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Trapping and Stabilization of Integral Membrane Proteins by Hydrophobically Grafted Glucose-Based Telomers Paola Bazzacco,†,‡ K. Shivaji Sharma,†,§,| Gre´gory Durand,*,§ Fabrice Giusti,‡ Christine Ebel,⊥,# Jean-Luc Popot,*,‡ and Bernard Pucci*,§ Laboratoire de Physico-Chimie Mole´culaire des Prote´ines Membranaires, UMR 7099, CNRS and Universite´ Paris-7, Institut de Biologie Physico-Chimique, 13, rue Pierre-et-Marie-Curie, F-75005 Paris, France, Laboratoire de Chimie Bioorganique et des Syste`mes Mole´culaires Vectoriels, Universite´ d’Avignon et des Pays de Vaucluse, Faculte´ des Sciences, 33 rue Louis Pasteur, F-84000 Avignon, France, CEA, IBS, Laboratoire de Biophysique Mole´culaire, F-38054 Grenoble, France, CNRS, UMR5075, F-38027 Grenoble, France, and Universite´ Joseph Fourier, F-38000, Grenoble, France Received August 18, 2009; Revised Manuscript Received October 30, 2009
Amphipols (APols) are short amphipathic polymers designed to adsorb onto the transmembrane surface of membrane proteins, keeping them water-soluble in the absence of detergent. Current APols carry charged groups, which is a limitation for certain types of applications. This has prompted the development of totally nonionic amphiphols (NAPols). In a previous work, glucose-based NAPols synthesized by free-radical cotelomerization of hydrophilic and amphiphilic monomers proved to be able to keep membrane proteins soluble (Sharma et al. Langmuir 2008, 24, 13581-13590). This provided a proof of principle, but the cumbersome synthesis prevented large-scale production and any detailed biochemical studies. In the present work, we describe a new synthesis route for NAPols based on grafting alkyl chains onto a glucosylated homotelomer. The NAPols thus prepared are highly water soluble. In aqueous solutions, they assemble into small, homogeneous particles similar to those formed by ionic APols. Two model membrane proteins, bacteriorhodopsin and the transmembrane domain of OmpA, form with NAPols small, well-defined water-soluble complexes whose size is comparable to that observed with ionic APols. Complexation by NAPols strongly stabilizes bacteriorhodopsin against denaturation. Glucosylated NAPols thus appear as a promising alternative to ionic APols for such applications as ion-exchange chromatography, isoelectrofocusing, and, possibly, structural approaches such as NMR and crystallography.
Introduction In vitro studies are essential to understanding the structure and function of membrane proteins (MPsa). They are, however, complicated by the insolubility of these proteins in aqueous solutions. Detergents, which are used traditionally to solubilize MPs,1,2 tend to be inactivating (see, e.g., refs 2-7). Specially * To whom correspondence should be addressed. E-mail:
[email protected] (J.-L.P.);
[email protected] (G.D.);
[email protected] (B.P.). † These two authors contributed equally. ‡ Institut de Biologie Physico-Chimique. § Universite´ d’Avignon et des Pays de Vaucluse. | Present address: De´partement de Chimie et Faculte´ de Pharmacie, Universite´ de Montre´al, Pavillon J. A. Bombardier, CP 6128 Succursale Centre Ville, Montreal QC H3C 3J7, Canada. ⊥ Institut de Biologie Structurale. # Universite´ Joseph Fourier. a Abbreviations: AIBN, R,R′-azobisisobutyronitrile; APol, amphipol; ASEC, aqueous size exclusion chromatography; AUC, analytical ultracentrifugation; BR, bacteriorhodopsin; C8E4, tetraethylene glycol monooctyl ether; cmc, critical micellar concentration; DABCO, 1,4-diazabicyclo[2.2.2]octane; DLS, dynamic light scattering; DMF, dimethylformamide; DPn, number-average degree of polymerization; M, N-[1,1-bishydroxymethyl 1-[(2′,3′,4′,6′-tetraO-acetyl-β-D-glucopyranosyl)-oxymethyl]-methyl]-acrylamide; Ma, molecular mass of NAPol particles; MP, membrane protein; 〈Mn〉, number-average molecular weight; 〈Mw〉, weight-average molecular weight; MW, molecular weight; NAPol, nonionic amphipol; OTG, n-octyl-β-D-thioglucopyranoside; p-GHT, protected grafted homotelomer; p-HT, protected homotelomer; PMMA, poly(methyl methacrylate); SEC, size exclusion chromatography; SV, sedimentation velocity; TA, telogen agent; THAM, tris(hydroxymethyl)acrylamidomethane; THF, tetrahydrofuran; TLC, thin-layer chromatography; tOmpA, transmembrane domain of Escherichia coli’s outer membrane protein A; Buffers: Tris buffer, 100 mM NaCl, 20 mM Tris/HCl, pH 8.0; Phosphate buffer, 100 mM NaCl, 20 mM sodium phosphate, pH 7.0.
