MBP-A2AR-BRIL- C con- the sacrifice-replacement strategy (Figure 1), the selected CCD Δ struct was designed based on the sequence of the A2AR-BRIL- C extracts MPs from biological membranes and then is readily construct18 (PDB ID: 4EIY) by adding maltose-binding protein replaced with new detergents or lipids by a mild decom- (MBP) as a fusion protein at the N-terminal. The modified position. Using this strategy, we successfully conducted an thermostabilizing apocytochrome b562RIL (BRIL) acting as a fusion economical detergent screening for the A2A adenosine receptor partner was inserted into the third intracellular loop (IL3) between (A R) and established a new nanodisc preparation protocol Lys209 and Gly218. A recombinant baculovirus was made from 2A Δ pFastbac1-MBP-A2AR-BRIL- C using the Bac-to-Bac system (In- that facilitates further electron microscopy (EM) analysis. 19 Δ These proof-of-concept applications demonstrate the signifi- vitrogen). The MBP-A2AR-BRIL- C protein was expressed in baculovirus-infected Sf 9 insect cells as described previously.20 Sf 9 cant potential of this chemical methodology in reconstituting insect cells were prepared at a density of 2 × 106 cells/mL with P1 MPs for various applications. virus at a multiplicity of infection of 5 and suspended in 5 L of the IPL-41/SF900 II complex media or ESF921 insect media. After 48 h, ■ EXPERIMENTAL SECTION the remaining cells were centrifuged at 6000g for 10 min and Synthesis and Characterization of CCDs. Starting from the harvested. The other engineered constructs of GPCRs in this context 18,21,22 fi commercially available peracetylated maltosylbromide 1, CCDs with a were expressed as previously described. In brief, for puri cation, ff variety of lengths (CCD-1−4) were easily obtained by a scalable cell pellets were homogenized by douncing them in a hypotonic bu er three-step procedure with a total yield of 51−63% (Scheme S1). (10 mM MgCl2, 20 mM KCl, protease inhibitor cocktail tablets Nuclear magnetic resonance (NMR) spectra were recorded on a (Roche), 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid Bruker AVANCE III 500 or 800 spectrometer (FT, 500/800 MHz for (HEPES), pH 7.5), and then precipitated by centrifugation at 35 000 1H NMR; 126/201 MHz for 13C NMR) at room temperature with rpm for 30 min. Repeat the above homogenization procedures again in hypotonic buffer and three times in osmotic buffer (1 M NaCl, 10 CDCl3 or CD3OD as the solvent. High-resolution mass spectra were recorded on an Agilent 6230 mass spectrometer using electrospray mM MgCl2, 20 mM KCl, 10 mM HEPES, pH 7.5) to give GPCR- ionization. Synthetic procedures, characterization data, and NMR containing membrane preparations. Membrane preparations were ff spectra of all CCDs are shown in the Supporting Information. All resuspended in suspension bu er (10 mM MgCl2, 20 mM KCl, 30% μ other reagents applied in this context were obtained from commercial (v/v) glycerol, 100 M ligand, 2 mg/mL iodoacetamide, 10 mM ° suppliers (Adamas, Anatrace, Accela, Aladdin, J&K, Sigma, etc.) and HEPES, pH 7.5) and incubated at 4 C for 1 h before being used without further purification. solubilized in buffer (800 mM NaCl, 1% (w/v) detergent, 50 mM Protein Expression and Purification. Membrane Scaffold HEPES, pH 7.5) for 3 h. Supernatants were isolated by ultra- Protein. The membrane scaffold protein (MSP) MSP2N2, which centrifugation and incubated overnight in 20 mM imidazole with the contains a 7-His-tag and a TEV protease site on the N-terminus, is TALON immobilized metal affinity chromatography (IMAC) resin expressed and purified as previously described.16,17 The plasmid (Clontech). The protein-bounded resin was sequentially washed with pMSP2N2 (AddGene) was transformed into Escherichia coli the wash buffers: (i) 800 mM NaCl, 10% glycerol, 8 mM ATP, 10 μ × BL21GOLD (DE3) and the related protein expression was induced mM MgCl2, 20 mM imidazole, 100 M ligand, 10 critical in Terrific Broth by addition of isopropyl β-D-1-thiogalactopyranoside concentration (CMC) detergent, 50 mM HEPES, pH 7.5; (ii) 500 μ to 1 mM at an OD600 of 1. After about 3 h, cells were harvested by mM NaCl, 10% glycerol, 10 mM MgCl2, 20 mM imidazole, 50 M

