Hydrogenated diglucose detergents for membrane-protein extraction

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Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins, Polymers

Hydrogenated diglucose detergents for membrane-protein extraction and stabilization Pierre Guillet, Florian Mahler, Kelly Garnier, Gildas NYAME MENDENDY BOUSSAMBE, Sébastien Igonet, Carolyn Vargas, Christine Ebel, Marine Soulié, Sandro Keller, Anass Jawhari, and Grégory Durand Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b02842 • Publication Date (Web): 15 Feb 2019 Downloaded from http://pubs.acs.org on February 16, 2019

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Hydrogenated diglucose detergents for membrane-protein extraction and stabilization Pierre Guillet,a,b Florian Mahler,c Kelly Garnier,b,e Gildas Nyame Mendendy Boussambe,a,b Sébastien Igonet,b,e Carolyn Vargas,c Christine Ebel,d Marine Soulié,a,b Sandro Keller,c* Anass Jawhari,b,e* Grégory Duranda,b* aInstitut

des Biomolécules Max Mousseron (UMR 5247 UM-CNRS-ENSCM) & Avignon University, Equipe

Chimie Bioorganique et Systèmes amphiphiles, 301 rue Baruch de Spinoza – 84916 AVIGNON cedex 9 (France); bCHEM2STAB, cMolecular

301 rue Baruch de Spinoza – 84916 AVIGNON cedex 9 (France);

Biophysics, Technische Universität Kaiserslautern (TUK), Erwin-Schrödinger-Str. 13, 67663 Kaiserslautern, Germany; dUniv.

Grenoble Alpes, CNRS, CEA, CNRS, IBS, F-38000 Grenoble;

eCALIXAR,

60 Avenue Rockefeller – 69008 Lyon (France).

*

Corresponding Authors. Grégory Durand. E-mail: [email protected]; Phone: +33 (0)4 9014 4445. Anass Jawhari. E-mail: [email protected]; Phone: +33 (0)4 81 07 64 63; Sandro Keller. E-mail: [email protected]; Phone: +49 631 205 4608.

ACS Paragon Plus Environment

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Abstract We report herein the design and synthesis of a novel series of alkyl glycosides detergents consisting of a nonionic polar headgroup that comprises two glucose moieties in a branched arrangement (DG), onto which octane-, decane- and dodecanethiols were grafted leading to ODG, DDG, and DDDG detergents. Micellization in aqueous solution was studied by isothermal titration calorimetry (ITC), 1H NMR spectroscopy, and surface tensiometry (SFT). Critical micellar concentration values were found to decrease by a factor of ~10 for each pair of methylene groups added to the alkyl chain, ranging from ~0.05 mM to 9 mM for DDDG and ODG, respectively. Dynamic light scattering (DLS) and analytical ultracentrifugation sedimentation velocity (AUC-SV) experiments were used to investigate the size and the composition of the micellar aggregates, showing that the aggregation number significantly increased from ~40 for ODG to ~80 for DDDG. All new compounds were able to solubilize MPs from bacterial membranes, insect cells, as well as Madin Darby Canine Kidney (MDCK) cells. In particular, native human adenosine receptor (A2AR) and bacterial transporter BmrA were solubilized efficiently. A striking thermostability improvement of +13 and +9°C when ODG and DDG were respectively applied to wild type and full length A2AR. Taken together, this novel detergent series shows promising detergent potency for solubilization and stabilization of MPs and, thus, makes a valuable addition to the chemical toolbox available for extracting and handling these important but challenging membrane protein targets.


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Introduction Membrane proteins (MPs) represent 20–30% of all human proteins. They are crucial for cellular physiology as they are directly involved in a large spectrum of cellular processes and represent 60% of therapeutic targets.1 To study membrane proteins and to use them as drug-discovery targets, biochemists and structural biologists need to solubilize them from the membrane bilayer. Thus, it is essential to develop tools for membrane-protein solubilization and stabilization to unlock structural and functional details as well as for drug discovery.2 A suitable detergent has to fulfill two principal criteria, namely, (i) efficient solubilization and (ii) conservation of native structural and functional properties of MPs. Conventional head-andtail detergents comprise well-segregated water-soluble, polar and hydrophobic, nonpolar domains, which are usually referred to as the headgroup and the tail, respectively. Detergents exhibit both surface-active and self-aggregation properties, that is, they adsorb to interfaces and form micellar aggregates above their critical micellar concentration (CMC). The most commonly used detergents for membrane-protein structural and functional studies are alkyl glycosides such as the maltoside and glucoside series. Because they tend to disrupt protein/lipid and lipid/lipid interactions rather than protein/protein interactions, they are widely considered non-denaturing. More quantitatively, the absence of denaturing properties—which is also referred to as the “mildness” of a detergent— is usually more pronounced for long-chain derivatives (i.e., C12–C14) than for short-chain analogs (i.e., C7–C10).3-4 Therefore, n-dodecyl-β-D-maltoside (DDM) is often considered the gold standard because it combines good solubilization properties with mildness towards many MPs and, therefore, has been successfully employed for a large variety of proteins. On the contrary, (zwitter)ionic detergents, while efficiently extracting proteins from membranes, also disrupt protein/protein interactions and, therefore, tend to be denaturing. Sodium dodecyl sulfate (SDS), for instance, is commonly used as a protein denaturant in gel electrophoresis5 and in protein unfolding/folding studies.6-7 Furthermore, a detergent that works for one membrane protein may not be suitable for a different membrane protein. This has motivated a number of recent efforts in designing milder