designed amphipathic polymers called ‘amphipols’ (APols) can substitute for detergents.8 APols adsorb onto the strongly hydrophobic transmembrane surface of MPs.9,10 Thereby, they make them water-soluble, while stabilizing them biochemically (reviewed in refs 6, 11-13). APol-trapped MPs are amenable to structural and functional studies by such methods as absorption or fluorescence spectroscopy,14-16 NMR,9,10,17 or, after APol-mediated immobilization onto solid supports, surface plasmon resonance and other approaches to detecting the binding of ligands.16 In addition, APols can be used to fold to their native state MPs that have been expressed under an inactive form.18,19 Because APols carry multiple hydrophobic chains, their solubility depends on the presence of highly hydrophilic moieties. The high aqueous solubility of the most extensively studied APol to date, APol A8-35, results from the presence of carboxylates.8,20,21 The charges thus conferred to MP/A8-35 complexes prevent the use of such techniques as ion exchange chromatography and isoelectric focusing to analyze or purify them. Neither are they a favorable factor for their crystallization. Furthermore, A8-35 cannot be used at pH values 100 g · L-1). In aqueous buffers, they
form small, compact, globular, well-defined particles whose size, as determined by ASEC, DLS, and AUC, is comparable to that of the particles formed by A8-35,21 namely, a hydrodynamic radius close to 3 nm, whatever the polymer length. The polydispersity of the particles, as reflected in the width of the SEC peaks, is also similar to that observed with A8-35. It is remarkable that both the hydrodynamic radius and the molecular mass of the particles, 72-75 kDa, are independent of the length of the polymer (from DPn ) 30-60). The particles formed by the longer polymers, for example, NA25-75b, therefore comprise about three 25 kDa molecules versus about six 12.5 kDa ones for the shorter NAPol, NA12.5-75. A8-35 particles, as a comparison, have a mass of ∼40 kDa and comprise about four 9-10 kDa molecules.21 Because NA25-75b carries an average of 16 undecyl chains per molecules and NA12.5-75 carries about 8, the hydrophobic core of their particles comprises, on average, ∼36 undecyl chains versus ∼80 octyl chains in the case of A835. This smaller volume of the hydrophobic core is likely due to the bulk of the hydrophilic moieties in glucosylated NAPols,
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Figure 7. UV/visible absorption spectrum of BR after trapping with various APols. BR in 18 mM OTG in phosphate buffer was supplemented with NAPols (NA25-75b or NA12.5-75) or A8-35 (batch FGH20) at the BR/APol mass ratios indicated in the inset and, after dilution under the cmc and a 20 min incubation at 4 °C, centrifuged for 20 min at 200000 × g, and the spectrum of the supernatant recorded.
Figure 8. Biochemical stability of BR after complexation by APols. Complexes were prepared as described in the legend to Figure 5 and stored in phosphate buffer at 4 °C in the dark for up to 15 days. The evolution of the concentration of native BR over time was determined from the absorbance of the solutions at 554 nm. The drop seen for BR/NAPol preparations after centrifugation is due to the sedimentation of part of the complexes (see Figure 5), due to their higher density (see Discussion). The evolution over time of preparations of BR trapped with A8-35 or kept in 18 mM OTG is shown for comparison. Inset: color code and BR/APol mass ratios used for trapping.