4320 DOI: 10.1021/acs.langmuir.8b04072 Langmuir 2019, 35, 4319−4327 Langmuir Article ligand, 10 × CMC detergent, 50 mM HEPES, pH 7.5; and (iii) 500 the following traditional physical protocols12,26 or the alternative mM NaCl, 10% glycerol, 20 mM imidazole, 100 μM ligand, 5 × CMC chemical protocols. detergent, 50 mM HEPES, pH 7.5) before GPCRs were eluted with Traditional Physical Protocols for Removal of Detergents. elute buffer (500 mM NaCl, 10% glycerol, 250 mM imidazole, 100 BioBeads SM-2 (Bio-Rad) was prepared by suspending in methanol, μM ligand, 2.5 × CMC detergent, 25 mM HEPES, pH 7.5) in a washing with several volumes of methanol, and rinsing with large minimal volume. The ligands for A2AR(A2A adenosine receptor), amounts of Milli-Q-treated water (Millipore). The above prepared − − GLP-1R (glucagon-like peptide-1 receptor), and SMO (smoothened BioBeads SM2 was added to the A2AR DDM mixture (0.2 0.3 g receptor) were ZM241385 (4-[2-[[7-amino-2-(2-furanyl)[1,2,4]- BioBead/mL mixture) and incubated at 4 °C overnight with constant triazolo[1,5-a][1,3,5]triazin-5-yl]amino]ethyl]), PF-06372222 rotation. Finally, BioBeads were removed by centrifugation. (N-{4-[(R)-(3,3-dimethylcyclobutyl)({6-[4-(trifluoromethyl)-1H- Chemical Protocols for Removal of Detergents. TCEP (5 equiv 15 − imidazol-1-yl]pyridin-3-yl} amino)methyl]benzene-1-carbonyl}- to the molar concentration of CCD-2 in the A2AR CCD2 mixture) beta-alanine), and LY2940680 (4-fluoro-N-methyl-N-[1-[4-(1-meth- was added to the protein mixture and incubated at 4 °C with constant yl-1H -pyrazol-5-yl)-1-phthalazinyl]-4-piperidinyl]-2- rotation for 2 h before further purification and analysis. (trifluoromethyl)benzamide), respectively. The detergent refers to n- The nanodisc-containing receptors were loaded onto a nickel NTA dodecyl-β-D-maltopyranoside (DDM) or CCDs. column and washed with buffer (25 mM HEPES, 150 mM NaCl, 20 Refolding of Outer Membrane Protein X (OmpX). Outer MP mM imidazole 100 μM ZM241385, pH 7.5). Then, the reconstitution X (OmpX) was expressed and purified in denature buffer (6 M urea, mixture was eluted by 10 column volumes of elute buffer (150 mM − 20 mM Tris, pH 8.5) according to previous reported procedures.23 25 NaCl, 200 mM imidazole 100 μM ZM241385, 25 mM HEPES, pH 1 μL denatured OmpX (∼20 mg/mL) was slowly diluted into 30 μL 7.5). The elution mixture was further purified by SEC (column: gel refolding buffer [1% (w/v) Fos-choline 12 (Fos12) or CCD-2, 50 filtration column superdex 200; buffer: 100 mM NaCl, 250 mM mM Tris-HCl, pH 9.0] at 4 °C. The refolding mixture was incubated EDTA, 20 mM Tris-HCl, pH 7.5). The fraction corresponding to Δ at 4 °C for 2 h and then heated at 70 °C with a water bath for 10 min. MBP-A2AR-BRIL- C reconstituted in lipid nanodiscs was collected After incubation at 4 °C for another 20 min, all OmpX samples were for further analysis by dynamic light scattering (DLS) and negative- loaded to a 12% polyacrylamide gel and analyzed by sodium dodecyl stained EM. (SDS)-gel electrophoresis in 2-(N-morpholino)ethanesulfonic Negative-Stained EM Data Acquisition and Analysis. Nano- acid buffer. The refolding yields of OmpX in each group were disc samples (5.0 μL) prepared as described above with the determined by band densitometry using ImageLab (Bio-Rad). concentration of 0.03 mg/mL were applied to a glow-discharged Thermal Shift Assay. Each sample comprised approximately 1 μg holey