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ABSTRACT: The use of fluorinated surfactants as efficient tools for handling membrane proteins in aqueous media
was demonstrated many years ago. The work reported herein deals with the synthesis of a new sugar-based surfactant bearing two glucose moieties and labelled F6-DigluM. The synthesis of F6-DigluM is based on a one-pot reduction/alkylation of a fluorinated thioacetate onto an acrylamido-type polar head
precursor, using NaBH4 in refluxing methanol. Its physical–chemical properties in aqueous solution were
studied by surface tension measurement, dynamic light scattering and analytical ultracentrifugation. F6-
DigluM exhibits a critical micellar concentration of �0.4 mM and self-assembles into small and welldefined
aggregates, very likely to globular micelles. Finally, the homogeneity and the stability of complexes of bacteriorhodopsin and F6-DigluM over time were observed, demonstrating that F6-DigluM
is a suitable tool for biochemical applications.
Journal of Fluorine Chemistry 05/2012; 134:63. · 2.03 Impact Factor
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Rita Rahmeh,
Marjorie Damian,
Martin Cottet,
Hélène Orcel,
Christiane Mendre,
Thierry Durroux,
K Shivaji Sharma,
Grégory Durand, Bernard Pucci,
Eric Trinquet,
Jurriaan M Zwier,
Xavier Deupi,
Patrick Bron,
Jean-Louis Banères,
Bernard Mouillac,
Sébastien Granier
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ABSTRACT: G protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters, representing the largest group of therapeutic targets. Recent studies show that some GPCRs signal through both G protein and arrestin pathways in a ligand-specific manner. Ligands that direct signaling through a specific pathway are known as biased ligands. The arginine-vasopressin type 2 receptor (V2R), a prototypical peptide-activated GPCR, is an ideal model system to investigate the structural basis of biased signaling. Although the native hormone arginine-vasopressin leads to activation of both the stimulatory G protein (Gs) for the adenylyl cyclase and arrestin pathways, synthetic ligands exhibit highly biased signaling through either Gs alone or arrestin alone. We used purified V2R stabilized in neutral amphipols and developed fluorescence-based assays to investigate the structural basis of biased signaling for the V2R. Our studies demonstrate that the Gs-biased agonist stabilizes a conformation that is distinct from that stabilized by the arrestin-biased agonists. This study provides unique insights into the structural mechanisms of GPCR activation by biased ligands that may be relevant to the design of pathway-biased drugs.
Proceedings of the National Academy of Sciences 04/2012; 109(17):6733-8. · 9.68 Impact Factor
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ABSTRACT: Structural studies of membrane protein are still challenging due to several severe bottlenecks, the first being the overproduction of well-folded proteins. Several expression systems are often explored in parallel to fulfil this task, or alternately prokaryotic analogues are considered. Although, mitochondrial carriers play key roles in several metabolic pathways, only the structure of the ADP/ATP carrier purified from bovine heart mitochondria was determined so far. More generally, characterisations at the molecular level are restricted to ADP/ATP carrier or the uncoupling protein UCP1, another member of the mitochondrial carrier family, which is abundant in brown adipose tissues. Indeed, mitochondrial carriers have no prokaryotic homologues and very few efficient expression systems were described so far for these proteins. We succeeded in producing UCP1 using a cell free expression system based on E. coli extracts, in quantities that are compatible with structural approaches. The protein was synthesised in the presence of a fluorinated surfactant, which maintains the protein in a soluble form. Further biochemical and biophysical analysis such as size exclusion chromatography, circular dichroism and thermal stability, of the purified protein showed that the protein is non-aggregated, monodisperse and well-folded.