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alternatives, such as lipopeptides,8 tripod 9, neopentyl glycol,10-11 branched,12 and cyclic-based amphiphiles,13-14, as well as fluorinated surfactants (FSs).15-17 Given the mildness of alkyl glycosides, we have developed a nonionic polar headgroup called DG that comprises two glucose moieties in a branched arrangement whose synthetic route is short and straightforward18 and onto which various apolar chains can be grafted.18-21 The advantages of having two glucoses in a branched arrangement lie in the good water solubility and the bulkiness it confers to the polar headgroup, which results in the formation of rather small and compact micelles.22 We have recently reported that several diglucoylated fluorinated detergents18-19 can solubilize lipid vesicles and extract a broad range of proteins from Escherichia coli membranes, thereby demonstrating their suitability as detergents.19 In this work, we have extended the use of the diglucosylated polar headgroup to hydrogenated alkyl chains in order to investigate the potency of these novel detergents. We have synthesized three new compounds named ODG, DDG, and DDDG that contain either an octyl, decyl, or dodecyl chain, respectively, O, D and DD refering to the alkyl chains. (Figure 1). Micellization in aqueous solution was studied by isothermal titration calorimetry (ITC), 1H NMR spectroscopy, and surface tensiometry (SFT). Dynamic light scattering (DLS) and analytical ultracentrifugation sedimentation velocity (AUC-SV) experiments were used to investigate the size and the shape of the micellar aggregates. The ability of the detergents to solubilize lipid vesicles as well as to extract different membrane proteins from various types of membranes was investigated. Finally, we demonstrated their great potential in stabilizing a pharmacologically relevant G-protein-coupled receptor (GPCR).


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Figure 1. Chemical structures of the three diglucosylated derivatives developed in this study.

Materials & Methods All starting materials were commercially available and were used without further purification. All solvents were of reagent grade and used as received unless otherwise indicated. MeOH was dried over Na under argon atmosphere. The progress of the reactions was monitored by thin layer chromatography. The compounds were detected either by exposure to ultraviolet light (254 nm) or by spraying with sulfuric acid (5% ethanol) and/or ninhydrin (5% ethanol), followed by heating at ~150°C. Size exclusion chromatography purifications were carried out on Sephadex LH-20 resin. 1H and

13

C NMR analysis were performed at 400 and 100 MHz,

respectively. Chemical shifts are given in ppm relative to the solvent residual peak as a heteronuclear reference for 1H and 13C. Abbreviations used for signal patterns are: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; dd, doublet of doublet; dt, doublet of triplet. HR-MS (ESI+) was determined on a QStar Elite mass spectrometer. The purity of the detergents was evaluated using a Shimadzu instrument equipped with a photodiode array detector, operating with an XTerra RP18 (100 × 2.1 mm, 5 μm) from Waters. The elution was done with a binary gradient of water and acetonitrile (complemented by 0.1% (v/v) of trifluoroacetic acid) as explained below: from 0 to 2 min, isocratic elution at 2% of acetonitrile, from 2 to 22 min, gradient elution from 2% to 80% of acetonitrile, from 22 to 26 min, isocratic elution at 80% of acetonitrile. Milli-Q water (resistivity of 18.2 MΩ cm, surface tension of 71.45 mN/m at 25°C) was employed for all physical chemical experiments.


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Synthesis of DigluM detergents. ODG

(N-(2-methyl-1,3-bis(O-β-D-glucose)propan-2-yl)-3-(octylthio)propanamide).

1-

octanethiol (0.33 g, 2.26 mmol, 1.23 equiv.) and NaBH4 (0.11 g, 2.91 mmol, 1.6 equiv.) were dissolved in 7 mL MeOH under stirring, and the mixture was heated at reflux of MeOH. After 15 min, a solution of N-tris[di(β-D-glucopyranosyl)oxymethyl]methylbut-3-enamide (0.89 g, 1.84 mmol, 1 equiv.) in 5 mL MeOH was added, and the reaction was stirred for 5 h. The reaction mixture was cooled down at room temperature and then concentrated under reduced pressure. The resulting crude compound was purified by size exclusion chromatography (MeOH) to give 0.88 g of ODG (1.40 mmol, 76%) as a white powder. 1H NMR (CD3OD, 400 MHz) δ 4.46 (dd, J = 7.8, 2.3 Hz, 2H), 4.12 (d, J = 9.9 Hz, 1H), 4.03 (d, J = 9.9 Hz, 1H), 3.95 – 3.70 (m, 4H), 3.69 (m, 2H), 3.40-3.25 (m, 6H), 3.24-3.17 (m, 2H), 2.73 (t, J = 7.3 Hz, 2H), 2.58 (t, J = 7.3 Hz, 2H), 2.48 (dt, J = 7.3, 1.8 Hz, 2H), 1.58 (q, J = 7.3 Hz, 2H), 1.56 (s, 3H), 1.26 (m, 10H), 0.89 (t, J = 6.8 Hz, 3H). 13C NMR (CD3OD, 100 MHz) δ 174.33, 104.81, 104.70, 78.07, 78.00, 77.96, 75.11, 75.06, 72.70, 72.36, 71.65, 62.75, 58.01, 38.27, 32.98, 32.89, 30.70, 30.36, 30.32, 29.91, 28.66, 23.69, 19.17, 14.41. HPLC (XTerra, Waters, RP18) tR (min): 12.9, purity 95% at 214 nm. HRMS (ESI+) calculated for C27H51NO13S ([M+H]+): 630.3159, found [M+H]+: 630.3162. DDG

(N-(2-methyl-1,3-bis(O-β-D-glucose)propan-2-yl)-3-(decylthio)propanamide).