carbon grid and stained with 2% (w/v) uranyl formate.27 The μ ff A2AR in a total volume of 50 Lbu er (500 mM NaCl, 10% glycerol, data of negative-stained samples were acquired on a transmission 50 μM ZM241385, 0.2% (w/v) detergent, 25 mM HEPES, pH 7.5). electron (FEI, USA) operated at 120 kV equiped with a The CPM (N-([4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]- 4k × 4k scintillator-based charge-coupled device camera (UltraScan λ λ fi maleimide) dye ( ex = 365 nm, em = 460 nm) was dissolved in 4000, Gatan). Images were recorded at a nominal magni cation of DMSO at 4 mg/mL and diluted into each sample with a final ×57 000 or ×73000 with a defocus range of −2.5 to −3.5 μm. A total μ ° of 118 micrographs with a magnification of ×73 000 (corresponding concentration of 2 g/mL. After incubation at 4 C for 15 min, all − samples were subjected to thermal denaturation with a ramp rate of 1 to 1.918 Å pixel 1 on the specimen) were used for the 2D °C/min from 25 to 90 °C in a Rotor-Gene Q spectrofluorometer classification of the MBP-A2AR-BRIL-ΔC-nanodisc samples. First (Qiagen). All data were processed with the GraphPad Prism program, of all, the CTF parameters of each image were determined by Gctf.28 ° and the melting temperature (Tm C) of each sample was determined Then, the particles were picked automatically by Gautomatch in terms of the Boltzmann sigmoidal fitted curve of the raw data. software and bad particles were excluded by manual selection. Detergent Screening. A R(∼0.1 mg/mL, 20 μL) in CCD-2 Finally, about 103 053 particles were used for 2D classification 2A 29 (0.05%, w/v) was added to 10 μL screening buffer (500 mM NaCl, analysis with Relion 2.0 software. 10% glycerol, 50 μM ZM241385, 20 mM or 20 × CMC detergent, 25 ° mM HEPES, pH 7.5) and incubated at 4 C for 1 h with constant ■ RESULTS AND DISCUSSION rotation. Tris(2-carboxyethyl)phosphine (TCEP) (10 mM, pH 7.5, 10 μL) was then added and the mixture was incubated for another 2 h Design and Physical Characterization of CCDs. Using before centrifugation at 14 000 rpm for 15 min. The homogeneity of the conventional detergent DDM as the template, we all A2AR samples was then evaluated through analytical size exclusion incorporated a disulfide bond between the hydrophilic maltose chromatography (aSEC, HPLC: Agilent, Bioinert 1100 series; and the hydrophobic alkyl chain (Figure 2). In this design, the column: Nanofilm SEC-250, 4.6 × 250 mm, 5 μm, Sepax Technologies Inc.), which run at 0.5 mL/min in SEC buffer (150 mM NaCl, 2% glycerol, 0.05% DDM, 0.01% CHS (cholesterol hemisuccinate), 25 mM HEPES, pH 7.5) from UV absorbance at 280 nm. All above steps were carried out at 4 °C. As for the thermal stability SEC assay, the above reconstitution samples were heated at 40 °C for another 3 min using a metal bath before the aSEC analysis. Pursuant to preliminary results (Figure S2), the final concentration of the test detergent was set at 5 mM (for detergents whose CMC is lower than 1 mM) or 5 × CMC (for detergents whose CMC is higher than 1 mM). Nanodisc Preparation. The elution mixture of MBP-A2AR-BRIL- ΔC was desalted by a 5 mL HiTrap desalting column (Amersham/ GE) with buffer (500 mM NaCl, 10% glycerol, 250 mM imidazole, 100 μM ZM241385, 3 × CMC detergent, 25 mM HEPES, pH 7.5). Δ The MBP-A2AR-BRIL- C protein (0.7 mg/mL) mixture was then incubated with MSP2N2 (∼17 mg/mL) and the lipid stock (POPC, 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine; POPS, 2-oleoyl-1- palmitoyl-sn-glycero-3-phospho-L-serine) at a molar ratio of 1:5:180:420 (A2AR/MSP2N2/POPC/POPS). The mixture was incubated at 4 °C for 1 h with constant rotation. Then, reconstitution Figure 2. Design of CCDs by the introduction of bioorthogonally was initiated by removal of detergents from the system according to cleavable disulfide bonds.