Biochimica et Biophysica Acta 03/2012; 1818(3):798-805. · 4.66 Impact Factor
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ABSTRACT: A novel type of nonionic amphipols for handling membrane proteins in detergent-free aqueous solutions has been obtained through free-radical homo-telomerization of an acrylamide-based monomer comprising a C(11) alkyl chain and two glucose moieties, using a thiol as transfer reagent. By controlling the thiol/monomer ratio, the number-average molecular weight of the polymers was varied from 8 to 63 kDa. Homopolymeric nonionic amphipols were found to be highly soluble in water and to self-organize, within a large concentration range, into small, compact particles of ~6 nm diameter with a narrow size distribution, regardless of the molecular weight of the polymer. They proved able to trap and stabilize two test membrane proteins, bacteriorhodopsin from Halobium salinarum and the outer membrane protein X of Escherichia coli, under the form of small and well-defined complexes, whose size, composition, and shape were studied by aqueous size-exclusion chromatography, analytical ultracentrifugation, and small-angle neutron scattering. As shown in a companion paper, nonionic amphipols can be used for membrane protein folding, cell-free synthesis, and solution NMR studies (Bazzacco et al. 2012, Biochemistry, DOI: 10.1021/bi201862v).
Langmuir 02/2012; 28(10):4625-39. · 4.19 Impact Factor
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ABSTRACT: Accurate determination of the free energy of transfer of a helical segment from an aqueous into a transmembrane (TM) conformation is essential for understanding and predicting the folding and stability of membrane proteins. Until recently, direct thermodynamically sound measurements of free energy of insertion of hydrophobic TM peptides were impossible due to peptide aggregation outside the lipid bilayer. Here, we overcome this problem by using fluorinated surfactants that are capable of preventing aggregation but, unlike detergents, do not themselves interact with the bilayer. We have applied the fluorescence correlation spectroscopy methodology to study surfactant-chaperoned insertion into preformed POPC (palmitoyloleoylphosphatidylcholine) vesicles of the two well-studied dye-labeled TM peptides of different lengths: WALP23 and WALP27. Extrapolation of the apparent free-energy values measured in the presence of surfactants to a zero surfactant concentration yielded free-energy values of -9.0±0.1 and -10.0±0.1 kcal/mol for insertion of WALP23 and WALP27, respectively. Circular dichroism measurements confirmed helical structure of peptides in lipid bilayer, in the presence of surfactants, and in aqueous mixtures of organic solvents. From a combination of thermodynamic and conformational measurements, we conclude that the partitioning of a four-residue L-A-L-A segment in the context of a continuous helical conformation from an aqueous environment into the hydrocarbon core of the membrane has a favorable free energy of 1 kcal/mol. Our measurements, combined with the predictions of two independent experimental hydrophobicity scales, indicate that the per-residue cost of transfer of the helical backbone from water to the hydrocarbon core of the lipid bilayer is unfavorable and is equal to +2.13±0.17 kcal/mol.
Journal of Molecular Biology 02/2012; 416(3):328-34. · 4.00 Impact Factor
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Paola Bazzacco,
Emmanuelle Billon-Denis,
K Shivaji Sharma,
Laurent J Catoire,
Sophie Mary,
Christel Le Bon,
Elodie Point,
Jean-Louis Banères,
Grégory Durand,
Francesca Zito, Bernard Pucci,
Jean-Luc Popot
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ABSTRACT: Nonionic amphipols (NAPols) synthesized by homotelomerization of an amphiphatic monomer are able to keep membrane proteins (MPs) stable and functional in the absence of detergent. Some of their biochemical and biophysical properties and applications have been examined, with particular attention being paid to their complementarity with the classical polyacrylate-based amphipol A8-35. Bacteriorhodopsin (BR) from Halobacterium salinarum and the cytochrome b(6)f complex from Chlamydomonas reinhardtii were found to be in their native state and highly stable following complexation with NAPols. NAPol-trapped BR was shown to undergo its complete photocycle. Because of the pH insensitivity of NAPols, solution nuclear magnetic resonance (NMR) two-dimensional (1)H-(15)N heteronuclear single-quantum coherence spectra of NAPol-trapped outer MP X from Escherichia coli (OmpX) could be recorded at pH 6.8. They present a resolution similar to that of the spectra of OmpX/A8-35 complexes recorded at pH 8.0 and give access to signals from solvent-exposed rapidy exchanging amide protons. Like A8-35, NAPols can be used to fold MPs to their native state as demonstrated here with BR and with the ghrelin G protein-coupled receptor GHS-R1a, thus extending the range of accessible folding conditions. Following NAPol-assisted folding, GHS-R1a bound four of its specific ligands, recruited arrestin-2, and activated binding of GTPγS by the G(αq) protein. Finally, cell-free synthesis of MPs, which is inhibited by A8-35 and sulfonated amphipols, was found to be very efficient in the presence of NAPols. These results open broad new perspectives on the use of amphipols for MP studies.