The

synthetic route was essentially the same as for ODG. 1-decanethiol (0.26 g, 1.50 mmol, 1.25 equiv.),

NaBH4

(0.07 g,

1.85 mmol,

1.5 equiv.)

and

N-tris[di(β-D-

glucopyranosyl)oxymethyl]methylbut-3-enamide (0.58 g, 1.20 mmol, 1 equiv.) were put in reaction. 0.47 g of DDG (0.71 mmol, 60%) were obtained as a white powder. 1H NMR (CD3OD, 400 MHz) δ 4.33 (dd, J = 7.8, 2.3 Hz, 2H), 4.12 (d, J = 9.8 Hz, 1H), 4.00 (d, J = 10 Hz, 1H), 3.92 – 3.74 (m, 4H), 3.67 (m, 2H), 3.38 – 3.25 (m, 6H), 3.24 – 3.17 (m, 2H), 2.73 (t, J = 7.3 Hz, 2H), 2.55 (t , J = 7.3 Hz, 2H), 2.45 (dt, J = 7.3, 1.8 Hz, 2H), 1.62 (q, J = 7.3 Hz, 2H), 1.39 (s, 3H), 1.30 (m, 14H), 0.90 (t, J = 6.9 Hz, 3H).

13

C NMR (CD3OD, 100 MHz) δ 174.34,


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104.82, 104.72, 78.08, 78.02, 77.98, 75.13, 75.07, 72.71, 72.37, 71.67, 71.65, 62.76, 58.02, 38.28, 33.06, 32.89, 30.71, 30.44, 30.37, 29.92, 28.67, 23.72, 19.17, 14.42. HPLC (XTerra, Waters, RP18) tR (min): 14.6, purity 94% at 214 nm. HRMS (ESI+) calculated for C29H55NO13S ([M+H]+): 658.3472 found [M+H]+: 658.3470. DDDG (N-(2-methyl-1,3-bis(O-β-D-glucose)propan-2-yl)-3-(dodecylthio)propanamide). The synthetic route was essentially the same as for ODG. 1-dodecanethiol (0.34 g, 1.68 mmol, 1.25 equiv.), NaBH4 (0.08 g, 2.11 mmol, 1.5 equiv.) and N-tris[di(β-D-glucopyranosyl)oxymethyl]methylbut-3-enamide (0.65 g, 1.34 mmol, 1 equiv.) were put in reaction. 0.51 g of DDDG (0.74 mmol, 56%) were obtained as a white powder. 1H NMR (CD3OD, 400 MHz) δ 4.36 (dd, J = 7.8, 2.3 Hz, 2H), 4.10 (d, J = 9.9 Hz, 1H), 3.98 (d, J = 9.9 Hz, 1H), 3.93 – 3.74 (m, 4H), 3.68 (m, 2H), 3.38 – 3.26 (m, 6H), 3.24 – 3.17 (m, 2H), 2.72 (t, J = 7.5 Hz, 2H), 2.54 (t, J = 7.3 Hz, 2H), 2.46 (dt, J = 7.5, 1.8 Hz, 2H), 1.58 (q, J = 7.3 Hz, 2H), 1.39 (s, 3H) 1.29 (m, 18H), 0.91 (t, J = 6.8 Hz, 3H). 13C NMR (CD3OD, 100 MHz) δ 174.34, 104.82, 104.71, 78.08, 78.01, 77.97, 75.12, 75.06, 72.71, 72.36, 71.66, 62.75, 58.01, 38.28, 33.06, 32.89, 30.77, 30.74, 30.72, 30.70, 30.46, 30.37, 29.92, 28.66, 23.72, 19.17, 14.43. HPLC (XTerra, Waters, RP18) tR (min): 16.1, purity 96% at 214 nm. HRMS (ESI+) calculated for C31H59NO13S ([M+H]+): 686.3785 found [M+H]+: 686.3792. Isothermal titration calorimetry. High-sensitivity microcalorimetry was performed at 25°C on an iTC200 (Malvern Instruments, Malvern, UK) for ODG and DDG and on a VP-ITC (Malvern) for DDDG. All solutions were prepared in water. For demicellization experiments, 1-µL aliquots of 55 mM ODG were titrated into 5 mM ODG, whereas 1-µL of 8 mM DDG or 10-µL of 0.7 mM DDDG were titrated into water. Time spacings between injections were chosen long enough to allow for complete re-equilibration. Baseline subtraction and peak integration were performed using NITPIC.23 All reaction heats were normalized with respect to the molar amount of detergent. Nonlinear least-squares fitting was performed in an Excel


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(Microsoft, Redmond, USA) spreadsheet using the Solver add-in (Frontline Systems, Incline Village, USA), as explained elsewhere.24 1

H NMR. 7 samples of each detergent at different concentrations were prepared from stock

solutions (0,63 g/L for ODG and 2,63 g/L for DDG). All samples were dissolved in D2O/H2O (10/90 (v/v)). 1-4 dioxane was used as an internal reference (10µL of a solution at 3 g/L were added). The signal of the terminal CH3 group of ODG and DDG was plotted as a function of the concentration to derive the CMC value by nonlinear least-squares fitting.25 It was, by contrast, not possible to determine the CMC of DDDG because it was below the limit of detection. Below the CMC, the observed chemical shift (δobs) is the chemical shift of the monomer (δmon), whereas above the CMC, δobs is the weighted average of the monomer and micelle chemical shifts assuming the exchange between the bulk solution and the micelle is fast on the NMR time scale. If the monomer concentration is constant above the CMC, the observed CMC

chemical shift can be written as follows: 𝛿obs = 𝛿mic − $

C

% (𝛿mic − 𝛿mon ).