4321 DOI: 10.1021/acs.langmuir.8b04072 Langmuir 2019, 35, 4319−4327 Langmuir Article mild maltose head and the alkyl tail were supposed to retain CCD-4 with one more atom, formed with the closest the nature of DDM, and the disulfide linker was expected to size to that of DDM. These observations could be well ascribed endow CCDs with the unique chemically cleavable property. to the distinct hydrophobicity and geometry of the S−S bond Among this series of CCDs, CCD-1, and CCD-2 were highly from the O−C bond. water soluble (>10%, w/v), while the other two detergents Thermal Stability of A2AR in CCDs. The preliminary bearing longer alkyl chains were of poor solubility (<1%, w/v). evaluation of CCDs for their stabilization on an engineered 18 Notably, the replacement of carbon with in CCD-3 A2AR was applied. A2AR is a class A GPCR with the dramatically decreased its solubility when compared to the characteristic seven transmembrane α-helices and has been conventional counterpart n-dodecyl-β-D-thiomaltoside identified as an important drug target for treating inflamma- (DTM). The CMCs of CCDs were then determined by the tion, cancer, Parkinson’s disease, and other neuronal fluorophore encapsulation assay and the micelle size was disorders.32 In recent decades, numerous in vitro studies measured through small-angle X-ray scattering (SAXS) Guinier have provided insights into the structure and function of analysis.30,31 As shown in Table 1, CMC values of CCDs 16,18,33,34 A2AR. The stability of A2AR in CCDs was assessed by a thermal Table 1. Molecular Weight (MW), CMCs, the Gyration shift assay.35 The transition temperature (T )reflects the R m Radii ( g) of Their Micelles, and Water Solubility of DDM, relative stabilization of diverse detergents to the protein. As DTM, and CCDs showed (Figure S3), all CCDs exhibited satisfying stabilization a b c d to A R when compared to DDM, except for CCD-1, which dets. MW (g/mol) CMC (mM) Rg (Å) Sol. (w/v, %) 2A ∼ ± appeared to be a moderately inferior stabilizing detergent (Tm DDM 510.1 0.17 31.7 0.21 >10 ° fi ° ∼ ± = 43.3 C). Signi cantly, CCD-3 and CCD-4 (Tm = 53.6 C DTM 526.7 0.05 37.7 1.63 >10 ° ∼ ± and 49.3 C, respectively) were superior to DDM (Tm = 47.4 CCD-1 516.7 0.50 23.8 0.19 >10 ° ∼ ± C). Overall, CCD-2 with comparable stabilization (Tm = 47.7 CCD-2 530.7 0.20 28.9 0.33 >10 ° CCD-3 544.7 ∼0.07 30.0 ± 3.18 <1 C), along with good solubility (>10%) and a similar CMC CCD-4 558.7 ∼0.02 31.2 ± 0.48 <1 value (0.2 mM) to DDM, was chosen as a promising candidate for further investigation of potential applications in MP studies. aMolecular weight. bCMC value of detergents was measured in fl 31 c Solubilization To Diverse GPCRs. To evaluate the ddH2O via the uorophore encapsulation assay. Gyration radius of ffi α micelles was calculated by SAXS Guinier analysis30 at 5 mM in solubilization e ciency of CCD-2 on -helix proteins, three d phosphate buffer (20 mM, pH 6.2, and 150 mM NaCl). Solubility in engineered GPCRs, including class A receptor A2AR(A2AR- Δ 18 ddH2O. BRIL- C), class B receptor GLP-1R (glucagon-like peptide- 1 receptor, GLP-1R-T4L),22 and class F receptor SMO 21 followed a negative trend from 0.50 to 0.02 mM, in line with (smoothened receptor, SMO-FLA), were solubilized and the general correlation of hydrophobicity to the alkyl chain purified by CCD-2. It turned out that CCD-2 was efficient in length. Meanwhile, the gyration radii (Rg) of micelles formed solubilizing A2AR with comparable yield and even superior by CCDs gradually increased with the elongation of the homogeneity when compared to DDM (Figure 3A). fi hydrophobic tail but were consistently smaller than DDM. Signi cantly, A2AR in CCD-2 remained consistently homoge- Intriguingly, CCD-2, the surrogate with one less atom, neous even after storage at 4 °C for 10 days, while obvious exhibited nearly an identical CMC value with DDM while aggregation was observed in the corresponding DDM sample

− fi Figure 3. Solubilization of diverse MPs using CCDs. (A C) Solubilization, puri cation of A2AR (A), GLP-1R (B), and SMO (C) using DDM (black) or CCD-2 (red). (D) SDS-gel electrophoresis of OmpX samples refolded in CCD-2 or Fos12. Denatured and folded OmpX were distinguished by migrating on the standard Laemmli gels at 18 and 16 kD, respectively.