Biochemistry 02/2012; 51(7):1416-30. · 3.42 Impact Factor
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ABSTRACT: A novel series of α-phenyl-N-tert-butyl nitrone derivatives, bearing a hydrophobic chain on the aromatic ring and three hydroxyl functions on the tert-butyl group, was synthesized through a short and convenient synthetic route based on a one-pot reduction/condensation of tris(hydroxymethyl)nitromethane with a benzaldehyde derivative. Because of the presence of hydroxyl functions on the tert-butyl group, an intramolecular Forrester-Hepburn reaction leading to the formation of an oxazolidine-N-oxyl compound was observed by electron paramagnetic resonance (EPR). The mechanism of cyclization was further studied by computational methods showing that intramolecular hydrogen bonding and high positive charge on the nitronyl carbon could facilitate the nucleophilic addition of a hydroxyl group onto the nitronyl carbon. At high nitrone concentrations, a second paramagnetic species, very likely formed by intermolecular nucleophilic addition of two nitrone molecules, was also observed but to a lesser extent. In addition, theoretical data confirmed that the intramolecular reaction is much more favored than the intermolecular one. These nitrones were also found to efficiently trap carbon-centered radicals, but complex spectra were observed due to the presence of oxazolidine-N-oxyl derivatives.
The Journal of Organic Chemistry 12/2011; 77(2):938-48. · 4.45 Impact Factor
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ABSTRACT: Solubilizing membrane proteins for functional, structural and thermodynamic studies is usually achieved with the help of detergents, which, however, tend to destabilize them. Several classes of non-detergent surfactants have been designed as milder substitutes for detergents, most prominently amphipathic polymers called 'amphipols' and fluorinated surfactants. Here we test the potential usefulness of these compounds for thermodynamic studies by examining their effect on conformational transitions of the diphtheria toxin T-domain. The advantage of the T-domain as a model system is that it exists as a soluble globular protein at neutral pH yet is converted into a membrane-competent form by acidification and inserts into the lipid bilayer as part of its physiological action. We have examined the effects of various surfactants on two conformational transitions of the T-domain, thermal unfolding and pH-induced transition to a membrane-competent form. All tested detergent and non-detergent surfactants lowered the cooperativity of the thermal unfolding of the T-domain. The dependence of enthalpy of unfolding on surfactant concentration was found to be least for fluorinated surfactants, thus making them useful candidates for thermodynamic studies. Circular dichroism measurements demonstrate that non-ionic homopolymeric amphipols (NAhPols), unlike any other surfactants, can actively cause a conformational change of the T-domain. NAhPol-induced structural rearrangements are different from those observed during thermal denaturation and are suggested to be related to the formation of the membrane-competent form of the T-domain. Measurements of leakage of vesicle content indicate that interaction with NAhPols not only does not prevent the T-domain from inserting into the bilayer, but it can make bilayer permeabilization even more efficient, whereas the pH-dependence of membrane permeabilization becomes more cooperative. This article is part of a Special Issue entitled: Protein Folding in Membranes.