Surface tension measurements. The surface activity of detergents in solution at the air/water interface was determined using a K100 tensiometer (Kruss, Hamburg, Germany). Surface tensions were determined by dilution of stock solutions (~5×CMC) using the Wilhelmy plate technique. In a typical experiment, 20–30 concentration steps were used with ~5–10 min between each concentration step. All measurements were performed at (25.0 ± 0.5)°C. ./01,3

Thermodynamic analysis. The CMC and the molar enthalpy of micelle formation, ∆H-

can be obtained directly from isothermal titration calorimetry demicellisation experiments. From the CMC, the mole fraction partition coefficient of detergent (Det) from the aqueous (aq) m/aq

aq

01

. into the micellar (m) phase is calculated as KDet = Xm Det ⁄XDet . Here, 𝑋9:; = 1 and 𝑋9:; = 01

01

𝑐9:; A(𝑐? + 𝑐9:; ) ≈ CMC⁄55.5 M denote the mole fractions of detergent in the micellar and 01

the aqueous phases, respectively, with 𝑐9:; being the concentration of detergent monomers in the aqueous phase and 𝑐? = 55.5 M the water concentration. Then, the standard molar Gibbs


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./01,°

free energy change accompanying micelle formation takes the form ∆𝐺9:; MNM

./01

./01,°

−𝑅𝑇 ln𝐾9:; = 𝑅𝑇 ln OO.O N and the entropic contribution is −𝑇∆𝑆9:; ./01,°

∆𝐻9:;

./01,°

, with ∆𝑆9:;

=

./01,°

= ∆𝐺9:;

−

denoting the standard molar entropy change on micelle formation.

Surface tension data were treated in terms of the Gibbs adsorption equation to determine the S

surface excess (Γmax) as 𝛤max = − RT

dγ d ln C

, and the surface area per molecule (Amin) at the

air/water interface from the slope of the surface tension curve as 𝐴min = U

S A Vmax

.

Dynamic light scattering. Hydrodynamic particle size distributions were determined on a Zetasizer Nano-S model 1600 (Malvern) equipped with a He–Ne laser (λ = 633 nm, 4.0 mW). Solutions ranging in detergent concentration from 1 to 57 mg/mL were prepared. The detergent solution was filtered (0.45 µm) before being transferred into a 1 mL low-volume quartz batch cuvette. The time-dependent correlation function of the scattered light intensity was measured at an angle of 173° (backscattering detection). The hydrodynamic diameter (DH) of the particles was estimated from their diffusion coefficient (D) using the Stokes–Einstein equation. CONTIN analysis was used for evaluating autocorrelation functions. All measurements were done at (25 ± 0.5)°C. Analytical ultracentrifugation sedimentation velocity. AUC-SV. Stock solutions of 20 mg/mL ODG and 10 mg/mL DDG or DDDG were prepared by diluting the powders in milliQ water, using a precision scale. The solution of ODG was shaken overnight to ensure complete solubilization. Stock solutions were used for further dilutions to 15 and 10, or 5 and 2 mg/mL with milliQ water using a precision scale. SV experiments were done at 42’000 rpm (130,000 g) and 20°C on an analytical ultracentrifuge XLI with an Anti-50 rotor (Beckman Coulter, Palo Alto, USA) and double-sector cells with optical path lengths of 12 or 3 mm equipped with Sapphire windows (Nanolytics, Potsdam, DE). The reference channel was filled with milliQ water. Acquisitions were made overnight using interference optics at time intervals of

1

min.

Data

were

processed

with

the


software

Redate

v 1.0.1

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(http://biophysics.swmed.edu/MBR/software.html) prior analysis with SEDFIT, v 15.01b (https://sedfitsedphat.nibib.nih.gov/software/)26

and

GUSSI

v 1.2.1

(http://biophysics.swmed.edu/MBR/software.html).27 We used partial specific volumes (v̅) of the detergents calculated from their chemical composition (Table 1) and tabulated values of viscosity, η = 1.035 mPa s, and density, ρ = 1.005 g/cm3, of water at 20°C. Each set of SV profiles was globally analyzed using the size distribution c(s) analysis26 and the non-interacting species analysis embedded in the SEDFIT software. c(s) analysis was performed with a 68% confidence level for regularization. We considered 200 particles with different sedimentation coefficients, s, and fitted a common frictional ratio, f/fmin (a mean operational value that is not further considered). The s values and signals in fringe of interference were determined by integrating the peaks of the c(s) distribution. From a linear fit of the signal (i.e., fringes of interference, normalized to an optical path length of 1 cm) measured by integrating the micelle contribution as a function of the total concentration, we derived the refractive index increment, (dn/dc), and the CMC. From a linear fit of the s-value as a function of the micelle concentration, the sedimentation at infinite dilution, s0, and concentration-dependence factors, k’s, were obtained: s = s0 (1-k's c). Molar masses, Mmic, thus aggregation numbers, Nagg, were derived from the non-interacting species analysis, from the independent estimates of the sedimentation and diffusion coefficients, s and D, combined in the Svedberg equation, with R the gas constant and T the absolute temperature: s = Mmic (1−𝜌v̅) D/RT. Mmic was also obtained by combining RH = DH/2 from DLS and s0 in the reformulated Svedberg equation, with NA the Avogadro’s number: 𝑠3 =

[\]^_``_ (Sabc̅) Ue fghij

Vesicle solubilization. POPC in powder form was suspended in phosphate buffer (50 mM Na2HPO4/NaH2PO4, 150 mM, pH 7.4) and vortexed for 10 min at 35°C. To obtain LUVs, the suspension was extruded at least 35 times through two stacked polycarbonate membranes with a nominal pore diameter of 100 nm using a LiposoFast extruder (Avestin, Ottawa, Canada).


