Structural insights into biased G protein-coupled receptor signaling revealed by fluorescence spectroscopy.

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.

Folding of diphtheria toxin T-domain in the presence of amphipols and fluorinated surfactants: Toward thermodynamic measurements of membrane protein folding.

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.

Production of UCP1 a membrane protein from the inner mitochondrial membrane using the cell free expression system in the presence of a fluorinated surfactant.

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.

Nonionic homopolymeric amphipols: application to membrane protein folding, cell-free synthesis, and solution nuclear magnetic resonance.

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.

Non-ionic amphiphilic homopolymers: synthesis, solution properties, and biochemical validation.

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).

Synthesis of tris-hydroxymethyl-based nitrone derivatives with highly reactive nitronyl carbon.

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.

A class of mild surfactants that keep integral membrane proteins water-soluble for functional studies and crystallization.

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-ß-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.

Stability study of the human G-protein coupled receptor, Smoothened.

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.

Propyl-ended hemifluorinated surfactants: synthesis and self-assembling properties.

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.

Inhibitory potential of chemical substitutions at bioinspired sites of ß-D-galactopyranose on neoglycoprotein/cell surface binding of two classes of medically relevant lectins.

Galactose is the key contact site for plant AB-toxins and the human adhesion/growth-regulatory galectins. Natural anomeric extensions and 3'-substitutions enhance its reactivity, thus prompting us to test the potential of respective chemical substitutions of galactose in the quest to develop potent inhibitors. Biochemical screening of a respective glycoside library with 60 substances in a solid-phase assay was followed by examining the compounds' activity to protect cells from lectin binding. By testing 32 anomeric extensions, 18 compounds with additional 3'-substitution, three lactosides and two Lewis-type trisaccharides rather mild effects compared to the common haptenic inhibitor lactose were detected in both assays. When using trivalent glycoclusters marked enhancements with 6- to 8-fold increases were revealed for the toxin and three of four tested galectins. Since the most potent compound and also 3'-substituted thiogalactosides reduced cell growth of a human tumor line at millimolar concentrations, biocompatible substitutions and scaffolds will be required for further developments. The synthesis of suitable glycoclusters, presenting headgroups which exploit differences in ligand selection in interlectin comparison to reduce cross-reactivity, and the documented strategic combination of initial biochemical screening with cell assays are considered instrumental to advance inhibitor design.

Structure and interactions of fish type III antifreeze protein in solution.

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.

Functional studies of membrane-bound and purified human Hedgehog receptor Patched expressed in yeast.

The Sonic Hedgehog (Shh) signalling pathway plays an important role both in embryonic development and in adult stem cell function. Inappropriate regulation of this pathway is often due to dysfunction between two membrane receptors Patched (Ptc) and Smoothened (Smo), which lead to birth defects, cancer or neurodegenerative diseases. However, little is known about Ptc, the receptor of the Shh protein, and the way Ptc regulates Smo, the receptor responsible for the transduction of the signal. To develop structure-function studies of these receptors, we expressed human Ptc (hPtc) in the yeast Saccharomyces cerevisiae. We demonstrated that hPtc expressed in a yeast membrane fraction is able to interact with its purified ligand Shh, indicating that hPtc is produced in yeast in its native conformational state. Using Surface Plasmon Resonance technology, we showed that fluorinated surfactants preserve the ability of hPtc to interact with its ligand after purification. This is the first report on the heterologous expression and the purification of a native and stable conformation of the human receptor Ptc. This work will allow the scale-up of hPtc production enabling its biochemical characterization, allowing the development of new therapeutic approaches against diseases induced by Shh signalling dysfunction.

Cholesterol-based α-phenyl-N-tert-butyl nitrone derivatives as antioxidants against light-induced retinal degeneration.

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.

Micellar and biochemical properties of (hemi)fluorinated surfactants are controlled by the size of the polar head.