Biochimica et Biophysica Acta 09/2011; 1818(4):1006-12. · 4.66 Impact Factor
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ABSTRACT: To prepare non-ionic amphiphilic polymers usable for solubilizing integral membrane proteins, hydrophilic and amphiphilic β-D-glucose-based tris(hydroxymethyl)acrylamidomethane monomers were synthesized and their radical homopolymerization rate constants (f k 2 p /k t) were determined. The polymerization kinet-ics, monitored by 1 H-NMR, were carried out in THF in the presence of AIBN as radical initiator. The plot of monomer conversion as a function of time followed Tobolsky's equation. The number of hydroxyl groups, as well as the nature of the substituents grafted onto the hydroxyl groups, was found to alter the rates of polymerization. While the steric hindrance significantly affected the kinetics, intermolecular hydro-gen bonds between hydroxyl groups, as demonstrated by NMR investigations at different concentrations and temperatures, may also play a role in the rate constants of polymerization. © Koninklijke Brill NV, Leiden, 2011
Designed Monomers & Polymers 09/2011; 14:499-513. · 1.44 Impact Factor
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ABSTRACT: Cell-free protein synthesis is a well-known technique for the roles it has played in deciphering the genetic code and in the beginnings of signal sequence studies. Since then, many efforts have been made to optimise this technique and, recently, to adapt it to membrane protein production with yields compatible with structural investigations. The versatility of the method allows membrane proteins to be obtained directly stabilised in surfactant micelles or inserted in a lipidic environment (proteoliposome, bicelle, and nanodisc) at the end of synthesis. Among the surfactants used, non-detergent ones such as fluorinated surfactants proved to be a good alternative in terms of colloidal stability and preservation of the integrity of membrane proteins, as shown for Escherichia coli homo-pentameric channel, MscL (Park et al., Biochem. J., 403: 183-187). Here we report cell-free expression of Escherichia coli leader peptidase (a transmembrane protease), Halobacterium salinarium bacteriorhodopsin (a transmembrane protein binding a hydrophobic cofactor) and E. coli MscL in the presence of non-detergent surfactants, amphipols and fluorinated surfactants in comparison to their expression in classical detergents. The results confirm the potentialities of fluorinated surfactants and, although pointing to limitations in using the first generations amphipols, results are discussed in the light of membrane protein refolding, especially in the case of bacteriorhodopsin. Preliminary experiments using new generations of amphipols supports choices made in developing new molecules.
New Biotechnology 04/2011; 28(3):255-61. · 2.76 Impact Factor
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ABSTRACT: The advantages of using hemifluorinated surfactants as an efficient alternative to detergents for manipulating membrane proteins in aqueous solution have been demonstrated in recent reports. However, the large-scale synthesis of these surfactants is still considered as a major matter and has limited their use for biochemical purposes. We report herein the synthesis of a novel series of perfluorohexane-based surfactants endowed with a short propyl hydrocarbon tip and whose polar head size is modulated by the presence of two or three glucose moieties. The synthetic route is based on the radical addition of two alkenes onto the 1,6-diiodoperfluorohexane using AIBN as a radical initiator, affording the surfactants in satisfactory overall yields. The self-assembling properties of these hemifluorinated surfactants were studied by surface tension measurements, dynamic light scattering, as well as their behavior upon reversed-phase chromatography and were compared with those of their perfluorinated analogues. Our findings strongly suggest the predominant influence of the propyl tip on both adsorption and micellization phenomena as well as on the hydrophobic character of the surfactants, whereas as previously observed, the shorter ethyl tip does not greatly affect these properties when compared to the perfluorinated analogues. Moreover, all the surfactants reported here self-assemble into small and monodisperse aggregates, a feature of crucial importance for biochemistry applications.
The Journal of Organic Chemistry 03/2011; 76(7):2084-93. · 4.45 Impact Factor
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Jens Hovers,
Meike Potschies,
Ange Polidori, Bernard Pucci,
Simon Raynal,
Françoise Bonneté,
Maria J Serrano-Vega,
Christopher G Tate,
Daniel Picot,
Yves Pierre,
Jean-Luc Popot,
Rony Nehmé,
Michel Bidet,
Isabelle Mus-Veteau,
Holger Busskamp,
Karl-Heinz Jung,
Andreas Marx,
Peter A Timmins,
Wolfram Welte
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ABSTRACT: Mixed protein-surfactant micelles are used for in vitro studies and 3D crystallization when solutions of pure, monodisperse integral membrane proteins are required. However, many membrane proteins undergo inactivation when transferred from the biomembrane into micelles of conventional surfactants with alkyl chains as hydrophobic moieties. Here we describe the development of surfactants with rigid, saturated or aromatic hydrocarbon groups as hydrophobic parts. Their stabilizing properties are demonstrated with three different integral membrane proteins. The temperature at which 50% of the binding sites for specific ligands are lost is used as a measure of stability and dodecyl-β-D-maltoside ('C12-b-M') as a reference for conventional surfactants. One surfactant increased the stability of two different G protein-coupled receptors and the human Patched protein receptor by approximately 10°C compared to C12-b-M. Another surfactant yielded the highest stabilization of the human Patched protein receptor compared to C12-b-M (13°C) but was inferior for the G protein-coupled receptors. In addition, one of the surfactants was successfully used to stabilize and crystallize the cytochrome b(6 )f complex from Chlamydomonas reinhardtii. The structure was solved to the same resolution as previously reported in C12-b-M.