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Unimodal size distribution was confirmed by DLS; LUVs had a hydrodynamic diameter of ~150 nm. A 0.2 mM stock solution of POPC LUVs and detergent were mixed in a 3 mm × 3 mm quartz glass cuvette before the light scattering intensity was monitored at 50°C using a Nano Zetasizer ZS90 (Malvern) equipped with a 633-nm He–Ne laser and a detection angle of 90°. To ensure quantitative comparability of scattering intensities, the attenuator was fixed to the maximum open position. Extraction of MPs from E. coli membranes. E. coli BL21(DE3) cells were transformed with an empty pET-24 vector and selected by kanamycin resistance. After incubation in lysogeny broth overnight at 37°C under permanent agitation, cells were harvested by centrifugation and washed twice with saline (154 mM NaCl). Cell pellets were resuspended in ice-cold buffer (100 mM NasCO3, pH 11.5) to a concentration below ~0.1 g mL–1 and ultrasonicated twice for 10 min in an S-250A sonifier (Branson Ultrasonics, Danbury, USA). To remove cell debris, the lysate was centrifuged at 4°C for 30 min at 1000 g. The supernatant was centrifuged at 4°C for 1 h at 150,000 g to separate membrane fragments from soluble and peripheral proteins. Membrane pellets were resuspended in buffer (50 mM Tris, 200 mM NaCl, pH 7.4) supplemented with complete protease inhibitor cocktail (Roche, Mannheim, Germany) to a final concentration of 100 mg wet-weight pellet per 1 mL of buffer and mixed in a 1:1 volume ratio with stock solutions of DDM or DG compounds in buffer. Detergent concentrations were chosen such as to arrive at the same micellar concentration cmic (i.e., ctotal = CMC + cmic) to ensure comparable extraction conditions. All samples were incubated for 16 h at 20°C under constant, gentle agitation. After ultracentrifugation at 4°C for 1 h at 100,000 g, the supernatant containing micelles was analyzed using SDS-PAGE. Extraction yields were then determined densitometrically using ImageJ gel analysis.28 Protein production and membrane fractionation of E.coli, insect Sf9 and MDCK cells. A2AR and M2 were produced using Sf9 and MDCK cells,29-30 respectively. BmrA was produced in E.coli.31-32 Cells were thawed on ice for 1 h with PBS (25 mM Na2HPO4, 150 mM


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NaCl, Protease Inhibitor Cocktail 1X). Protein production was performed as described previously for A2AR31 and M2.29 Mechanical cell lysis was performed on ice using a BeadBeater homogenizer with 0.1-mm diameter glass beads, including 5 pulses of 30 s with 2-min breaks in between. Membrane fractionation was carried out at 4°C by sequential centrifugations: 1000 g for 5 min, 15,000 g for 30 min, and 100,000 g for 45 min. Internal (15,000 g fraction) and plasma membranes (100,000 g fraction) were resuspended in PBS, PIC 1X, 20% glycerol to a final concentration of 10mg/mL, flash-frozen and stored at 80°C. Protein quantification and extraction. Total protein concentrations in the plasma and the internal membrane fractions were determined with the micro BCA protein assay kit (Pierce) using the bovine serum albumin (BSA) as a standard. For A2AR and BmrA solubilization, fractions of internal and plasma membrane were incubated for 2 h at 4°C at a final concentration of 5 mg/mL BCA in 1× PBS and 1× protease inhibitor cocktail, using 2, 5, 10 and 20 mM of DDM, ODG, DDG or DDDG. For M2 solubilization, 10×CMC of each detergent was used. Extraction without detergent and with SDS served as negative and positive controls, respectively. After solubilization samples were centrifuged at 100,000 g for 45 min at 4°C and an aliquot of the total extract, the pellet and the supernatant from each solubilization condition was analysed by stain-free SDS-PAGE and Western blots to evaluate total protein and specific protein of interest, respectively. SDS-PAGE and Western blotting. Protein samples was denatured with 6x Laemmli buffer (375 mM Tris-HCl (pH 6.8), 9% (w/v) SDS, 50% (w/v) glycerol, and 0.03% (w/v) bromophenol blue, and 30 mM TCEP) and incubated for 5 min at RT prior to analysis without heating to avoid aggregates formation. Proteins were separated by SDS-PAGE on a 4–15% acrylamide gel (Mini-PROTEAN TGX Stain-Free Gel, Bio-Rad) and subsequently immobilized by electrotransfer to PVDF membranes. Immunodetection of the proteins of interest was performed by using the SNAP i.d. system (Millipore) with primary goat anti-A2A and anti-M2


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antibodies. Due to overexpression of BmrA, stain free gels were used to monitor the solubilization of total proteins and BmrA. Silver Stain/Coomassie Stain, clear native PAGE, and Western blotting. SDS-PAGE were silver-stained using Dodeca Silver Stain Kit (Bio-Rad) following the supplier’s protocol or Coomassie-stained using the PageBlue protein staining solution. Non-denaturated proteins were separated by native-PAGE on a 4–15% acrylamide gel (Mini-PROTEAN TGX Stain-Free Gel, Bio-Rad) using 25 mM imidazole as anode buffer and 7.5 mM imidazole, 0.05% deoxycholate, 0.01% DDM as cathode buffer). Clear native PAGE gels ran for 90 min at 200 V and 4°C. Proteins were then immobilized by electro-transfer to PVDF membrane. The immunodetection of A2AR was performed by using the SNAP i.d. system (Millipore) with a primary A2AR antibody. Thermostability assay. Membranes of A2AR (2 mg/mL total protein) was solubilized in different conditions (see solubilization method above) for 2 h at 4°C. Solubilized fractions were obtained after 100’000 g ultracentrifugation for 1 h at 4°C. Solubilized fractions were split into 50-μL aliquots to be submitted to one temperature each as part of a temperature gradient ranging from 30 to 80°C using a PCR thermal cycler (PeqSTAR 2× gradient; Peqlab). Samples were then centrifuged 40 min at 20000 g before supernatants were analyzed by SDS-PAGE and Western blotting using an anti-A2AR antibody. The relative intensity of the target protein at each temperature was quantified on Western blots using Image Lab software 4.1 (Bio-Rad). Each condition was assayed twice. Intensity was plotted as a function of temperature, normalized, and fit to a generic sigmoidal function.33

Results & Discussion Synthesis. The new detergents were prepared by a thiolen Michael addition of the alkanethiols, that is, octanethiol, decanethiol, or dodecanethiol, onto the diglucoylated polar head as previsously described (Scheme 1).18, 34 Following this method, thiols were reacted with the


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polar headgroup in the presence of 1.5 equivalents of NaBH4 in refluxing methanol, which after purification afforded the final compounds in satisfactory yields (56–76%). The crude detergents were purified by Sephadex LH-20 size exclusion chromatography and then freeze-dried to give the pure detergents.