Surfactants with fluorinated and hemifluorinated alkyl chains have yielded encouraging results in terms of membrane protein stability; however, the molecules used hitherto have either been chemically heterogeneous or formed heterogeneous micelles. A new series of surfactants whose polar head size is modulated by the presence of one, two, or three glucose moieties has been synthesized. Analytical ultracentrifugation and small-angle neutron scattering show that fluorinated surfactants whose polar head bears a single glucosyl group form very large cylindrical micelles, whereas those with two or three glucose moieties form small, homogeneous, globular micelles. We studied the homogeneity and stability of the complexes formed between membrane proteins and these surfactants by using bacteriorhodopsin and cytochrome b(6)f as models. Homogeneous complexes were obtained only with surfactants that form homogeneous micelles. Surfactants bearing one or two glucose moieties were found to be stabilizing, whereas those with three moieties were destabilizing. Fluorinated and hemifluorinated surfactants with a two-glucose polar head thus appear to be very promising molecules for biochemical applications and structural studies. They were successfully used for cell-free synthesis of the ion channel MscL.

Hydrogenated and fluorinated surfactants derived from Tris(hydroxymethyl)-acrylamidomethane allow the purification of a highly active yeast F1-F0 ATP-synthase with an enhanced stability.

Loss of stability and integrity of large membrane protein complexes as well as their aggregation in a non-lipidic environment are the major bottlenecks to their structural studies. We have tested C(12)H(25)-S-poly-Tris-(hydroxymethyl)acrylamidomethane (H(12)-TAC) among many other detergents for extracting the yeast F(1)F(0) ATP-synthase. H(12)-TAC was found to be a very efficient detergent for removing the enzyme from mitochondrial membranes without altering its sensitivity towards specific ATP-synthase inhibitors. This extracted enzyme was then solubilized by either dodecyl maltoside (DDM), H(12)-TAC or fluorinated surfactants such as C(2)H(5)-C(6)F(12)-C(2)H(4)-S-poly-Tris-(hydroxymethyl)acrylamidomethane (H(2)F(6)-TAC) or C(6)F(13)-C(2)H(4)-S-poly-Tris-(hydroxymethyl)acrylamidomethane (F(6)-TAC), two surfactants exhibiting a comparable polar head to H(12)-TAC but bearing a fluorinated hydrophobic tail. Preparations from enzymes purified in the presence of H(12)-TAC were found to be more adapted for AFM imaging than ATP-synthase purified with DDM. Keeping H(12)-TAC during the Ni-NTA IMAC purification step or replacing it by DDM at low concentrations did not however allow preserving enzyme activity, while fluorinated surfactants H(2)F(6)-TAC and F(6)-TAC were found to enhance enzyme stability and integrity as indicated by sensitivity towards inhibitors. ATPase specific activity was higher with F(6)-TAC than with H(2)F(6)-TAC. When enzymes were mixed with egg phosphatidylcholine, ATP-synthases purified in the presence of H(2)F(6)-TAC or F(6)-TAC were more stable upon time than the DDM purified enzyme. Furthermore, in the presence of lipids, an activation of ATP-synthases was observed that was transitory for enzymes purified with DDM, but lasted for weeks for ATP-synthases isolated in the presence of molecules with Tris polyalcoholic moieties. Relipidated enzymes prepared with fluorinated surfactants remained highly sensitive towards inhibitors, even after 6 weeks.

Spin trapping and cytoprotective properties of fluorinated amphiphilic carrier conjugates of cyclic versus linear nitrones.

Nitrones have been employed as spin trapping reagent as well as pharmacological agent against neurodegenerative diseases and ischemia-reperfusion induced injury. The structure-activity relationship was explored for the two types of nitrones, i.e., cyclic (DMPO) and linear (PBN), which are conjugated to a fluorinated amphiphilic carrier (FAC) for their cytoprotective properties against hydrogen peroxide (H(2)O(2)), 3-morpholinosynonimine hydrochloride (SIN-1), and 4-hydroxynonenal (HNE) induced cell death on bovine aortic endothelial cells. The compound FAMPO was synthesized and characterized, and its physical-chemical and spin trapping properties were explored. Cytotoxicity and cytoprotective properties of various nitrones either conjugated and nonconjugated to FAC (i.e., AMPO, FAMPO, PBN, and FAPBN) were assessed using a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium (MTT) reduction assay. Results show that of all the nitrones tested, FAPBN is the most protective against H(2)O(2), but FAMPO and to a lesser extent its unconjugated form, AMPO, are more protective against SIN-1 induced cytotoxicity. However, none of the nitrones used protect the cells from HNE-induced cell death. The difference in the cytoprotective properties observed between the cyclic and linear nitrones may arise from the differences in their intrinsic antioxidant properties and localization in the cell.