4322 DOI: 10.1021/acs.langmuir.8b04072 Langmuir 2019, 35, 4319−4327 Langmuir Article

Figure 4. Bioorthogonal cleavage of CCD-2. (A) Chemical scheme of CCD-2 cleavage by the treatment with TCEP. (B) Complete decomposition of CCD-2 detected by ELSD-HPLC. CCD-2 (0.5 mM, in 20 mM HEPES, pH 7.5) was incubated with TCEP (2.5 mM) at 4 °C for 2 h. (C) ° Homogeneity of A2AR in DDM (0.025%) before (black column) and after (red column) CCD-2 cleavage at 4 C for 2 h. (D) OmpX refolding in 1% Fos12 before (black column) and after (red column) the treatment of CCD-2 cleavage at 4 °C for 3 h.

fi Figure 5. Detergent screening for A2AR using the sacri ce-replacement strategy. A2AR solubilized in CCD-2 (black) was reconstituted in selected detergents at 4 °C (blue) and then subjected to thermal denaturation by heating at 40 °C for 3 min (red) before aSEC analysis.

(Figure S4). Moreover, the other two engineered GPCRs comparable efficacy (73%) to its favorable detergent Fos12 (GLP-1R-T4L and SMO-FLA) that were extracted from the (67%), indicating that CCD-2 is also amenable to the β-barrel membrane by CCD-2 also exhibited comparable protein yield protein research. and homogeneity to that of DDM (Figure 3B,C), implying that Chemical Cleavage of CCD-2. According to the design, CCD-2 could be a perfect surrogate of DDM for solubilization the disulfide bond of CCDs that locates close to the of various GPCRs. hydrophilic area would be liable to aqueous reductants. Refolding of OmpX. To further explore the versatility of Among various common reductive conditions (Table S1), CCD-2 for β-barrel MP, the bacterial OmpX was applied CCD-2 was found to be readily decomposed by the treatment (Figure 3D). OmpX is an eight-stranded β-barrel MP that of TCEP. The exhaustive cleavage of CCD-2 (Figure 4A) was plays an important role in bacterial interactions with the confirmed in the HPLC profiles detected by an evaporative host.24,25,36,37 Well-folded OmpX can be conveniently light-scattering detector (ELSD) (Figure 4B) and led to the distinguished from the denatured one in standard gel complete disappearance of micelles, as detected by the electrophoresis, based on which the refolding efficacy in fluorophore encapsulation assay (Figure S5A) and DLS diverse detergents can be readily analyzed by the readout of (Figure S5B). Moreover, under the optimal cleavage condition SDS-polyacrylamide gel electrophoresis.38 Accordingly, refold- (entry 10, Table S1), complete degradation of CCD-2 was also ing of denatured OmpX in 1% CCD-2 accomplished a achieved in the presence of commercial detergents (lauryl

4323 DOI: 10.1021/acs.langmuir.8b04072 Langmuir 2019, 35, 4319−4327 Langmuir Article

Figure 6. A2AR reconstitution into nanodiscs for EM studies. (A) Empty nanodisc assembly and (B) A2AR-reconstituted nanodisc assembly using the chemical cleavage approach (CCD-2/TCEP) or traditional physical adsorption (DDM/BioBeads), respectively. (C) Raw images of negative- stained A2AR-reconstituted nanodiscs. maltose neopentyl glycol, LMNG; DTM and n-octyl-β-D- aSEC. As a result, 7 nonionic maltoside detergents (DDM, gluopyranoside) or the lipid (POPC) and resulted in a unitary LMNG, DTM, decyl maltose neopentyl glycol (DMNG), n- mimetic system (Figures S6 and S7). undecyl-β-D-maltopyranoside (UDM), n-decyl-β-D-maltopyra- Of note, TCEP, as well as the thiol debris, showed minimal noside (DM), and CYMAL-7) stood out from 23 commercial interference on the property of commercial , as detergents for the maintenance of A2AR(Figure 5 and S12), exemplified by the consistent CMC value of DDM under which suggests that the neutral maltose group in detergents ff di erent conditions (Figure S8). More importantly, the whole plays a key role in the stabilization of A2AR. In comparison, cleavage process of CCD-2 was also well compatible with β A2AR in the other detergents, including glucosides (n-nonyl- - various tested MPs (Figures 4C,D, S9, and S10). All three D-glucopyranoside, NG), derivatives ∼ μ fragile GPCRs ( 5 M), notably including SMO-FLA with a (C12E8, tetraethylene glycol monooctyl ether, TX-100), cysteine-rich domain, were well preserved in DDM under 5 amino oxides (n-dodecyl-N,N-dimethylamine-N-oxide), and mM TCEP for 6 h (Figure S11). We speculated that these zwitterionic detergents (CHAPS, Tripao, Fos-Choline), critical disulfide bonds of MPs should be buried in the − displayed high