Scheme 1. Synthesis of the new diglucosylated detergents.

Micellization. The micellization processes of the three detergents were characterized by means of high-sensitivity ITC, solution 1H NMR, and surface tension such as that exemplified in Figure 2A-C from which we derived micellar parameters (Table 1). CMC values are in a very good agreement among the three techniques. While ODG exhibited a CMC slightly below 9 mM, the two-carbon longer chain derivative DDG had a CMC of about 0.6 mM. The addition of two more carbon atoms within the chain led to a CMC of about 0.05 mM for DDDG. Thus, the CMC decreased by a factor of ~10 for each pair of methylene groups added to the alkyl chain, as predicted by Traube’s rule.35 m/aq,°

m/aq,°

m/aq,°

The changes in Gibbs free energy ΔGDet , enthalpy ΔH9:; , and entropy −𝑇ΔSDet , accompanying the transfer of detergent monomers from the aqueous solution into micelles summarized in Table 1 showed that micellization was almost exclusively driven by entropy, with enthalpy making only a minor contribution that decreased and changed sign with increasing chain length. The Gibbs free energy increased in magnitude with chain length; each CH2 group accounted for ~–3.1 kJ/mol, which is in good agreement with the literature.36-37 SFT data were used to construct Gibbs adsorption isotherms (data not shown) to determine the


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surface excess concentration at surface saturation, Γmax. The values observed for DDG (2.51 × 10–12 mol/mm²) and DDDG (2.87 × 10–12 mol/mm²) were very similar, thus indicating similar packing of the two detergents at the air/water interface. From these values, the area occupied per detergent molecule at the air/water interface, Amin, was determined to be 66.2 and 57.9 Å² for DDG and DDDG, respectively.

Figure 2. (A) ITC data for DDG. Shown are an experimental isotherm (open symbols) and a fit based on a generic sigmoidal function (solid line). (B) 1H NMR peak chemical shift, δobs, versus DDG concentration. We followed the signal of the terminal CH3 group of the chain. The solid line represents the nonlinear fit of the experimental points. 25 (C) Surface tension versus DDG concentration. The solid line represents the nonlinear fit of the experimental points.25


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Table 1. Micellar properties of DG derivatives. Detergents Molecular Weight (g/mol)

NMR

TS

657.81

685.86

8.60 ± 0.04

0.69 ± 0.02

0.056 ± 0.011

–TΔS°mic (kJ/mol)b

−25.10 ± 0.01

−29.52 ± 0.07

−33.56 ± 0.55

ΔH°mic (kJ/mol)c

3.36 ± 0.01

1.53 ± 0.02

−0.67 ± 0.05

ΔG°mic (kJ/mol)d

−21.73 ± 0.01

−27.99 ± 0.06

−34.23 ± 0.50

CMC (mM)a

8.69 ± 0.47

0.56 ± 0.02

nd

CMC (mM)a

nd

0.55 ± 0.05

0.052 ± 0.013

γCMC (mN/m)e

nd

42.9 ± 0.0

42.5 ± 1.2

Γmax

*10-12

nd

2.51 ± 0.00

2.87 ± 0.12

Amin

(Å2)f

nd

66.2 ± 0.1

57.9 ± 2.5

nd

−28.56 ± 0.02

−33.44 ± 0.67

89.53

76.49

71.04

89.60

74.92

71.74

DH (nm)h

5.5

6.6

6.9

v̅ (mL/g)i

0.778

0.794

0.808

∂n/∂c (mL/g)j

0.140

0.140

0.140

2.2

2.6

3.15

7.0

5.0

5.4

27

40

58

42

61

84

11.5

0.83

0.31

D

(mol/mm2)f

(µm²/s)g

D0

s0 AUC SV

DDDG

629.76

ΔG°mic (kJ/mol)d DLS

DDG

(mM)a

CMC ITC

ODG

(µm²/s)g

(S)k

k's (mL/g)l Micelle mass Nagg

n

CMC (mM)o

(kDa)m

are averages of at least two experiments. ± indicates standard errors from at least two experiments. contribution to micelle formation. cEnthalpic contribution to micelle formation. dGibbs free energy of micellization. eSurface tension attained at the CMC. fThe surface excess (Γ max) and the surface area per molecule (Amin) were estimated from the slope of the surface tension curve. gDiffusion coefficients determined from Stokes Einstein equation. hHydrodynamic diameter calculated from extrapolation to zero concentration of diffusion coefficients. iPartial specific volume estimated by calculation from chemical composition according to reference by Durchschlag et al.38 jRefractive index increment (∂n/∂c) from linear fit in Figure S15. kSedimentation coefficient at infinite dilution (s ) in water at 20°C. 0 lConcentration dependence factor k’ from linear fits in Figure S15. Errors for k’ are ± 3 mL/g. s s kMolar mass of the micelles from the non-interacting species analysis, error is estimated at 5%. nNumber of aggregation (N ) obtained from micelle and detergent masses. agg oFrom linear fit in Figure S15. aData

bEntropic

We next investigated the self-assembly properties of the detergents in water using dynamic light scattering (DLS). Volume-weighted particle size distributions for ODG, DDG, and DDDG revealed unimodel distributions with average hydrodynamic diameters ranging from 5.5 nm for ODG to 6.9 nm for DDDG (Figure 3A). Upon dilution, no significant difference in the volumeweighted distributions was observed for both compounds (Figure 3B). Number-weighted


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particle size distributions also revealed unimodel distributions for the three compounds while a bimodal pattern was observed in intensity-weighed particle size distributions, for ODG and DDDG, with a second peak of average hydrodynamic diameter around 100 and 200 nm, respectively (Figure S14). However, these larger particles accounted for only a small fraction of the total material present in the samples.