Molecular Membrane Biology 02/2011; 28(3):171-81. · 2.86 Impact Factor
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ABSTRACT: The precise mechanism-of-action of thalidomide remains uncertain and might differ between diseases and under different clinical condition. With implications in the treatment of a variety of inflammatory and autoimmune diseases, as well as for use as an anticancer agent, alone or in combination with established therapeutics, it is clear that thalidomide and its derivatives deserve further scrutiny. In particular, thalidomide was shown to be effective in a mouse model of multiple sclerosis (MS), an autoimmune inflammatory disorder, called experimental autoimmune encephalomyelitis (EAE). Herein, we describe the synthesis and preliminary biological evaluation of new macromolecular prodrugs of thalidomide bearing an aminoalkyl group on the phthalimide ring. The effectiveness of these compounds to limit EAE was investigated, and it was shown that, at 100 mg kg⁻¹ thalidomide-equivalent dose, they abrogated the clinical and pathological features of EAE.
ChemMedChem 10/2010; 5(12):2057-64. · 3.15 Impact Factor
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ABSTRACT: Two cholesterol-based α-phenyl-N-tert-butyl nitrone derivatives were synthesized as antioxidants against light-induced retinal degeneration. Whereas nitrone 10 significantly protected retina against bright fluorescent light exposure when injected into the vitreous at 1 mM, no protection was observed with nitrone 6. The parent compound α-phenyl-N-tert-butyl nitrone also exhibited protective activity at 9 mM but not at 1 mM. This suggests that nitrone 10 may be a candidate for the treatment of retinal diseases.
Bioorganic & medicinal chemistry letters 10/2010; 20(24):7405-9. · 2.65 Impact Factor
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ABSTRACT: It has been suggested that above a critical protein concentration, fish Type III antifreeze protein (AFP III) self-assembles to form micelle-like structures that may play a key role in antifreeze activity. To understand the complex activity of AFP III, a comprehensive description of its association state and structural organization in solution is necessary. We used analytical ultracentrifugation, analytical size-exclusion chromatography, and dynamic light scattering to characterize the interactions and homogeneity of AFP III in solution. Small-angle neutron scattering was used to determine the low-resolution structure in solution. Our results clearly show that at concentrations up to 20 mg mL(-1) and at temperatures of 20 degrees C, 6 degrees C, and 4 degrees C, AFP III is monomeric in solution and adopts a structure compatible with that determined by crystallography. Surface tension measurements show a propensity of AFP III to localize at the air/water interface, but this surface activity is not correlated with any aggregation in the bulk. These results support the hypothesis that each AFP III molecule acts independently of the others, and that specific intermolecular interactions between monomers are not required for binding to ice. The lack of attractive interactions between monomers may be functionally important, allowing for more efficient binding and covering of the ice surface.
Biophysical Journal 07/2010; 99(2):609-18. · 3.65 Impact Factor
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ABSTRACT: Our group has demonstrated that the amphiphilic character of alpha-phenyl-N-tert-butyl nitrone based agents is a key feature in determining their bioactivity and protection against oxidative toxicity. In this work, we report the synthesis of a new class of amphiphilic amide nitrones. Their hydroxyl radical scavenging activity and radical reducing potency were shown using ABTS competition and ABTS(+) reduction assays, respectively. Cyclic voltammetry was used to investigate their redox behavior, and the effects of the substitution of the PBN on the charge density of the nitronyl atoms, the electron affinity, and the ionization potential were computationally rationalized. The protective effects of amphiphilic amide nitrones in cell cultures exposed to oxidotoxins greatly exceeded those exerted by the parent compound PBN. They decreased electron and proton leakage as well as hydrogen peroxide formation in isolated rat brain mitochondria at nanomolar concentration. They also significantly enhanced mitochondrial membrane potential. Finally, dopamine-induced inhibition of complex I activity was antagonized by pretreatment with these agents. These findings indicate that amphiphilic amide nitrones are much more than just radical scavenging antioxidants but may act as a new class of bioenergetic agents directly on mitochondrial electron and proton transport.