which works against the formation of particles with a large radius of curvature.44 The small size of NAPol particles is a favorable feature for biophysical applications because it is expected to favor the formation of small MP/NAPol complexes, as was actually found. Biochemical Properties. In keeping with previous data,24 glucosylated NAPols efficiently kept water-soluble, in the absence of micellar detergent, the two model MPs tested, BR and tOmpA. With the larger batches yielded by the new synthesis protocol, a first investigation could be carried out of the properties of MP/NAPol complexes. Upon SEC, tOmpA/ NAPol and BR/NAPol complexes migrated as small, welldefined particles, with an apparent size and dispersity comparable to those of tOmpA/A8-35 and BR/A8-35 complexes, respectively. While the apparent size of MP/APol complexes, as determined by SEC, must be considered with some caution (see refs 9, 15, 39), particularly when comparing particles with very different charge densities, this observation suggests that MP/NAPol complexes are suitable for applications where the overall size of the particles matters, such as NMR or crystallization. A notable difference between the MP/NAPol and MP/
A8-35 complexes is that, under our standard ultracentrifugation conditions (20 min at 200000 × g), the retention in solution of BR/NAPol and tOmpA/NAPol complexes after elimination of the detergent was significantly less than observed with A8-35 (typically ∼60 vs ∼95%). This behavior could reflect the presence of very large complexes, if not of aggregates. This hypothesis, however, is not consistent with SEC analyses, which reveal the presence of small particles only, nor with the absence of a protein pellet at the bottom of the centrifugation tubes. On the other hand, BR/NAPol complexes, whose purple color facilitates the observation, were seen to form a gradient of concentration during centrifugation, which accounts for the loss of protein in the sample recovered from the supernatant. This was not observed with BR/A8-35 complexes, whose distribution remained, to the eye, homogeneous. This difference in behavior is probably due to the higher density of MP/NAPol complexes. Sedimentation velocity is proportional to the buoyancy term, the molar mass, and the inverse of the hydrodynamic radius. The buoyancy term is (1 - FVj), with F the solvent density and Vj the partial specific volume (for A8-35, which is a polyelectrolyte, the expression uses φ′ instead of Vj, with φ′ ) 0.866
Grafted Glucose-Based Amphipols
mL · g-1; see ref 20). The buoyancy term for NAPols, whose Vj is 0.732-0.735 mL · g-1 (see Experimental Section), is twice that for A8-35, so that complexes of similar composition and shape are expected to sediment more quickly if comprising NAPols than A8-35, for example, ∼1.6× faster for complexes comprising ∼2 g APol per g protein.15 A more detailed analysis of the composition, size, shape, and hydrodynamic properties of MP/NAPol complexes is beyond the scope of the present report. The major incentive behind the substitution of APols to detergents is to protect MPs from detergent-induced denaturation. The ability of glucosylated NAPols to stabilize MPs was tested using BR. Under the conditions of salt and pH used in the present work, BR, upon storage for two weeks at 4 °C in the dark, denatured by ∼50% when kept in detergent solution. NAPol-trapped BR was found not to inactivate to any extent over the same length of time. Over this period and under these conditions, there was no detectable difference in stability whether BR was complexed by A8-35 or by NAPols, the protein remaining perfectly stable in both environments. However, it is well-known that charged detergents tend to be more aggressive toward MPs than electrically neutral ones (see, e.g., ref 45). There are therefore reasons to expect that NAPols may provide an even milder environment to MPs than A8-35 does.
Conclusion In summary, the present work has established that glucosylated NAPols can be obtained by a simpler synthetic route than was previously available, yielding amounts suitable for biological applications. The new route relies on the homotelomerization of a glucosylated acrylamide monomer onto which alkyl chains are subsequently grafted through a urethane bond. Physicalchemical investigations showed that, regardless of the synthetic route used for their preparation, glucosylated NAPols are highly water-soluble and self-organize into small particles similar in size to those formed by ionic APols. They associate with MPs to form small, well-defined, water-soluble complexes and appear to provide them with a strongly stabilizing environment. Acknowledgment. Particular thanks are due to T. Dahmane for her participation in early experiments, as well as to the Analytical Ultracentrifugation AUC platform of the PBS, Grenoble (France). The authors acknowledge the financial support of the CNRS, Universite´ Paris-7, the CEA, Universite´ d’Avignon et des Pays du Vaucluse, Universite´ Joseph Fourier, ANR grant Hemisurf, and E.C. Specific Targeted Research Project 5137770 Innovative tools for membrane structural proteomics (IMPS). P.B. was the recipient of a Marie Curie Early Stage Training fellowship awarded by the BioMem E.C. training network. K.S.S. was supported by IMPS. Supporting Information Available. Materials, general procedures, and instrumentation for the synthesis; UV-visible spectra of TA, p-HT, and p-GHT of NA25-75b; particle size distribution of NAPols obtained by DLS at various concentrations and temperatures; ASEC profiles of NA25.5-67 at various wavelengths; average molecular weight, obtained by UV spectroscopy or SEC, of various protected homotelomers and grafted homotelomers; particle size distribution parameters (from DLS and ASEC) of various NAPols in aqueous solution at several concentrations. This material is available free of charge via the Internet at http://pubs.acs.org.
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