aggregation peaks in aSEC (Figure S12), hydrophobic interior of protein detergent complexes, which is indicating that these detergents are either too weak to stabilize hardly accessible to the aqueous reductant. In all, the chemical A2AR or so harsh that they denatured the fragile protein. cleavage of CCD-2 is rapid, complete, and biorthogonal and For a further comparison, these optimal A R samples, should facilitate a chemical methodology for in situ detergent 2A encapsulated in the 7 promising maltoside detergents, were replacement in diverse MP studies. subjected to thermal denaturation by heating at 40 °C for 3 Detergent Screening for the Stabilization of A R. It 2A min before aSEC analysis. As a result, DDM and its thio- is well known that the stability of MPs and the compatibility on the biophysical studies are highly dependent on the glycosylated homolog, DTM, outperformed the other detergents used.3,8 There are nearly 100 commercially available detergents by maintaining more than half the A2AR in a native detergents (www.anatrace.com), which is ever-expanding as a monotropic state (Figure 5 and Table S3). Another commonly result of ongoing research. However, a rational design or used detergent, LMNG, a seemingly dimerized homolog of selection of detergents for a given MP has been difficult DDM, also performed well with about 50% of A2AR remaining because the understanding of the fundamental behavior of in the monomer after heating. In comparison, A2AR in shorter- detergents around MPs is still limited, though the recent efforts chained maltosides, both traditional DM and two-head two- have pushed forward in this field.17,39,40 On the basis of our tailed DMNG, almost totally denatured after thermal treat- sacrifice-replacement strategy, we set out to conduct a ment. In addition, the cyclohexanyl-terminated detergent comprehensive detergent screening on the stabilization of CYMLA-7 apparently exhibited comparable stabilization to A2AR. the linear chained maltoside detergent UDM (Figure 5). These fi Accordingly, the A2AR protein puri ed by CCD-2 was screening results were in good agreement with the detergent successfully reconstituted into a group of selected detergents priority of previous structural biology studies on A2AR, (Table S2) by the chemical replacement strategy and the indicating that this facile and economical detergent-screening monodispersity of A2AR samples was then evaluated through methodology could provide reliable guidance for the selection

4324 DOI: 10.1021/acs.langmuir.8b04072 Langmuir 2019, 35, 4319−4327 Langmuir Article of proper amphiphile candidates for MP structure determi- barrel MPs while readily decompose for removal under mild nation. conditions. Based on this first proof-of-principle, other Nanodisc Preparation Using CCD-2 as the Sacrifice biocompatible bond-cleavage chemistry along with diverse Detergent. The nanodisc is a discoidal lipid nanostructure triggering stimuli,43 such as other reduction-induced cleavage where phospholipid bilayers are encircled by two MSPs aligned reactions,44 photo-induced decaging reactions,45 metal-in- − in double belt formation. Thanks to their well-defined size, duced deallylation and depropargylation,46 48 could poten- good stability, and more native-like lipid environment when tially be used to expand the CCD repertoire. Compared to compared to other membrane mimics, nanodiscs have recently traditional physical methods, this chemical degradation is more been recognized as a versatile tool that has enabled structure− a quantitative approach that facilitates MP reconstitution. In function investigations of MPs in .41 However, addition to detergent screening and nanodisc preparation, nanodiscs are usually unable to extract MPs from the biological other speculative applications could also be investigated. For membrane. Traditional protocols for incorporating a target MP example, CCD-2 might be amenable to the precise bottom-up in nanodiscs commence with the gradual removal of initial proteomic studies of integral MPs by detergents through BioBeads adsorption or dialysis, which prompting full coverage .49,50 The CCD-based usually need laborious optimization in a preliminary strategy should in the future speed up such innovation or stage.12,13,42 Because CCD-2 can efficiently solubilize and upgrading of biophysics studies. stabilize diverse GPCRs and is readily degraded, we