Trapping and stabilization of integral membrane proteins by hydrophobically grafted glucose-based telomers.

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.

Preliminary biological evaluations of new thalidomide analogues for multiple sclerosis application.

The present work deals with the synthesis of a new series of thalidomide derivatives for therapeutic applications. These compounds were evaluated in vitro on a human endothelial cell line EA.hy926 for their antiproliferative potential and in vivo on an experimental animal multiple sclerosis model called EAE as angiogenesis inhibitors. The preliminary results obtained on EAE assays seem to validate that anti-angiogenesis compounds could be promising tools for the treatment of MS.

FCS study of the thermodynamics of membrane protein insertion into the lipid bilayer chaperoned by fluorinated surfactants.

Experimental determination of the free energy (DeltaG) stabilizing the structure of membrane proteins (MPs) in their native environment has been hampered by the aggregation and precipitation of MPs outside the lipid bilayer. We recently demonstrated that the latter process can be prevented by the use of fluorinated surfactants, FTACs, that act as chaperones for MP insertion without partitioning in the membrane themselves. Here we combine the advantages of the chaperone-like ability of FTACs with the sensitivity of fluorescence correlation spectroscopy measurements to determine DeltaG of bilayer insertion of model MPs. First, we calibrate our approach by examining the effects of chaperoned insertion on DeltaG of transmembrane insertion of Annexin B12. We find that a shorter-chained surfactant, FTAC-C6, for which the working concentration range of 0.05-0.2 mM falls below CMC = 0.33 mM, has a mild effect on an apparent DeltaG. In contrast, additions of a longer-chained FTAC-C8 (CMC = 0.03 mM) result in a steep and nonlinear concentration dependence of DeltaG. We then apply the same methodology to the pH-triggered insertion of diphtheria toxin T-domain, which is known to be affected by nonproductive aggregation in solution. We find that the correction of the DeltaG value needed to compensate for unchaperoned insertion of the T-domain exceeds 3 kcal/mole. A relatively shallow and linear dependence of the DeltaG for Annexin B12 and T-domain insertion on FTAC-C6 concentration is encouraging for future applications of this surfactant in thermodynamic studies of the stability of other MPs.

Interactions of fluorinated surfactants with diphtheria toxin T-domain: testing new media for studies of membrane proteins.

The principal difficulty in experimental exploration of the folding and stability of membrane proteins (MPs) is their aggregation outside of the native environment of the lipid bilayer. To circumvent this problem, we recently applied fluorinated nondetergent surfactants that act as chemical chaperones. The ideal chaperone surfactant would 1), maintain the MP in solution; 2), minimally perturb the MP's structure; 3), dissociate from the MP during membrane insertion; and 4), not partition into the lipid bilayer. Here, we compare how surfactants with hemifluorinated (HFTAC) and completely fluorinated (FTAC) hydrophobic chains of different length compare to this ideal. Using fluorescence correlation spectroscopy of dye-labeled FTAC and HFTAC, we demonstrate that neither type of surfactant will bind lipid vesicles. Thus, unlike detergents, fluorinated surfactants do not compromise vesicle integrity even at concentrations far in excess of their critical micelle concentration. We examined the interaction of surfactants with a model MP, DTT, using a variety of spectroscopic techniques. Site-selective labeling of DTT with fluorescent dyes indicates that the surfactants do not interact with DTT uniformly, instead concentrating in the most hydrophobic patches. Circular dichroism measurements suggest that the presence of surfactants does not alter the structure of DTT. However, the cooperativity of the thermal unfolding transition is reduced by the presence of surfactants, especially above the critical micelle concentration (a feature of regular detergents, too). The linear dependence of DTT's enthalpy of unfolding on the surfactant concentration is encouraging for future application of (H)FTACs to determine the stability of the membrane-competent conformations of other MPs. The observed reduction in the efficiency of Förster resonance energy transfer between donor-labeled (H)FTACs and acceptor-labeled DTT upon addition of lipid vesicles indicates that the protein sheds the layer of surfactant during its bilayer insertion. We discuss the advantages of fluorinated surfactants over other types of solubilizing agents, with a specific emphasis on their possible applications in thermodynamic measurements.