Figure 3. (A) Normalized volume-weighted particle size distributions for ODG, DDG, and DDDG. (B) Dependence of the intensity-weighted hydrodynamic diameter on detergent concentration. (C) Superposition of experimental and calculated SV profiles for DDG at 7.5 mM at 130.000 g every 10 min, over 983 min in a centerpiece of 3 mm optical pathlength. (D) Superposition of c(s) for DDG at 15.4 mM (green), 7.5 mM (blue), and 3.1 mM (black).

To further characterize the aggregates formed above the CMC, AUC-SV experiments were performed at 20°C in water at three detergent concentrations. From the c(s) analysis (Figure 3 and Figure S15), we observed a boundary representing micelles at ~2 S for ODG, ~2.5 S for


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DDG, and ~3 S for DDDG. The s-value of the micelles depended on the detergent type and decreased slightly with concentration, as expected for excluded-volume effects. We used dilution series (Figure S16) to determine, from the micelle signals versus concentration, the refractive index increment (dn/dc) and the CMC, as well as, from the s-values, the s-value at infinite dilution (s0) and concentration-dependence factors (k’s) (Table 1). For the three detergents, AUC provided CMC values larger than those obtained from NMR, SFT, and ITC (Table 1) as previously observed for other detergents,19 the difference between the values determined by AUC and the other techniques being increased as the CMC decreased. The molar masses, Mmic, and, thus, the aggregation numbers, Nagg, of the micelles obtained by the non-interacting species analysis of AUC-SV profiles are reported in Table 1. Nagg values increased with the length of the alkyl chain from 42 for ODG to 61 for DDG and to 84 for DDDG, which is in line with what is generally observed for hydrogenated

39

and fluorinated

detergents.19 Slightly larger Nagg values of 50, 70, and 92 for ODG, DDG, and DDDG respectively, were obtained by combining the hydrodynamic diameter from DLS and s0 from SV-AUC in the Svedberg equation. Detergency. The detergency of a detergent reflects its ability both to solubilize (artificial) lipid bilayers and to extract membrane proteins. To study the detergency of the new DG derivatives, we investigated the solubilization of large unilamellar vesicles (LUVs) composed of 1-plamitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC) into mixed micelles with the aid of light scattering measurements.40 The solubilization of 0.1 mM POPC in the form of LUVs was complete within ~6 h for ODG and in ~2 h for DDG and DDDG, each at 5 mM detergent above the respective CMC value (Figure S17). We next investigated the extraction of integral membrane proteins from E. coli membranes in terms of SDS-PAGE band patterns and overall amounts of extracted proteins. We compared the protein-extraction yields thus obtained with those afforded by the common detergent n-dodecyl-ß-D-maltopyranoside (DDM). As can be seen from Figure 4A, each derivative was


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able to extract MPs spanning a broad range of molecular weights. The overall band patterns of the three derivatives showed only minor differences; for example, ODG was not able to solubilize MPs below 10 kDa, which contrasted with DDG and DDDG. DDM was by far the most effective detergent in extracting outer membrane protein A, OmpA (~35 kDa).41 Figure 4B shows the overall protein-extraction yields relative to the value obtained using a negative control (i.e., buffer without any detergent) in dependence of the concentration of micellar detergent (i.e., total detergent concentration minus CMC). As expected, all proteinextraction yields were concentration-dependent, but this dependence varied clearly among the three DG compounds. At a concentration of 1 mM above the respective CMC, each of the three derivatives performed better than DDM, with ODG and DDG being the most efficient protein solubilizers. While for the whole concentration range a slight decrease was observed for ODG, the extraction yields of DDG and DDDG increased significantly with increasing concentration up to 5 mM above the CMC. Further increasing the concentration to 10 mM above the CMC did not result in a further enhancement in protein-extraction yields for DDG and DDDG, whereas the protein-extraction yield of DDM continued to increase steeply and, therefore, outperformed that of the DG derivatives at very high detergent concentrations.

Figure 4. (A) SDS-PAGE of E. coli extracts upon exposure to different detergents at increasing micellar concentration (i.e., total detergent concentration minus CMC). The arrow indicates the position of the abundant outer-membrane porin OmpA, which was extracted extraordinarily well by DDM. (B) Protein-extraction yield as functions of the micellar concentration of ODG, DDG, DDDG, or DDM. Extraction yields are reported relative


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to the yield obtained when only buffer was used as negative control. Error bars indicate standard deviation for 2 experiments for the DG derivatives and 6 for DDM.

In order to evaluate if the solubilizing properties of the newly developed molecules can be extended to other membranes and other membrane proteins, we applied them to other membrane proteins expressed in E. coli and insect cells (Sf9). 2, 5, 10 and 20 mM of each detergent was used to evaluate solubilization efficiency on two membrane proteins, namely, bacterial transporter (BmrA) and native adenosine receptor (A2AR). These two targets represent two important but distinct classes of membrane proteins, that is, transporters and G-proteincoupled receptors (GPCRs) , respectively. Solubilization efficiency was assessed using stainfree SDS-PAGE and Western blots for total and target proteins (i.e., BmrA and A2AR), respectively. As shown in Figure 5A, ODG was as good as DDM for solubilization of total membrane proteins from Sf9 cells. DDG and DDDG solubilized more efficiently than DDM from Sf9 membranes (compare lne 11 vs 3 and 15 to 3). The same observation was made when A2AR signal (including truncated and full length populations of the protein, analysed by Western blot, Figure 5B). DDG and DDDG seem also superior to ODG in term of solubilization. The same tendencies were also obtained when BmrA was solubilized (Figure 5C). ODG, DDG and DDDG were also able to solubilize Matrix 2 ion channel from MDCK cells (Figure S18).