Journal of Medicinal Chemistry 07/2010; 53(13):4849-61. · 4.80 Impact Factor
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ABSTRACT: Due to the dual property of synthetic nitroxide compounds to either act as probe or antioxidant, efforts toward their selective targeting using specific ligands have been extensively explored. Herein, we report the synthesis of novel amphiphilic nitroxides in which the nitroxyl group is grafted onto an amphiphilic carrier comprising a lactobionamide polar group, a non-polar alkyl chain and an amino acid as scaffold. Piperidine and pyrrolidine nitroxides such as 4-amino-TEMPO (4-AT) and 3-carboxyproxyl (3-CP), respectively, were grafted onto the amphiphilic carriers. To further investigate the effect of the nature of the chain on the physical-chemical and biological properties of nitroxides, hydrogenated or perfluorinated alkyl chains were used. The self-aggregation properties in aqueous media of these surfactant-like nitroxides were confirmed by dynamic light scattering (DLS) as well as electron paramagnetic resonance (EPR) spectroscopy, and were correlated with their respective lipophilicity. The effect of the carrier groups on the electrochemical property of nitroxides was investigated using cyclic voltammetry, and the rates of reduction using ascorbate as reducing agent were measured. Finally, their cytoprotective property against toxic concentrations of hydrogen peroxide using bovine aortic endothelial cells was also investigated.
New Journal of Chemistry 03/2010; 34:1909. · 2.61 Impact Factor
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ABSTRACT: Smoothened is a member of the G-protein coupled receptor (GPCR) family responsible for the transduction of the Hedgehog signal to the intracellular effectors of the Hedgehog signaling pathway. Aberrant regulation of this receptor is implicated in many cancers but also in neurodegenerative disorders. Despite the pharmacological relevance of this receptor, very little is known about its functional mechanism and its physiological ligand. In order to characterize this receptor for basic and pharmacological interests, we developed the expression of human Smoothened in the yeast Saccharomyces cerevisiae and Smoothened was then purified. Using Surface Plasmon Resonance technology, we showed that human Smoothened was in a native conformational state and able to interact with its antagonist, the cyclopamine, both at the yeast plasma membrane and after purification. Thermostability assays on purified human Smoothened showed that this GPCR is relatively stable in the classical detergent dodecyl-beta-d-maltoside (DDM). The fluorinated surfactant C(8)F(17)TAC, which has been proposed to be less aggressive towards membrane proteins than classical detergents, increased Smoothened thermostability in solution. Moreover, the replacement of a glycine by an arginine in the third intracellular loop of Smoothened coupled to the use of the fluorinated surfactant C(8)F(17)TAC during the mutant purification increased Smoothened thermostability even more. These data will be very useful for future crystallization assays and structural characterization of the human receptor Smoothened.
Biochimica et Biophysica Acta 02/2010; 1798(6):1100-10. · 4.66 Impact Factor
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ABSTRACT: Handling integral membrane proteins in aqueous solutions traditionally relies on the use of detergents, which are surfactants capable of dispersing the components of biological membranes into mixed micelles. The dissociating character of detergents, however, most often causes solubilized membrane proteins to be unstable. This has prompted the development of alternative, less-aggressive surfactants designed to keep membrane proteins soluble, after they have been solubilized, under milder conditions. A short overview is presented of the structure, properties, and uses of two families of such surfactants: amphiphilic polymers ("amphipols") and fluorinated surfactants.
Methods in molecular biology (Clifton, N.J.) 01/2010; 601:219-45.