envisioned that CCD-2 would be an ideal initial detergent from which a ■ ASSOCIATED CONTENT simple and convenient protocol for the preparation of GPCR- *S Supporting Information embedded nanodiscs could be established. The Supporting Information is available free of charge on the First, we investigated the self-assembly of empty nanodiscs ACS Publications website at DOI: 10.1021/acs.lang- with different detergent removal methodologies. As showed in muir.8b04072. Figure 6A, the chemical cleavage methodology turned out to Supplementary figures, synthetic procedures, and be perfectly compatible with the nanodisc assembly and supporting spectra (PDF) yielded equal amounts of empty nanodiscs to that of the control detergent-free group. By contrast, the traditional physical adsorption method seemingly brought in slightly ■ AUTHOR INFORMATION more MSP aggregation and resulted in a lower nanodisc yield. Corresponding Authors * Subsequently, an engineered construct of A2AR (MBP-A2AR- E-mail: [email protected] (Z.L.). BRIL-ΔC) was applied to investigate the potential utility of *E-mail: [email protected] (H.T.). this chemical strategy to deliver GPCRs into nanodiscs for EM ORCID Δ studies. Because MBP-A2AR-BRIL- C appeared to be less Zhong-Xing Jiang: 0000-0003-2601-4366 stable than the previous crystallization construct (A2AR-BRIL- Houchao Tao: 0000-0001-7598-0275 ΔC), 20% CHS was applied to improve its thermostability during protein purification.5 Accordingly, both DDM/CHS Author Contributions and CCD-2/CHS yielded highly homogeneous monotropic The manuscript was written through contributions of all authors. All authors have given approval to the final version of A2ARsamples(Figure S13). Following the complete decomposition of CCD-2 or physical adsorption of DDM, the manuscript. Notes A2AR was in situ reconstituted into nanodiscs. The empty nanodiscs, redundant reagents as well as cleavage debris, were The authors declare no competing financial interest. removed via IMAC and the resulting reconstitution mixture was fractionated on SEC before EM analysis. Compared to the ■ ACKNOWLEDGMENTS traditional physical protocol, the new reconstitution strategy The authors (ZL and HT) are thankful to Shanghai Municipal produced a slightly better monodispersed peak of A2AR- Government and ShanghaiTech University for startup financial incorporated nanodiscs in FPLC profiles (Figure 6B). Of note, support. This work was also supported by NSFC (21672147, nanodiscs prepared according to the chemical protocol, both HT) and partially from the National Key R&D Program of with and without the incorporated protein, showed the China (2018YFA0507000). We thank the staff of BL19U2 consistent size with those prepared according to the traditional beamline at the National Facility for Protein Science Shanghai physical method (Table S4). Furthermore, these A2AR- (NFPS) and Shanghai Synchrotron Radiation Facility, incorporated nanodiscs prepared using the chemical approach Shanghai, People’s Republic of China, for assistance during were displayed as highly homogeneous particles in negative- data collection. We thank J. Liu, Q. Shi, Y. Xu, Q. Tan, Q. Sun, stained EM (Figure 6C) and could be further clearly and L. Yang for assistance in the membrane preparation, assay differentiated into nine representative classes with different performance, plasmid preparation, EM data acquisition, and orientations by 2D classification (Figure S14). So far, the NMR data collection, respectively, at the Core Facility of the chemical replacement strategy has proven to be a perfect iHuman Institute. We thank R. C. Stevens, G. Zhong, G. Song, complementary method to the existing physical approaches. D. Wu, D. Liu, J. Cheng, and C. Zhang for their insightful discussion and assistance in manuscript preparation. ■ CONCLUSIONS In summary, we present here an alternative detergent ■ ABBREVIATIONS replacement for MP reconstitution mediated by a class of MP, membrane protein; CCD, chemically cleavable detergent; chemically cleavable detergents. Thanks to the unique property DDM, n-dodecyl-β-D-maltopyranoside; GPCR, G protein- of disulfide bonds, CCDs derived from DDM largely maintain coupled receptors; OmpX, outer membrane protein X; the excellent capability in the manipulation of α-helix and β- NMR, nuclear magnetic resonance; HRMS, high-resolution

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