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Figure 5. Solubilization of two different membrane proteins, namely, wild-type, full-length A2AR (A and B) and BmrA (C) produced in insect cells (Sf9) and E.coli, respectively. (-) and SDS correspond to negative (buffer) and positive controls, respectively. M corresponds to molecular weight marker. 2, 5, 10 and 20 mM of ODG, DDG and DDDG were used for solubilization. The resulting solubilized fractions were loaded into SDS-PAGE. A2AR solubilization was monitored by stain free gel (A) and western blot against his-tag (B). Solubilization of BmrA was monitored by stain free gel only (C) since the protein was overexpressed in comparison to A2AR.


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Stabilizing properties. We finally evaluated the effect of the new detergents on A2AR stability by means of a thermal-shift assay that was established to compare solubilizing properties of detergents at 10 CMC.30 Since DDDG was not able to solubilize A2AR at 10 CMC (data not shown) but required higher concentration (Figure 5), only ODG and DDG were used in this assay. We validated that at 10 CMC both ODG and DDG were able to solubilize A2AR. A2AR is a good protein target since it is known to be rather unstable when expressed and solubilized as full length without truncation and mutations…We first assessed the aggregational state of A2AR after solubilization by means of native PAGE (Figure 6A). We used DDM/Cholesterol Hemisuccinate (CHS) as a solubilization reference for A2AR.33 DDM/CHS led to the formation of A2AR aggregates that did not enter the gel (Figure 6A, lane 1). Interestingly, solubilization by ODG and DDG significantly improved A2AR migration on native PAGE, with DDG exhibiting the best profile. The better behavior in solution might be due to a potential stabilizing effect of DDG and ODG. To test this hypothesis, we investigated the stability of A2AR after solubilization using a Western-blot-based thermal shift assay.33 This assay relies on the assumption that unstable heated proteins will aggregate and that after ultracentrifugation and western blot the band intensity corresponding to the protein will decay proportionally to its instability. Previous studies have shown that the native form of A2AR is notoriously unstable and that the only relatively stable condition in the presence of DDM/CHS was achieved by truncation of the C-terminus as well as mutagenesis of eight amino acid residues.42-44 Intriguingly, we observed a clear upshift in the midpoint temperature of thermal denaturation, Tm, for both ODG and DDG as compared with DDM/CHS (Figure 6B): DDG improved A2AR stability by 9°C, whereas ODG showed a striking increase in Tm by 13°C in comparison with DDM/CHS (Figure 6C). Similar results were recently reported using glycosyl-substituted dicarboxylates45 or calixarene-based detergent30. Using stabilizing detergents like DDG or ODG represent an important alternative to classical mutagenesis, truncation and fusion


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approaches used to stabilize MPs. Differences in the degree of cooperativity of the curves were noticed. However, further studies are required to confirm and provide an explanation.

Figure 6. Homogeneity and stability of solubilized A2AR using DDM/CHS, ODG, or DDG. (A) Solubilized A2AR was loaded on a 4–15% Tris-glycine clear native PAGE and detected by A2AR antibody. (B) Solubilized A2AR was submitted to heat treatment (30–80°C), followed by ultracentrifugation before loading of the supernatant onto SDS-PAGE and Western blotting. (C) A2AR gel bands were quantified to derive Tm.


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Conclusions We have designed a series of detergents called ODG, DDG, and DDDG whose polar headgroup consists of two glucose moieties and whose hydrophobic tail is made, respectively, of octane, decane, and dodecane thiol attached to the headgroup by a Michael reaction. Formation of micelles in water occurred over a large range of concentrations as governed by the length of the hydrophobic chain. The detergents self-assembled into compact and well-defined globular micelles of 6–8 nm diameter with aggregation numbers that increase with chain length from ~40 to ~80. Their potency to act as detergents was first demonstrated using synthetic lipid vesicles and was further confirmed through the extraction of membrane proteins from bacterial, insect, and MDCK membranes. The three ODG, DDG and DDDG showed detergency however DDG and DDDG exhibited better solubilizing properties than ODG as observed in terms of lipid solubilization (Figure S17) and membrane-protein extraction (Figures 4, 5, and S18), with DDG being efficient at lower concentrations than DDDG. The stabilizing properties of ODG and DDG were finally demonstratred by a striking thermostability improvement of 13 and 9°C, respectively (Figure 6). Therefore, the combination of a diglucose branched polar head with a 10C chain through a thioether bond led to the optimized DDG detergent with good solubilizing and stabilizing properties. Interestingly, the shorter octyl analog ODG exhibited even improved stabilizing properties but lower solubilizing potency. Taken together, these findings support the usefulness of this novel series of diglucosylated detergents as promising solubilizing and stabilizing agents for membrane proteins.

Acknowledgements. This work was supported by the Agence Nationale de la Recherche (ANR) through grants no. ANR-14-LAB7-0002 and by the Deutsche Forschungsgemeinschaft (DFG) through grant no. KE 1478/7-1. This work used the platforms of the Grenoble InstructERIC Center (ISBG: UMS 3518 CNRS-CEA-UGA-EMBL) with support from FRISBI (ANR10-INSB-05-02) and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB). Thanks are due for the excellent technical assistance of Anaïs


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Marconnet in the synthesis of the compounds and of Aline Le Roy in AUC experiments. We thank Elodie Desuzinges for her help with producing BmrA and M2 membranes. The authors thank Emmanuel Dejean for his continuous support.

Supplementary material available. 1H and

13

C NMR spectra, mass spectrometry data and

HPLC chromatogram of ODG, DDG and DDDG. 1H NMR chemical shift dependence with the concentration for ODG; DLS particle size distributions for ODG, DDG, and DDDG, sedimentation velocity profiles of ODG and DDDG; vesicle solubilization by ODG, DDG, and DDDG. Conflict of interest. ODG and DDG molecules are directly commercially available from CALIXAR (www.calixar.com) and other distributors worldwide. DDDG is available from Molecular Dimension (www.moleculardimensions.com).


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Hydrogenated diglucose detergents for membrane-protein extraction

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