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 b₆f 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.     

In vitro synthesis and oligomerization of the mechanosensitive channel of large conductance, MscL, into a functional ion channel

Elucidation of high-resolution structures of integral membrane proteins is drastically lagging behind that of cytoplasmic proteins. In vitro synthesis and insertion of membrane proteins into synthetic membranes could circumvent bottlenecks associated with the overexpression of membrane proteins, producing sufficient membrane-inserted, correctly folded protein for structural studies. Using the mechanosensitive channel of large conductance, MscL, as a model protein we show that in vitro synthesized MscL inserts into YidC-containing proteoliposomes and oligomerizes to form a homopentamer. Using planar membrane bilayers, we show that MscL forms functional ion channels capable of ion transport. These data demonstrate that membrane insertion of MscL is YidC mediated, whereas subsequent oligomerization into a functional homopentamer is a spontaneous event.     

Cell-free synthesis of a functional ion channel in the absence of a membrane and in the presence of detergent

We have investigated the possibility of cell-fee synthesis of membrane proteins in the absence of a membrane and in the presence of detergent. We used the bacterial mechanosensitive channel MscL, a homopentamer, as a model protein. A wide range of nonionic or zwitterionic detergents, Triton X-100, Tween 20, Brij 58p, n-dodecyl beta-D-maltoside, and CHAPS, were compatible with cell-free synthesis, while n-octyl beta-D-glucoside and deoxycholate had an inhibitory effect. In vitro synthesis in the presence of Triton X-100 yielded milligram amounts of MscL per milliliter of lysate. Cross-linking experiments showed that the protein was able to oligomerize in detergents. When the purified protein was reconstituted in liposomes and studied by the patch-clamp technique, its activity at the single-molecule level was similar to that of the recombinant protein produced in Escherichia coli. Cell-free synthesis of membrane proteins should prove a valuable tool for the production of membrane proteins whose overexpression in heterologous systems is difficult.     

Coupled cell-free synthesis and lipid vesicle insertion of a functional oligomeric channel MscL MscL does not need the insertase YidC for insertion in vitro

The mechanosensitive channel MscL of the plasma membrane of bacteria is a homopentamer involved in the protection of cells during osmotic downshock. The MscL protein, a polypeptide of 136 residues, was recently shown to require YidC to be inserted in the inner membrane of E. coli. The insertase YidC is a component of an insertion pathway conserved in bacteria, mitochondria and chloroplasts. MscL insertion was independent of the Sec translocon. Here, we report sucrose gradient centrifugation and freeze-etching microscopy experiments showing that MscL produced in a cell-free system complemented with preformed liposomes is able to insert directly in a pure lipid bilayer. Patch-clamp experiments performed with the resulting proteoliposomes showed that the protein was highly active. In vitro cell-free synthesis targeting to liposomes is a new promising expression system for membrane proteins, including those that might require an insertion machinery in vivo. Our results also question the real role of insertases such as YidC for membrane protein insertion in vivo.     

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.     

S. aureus MscL is a pentamer in vivo but of variable stoichiometries in vitro: implications for detergent-solubilized membrane proteins

While the bacterial mechanosensitive channel of large conductance (MscL) is the best studied biological mechanosensor and serves as a paradigm for how a protein can sense and respond to membrane tension, the simple matter of its oligomeric state has led to debate, with models ranging from tetramers to hexamers. Indeed, two different oligomeric states of the bacterial mechanosensitive channel MscL have been resolved by X-ray crystallography: The M. tuberculosis channel (MtMscL) is a pentamer, while the S. aureus protein (SaMscL) forms a tetramer. Because several studies suggest that, like MtMscL, the E. coli MscL (EcoMscL) is a pentamer, we re-investigated the oligomeric state of SaMscL. To determine the structural organization of MscL in the cell membrane we developed a disulfide-trapping approach. Surprisingly, we found that virtually all SaMscL channels in vivo are pentameric, indicating this as the physiologically relevant and functional oligomeric state. Complementing our in vivo results, we purified SaMscL and assessed its oligomeric state using three independent approaches (sedimentation equilibrium centrifugation, crosslinking, and light scattering) and established that SaMscL is a pentamer when solubilized in Triton X-100 and C(8)E(5) detergents. However, performing similar experiments on SaMscL solubilized in LDAO, the detergent used in the crystallographic study, confirmed the tetrameric oligomerization resolved by X-ray crystallography. We further demonstrate that this stoichiometric shift is reversible by conventional detergent exchange experiments. Our results firmly establish the pentameric organization of SaMscL in vivo. Furthermore they demonstrate that detergents can alter the subunit stoichiometry of membrane protein complexes in vitro; thus, in vivo assays are necessary to firmly establish a membrane protein's true functionally relevant oligomeric state.     

Gating of the mechanosensitive channel protein MscL: the interplay of membrane and protein

The mechanosensitive channel of large conductance (MscL) belongs to a family of transmembrane channel proteins in bacteria and functions as a safety valve that relieves the turgor pressure produced by osmotic downshock. MscL gating can be triggered solely by stretching of the membrane. This work reports an effort to understand this mechanotransduction by means of molecular dynamics (MD) simulation on the MscL of mycobacterium tuberculosis embedded in a palmitoyloleoylphosphatidylethanolamine membrane. Equilibrium MD under zero membrane tension produced a more compact protein structure, as measured by its radii of gyration, compared to the crystal structure, in agreement with previous experimental findings. Even under a large applied tension up to 1000 dyn/cm, the MscL lateral dimension largely remained unchanged after up to 20 ns of simulation. A nonequilibrium MD simulation of 3% membrane expansion showed a significant increase in membrane rigidity upon MscL inclusion, which can contribute to efficient mechanotransduction. Direct observation of channel opening was possible only when an explicit lateral bias force was applied to each of the five subunits of MscL in the radially outward direction. Using this force, open structures with a large pore of radius 10 Å could be obtained. The channel opening takes place in a stepwise manner and concurrently with the water chain formation across the channel, which occurs without direct involvement of protein hydrophilic residues. The N-terminal S1 helices stabilize the open structure, and the membrane asymmetry (different lipid density on the two leaflets of membrane) promotes channel opening.     

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.     

Protein-surfactant interactions: a tale of many states

The scientific study of protein surfactant interactions goes back more than a century, and has been put to practical uses in everything from the estimation of protein molecular weights to efficient washing powder enzymes and products for personal hygiene. After a burst of activity in the late 1960s and early 1970s that established the general principles of how charged surfactants bind to and denature proteins, the field has kept a relatively low profile until the last decade. Within this period there has been a maturation of techniques for more accurate and sophisticated analyses of protein-surfactant complexes such as calorimetry and small angle scattering techniques. In this review I provide an overview of different useful approaches to study these complexes and identify eight different issues which define central concepts in the field. (1) Are proteins denatured by monomeric surfactant molecules, micelles or both? (2) How does unfolding of proteins in surfactant compare with "proper" unfolding in chemical denaturants? Recent work has highlighted the role of shared micelles, rather than monomers, below the critical micelle concentration (cmc) in promoting both protein denaturation and formation of higher order structures. Kinetic studies have extended the experimentally accessible range of surfactant concentrations to far above the cmc, revealing numerous different modes of denaturation by ionic surfactants below and above the cmc which reflect micellar properties as much as protein unfolding pathways. Uncharged surfactants follow a completely different denaturation strategy involving synergy between monomers and micelles. The high affinity of charged surfactants for proteins means that unfolding pathways are generally different in surfactants versus chemical denaturants, although there are common traits. Other issues are as follows: (3) Are there non-denaturing roles for SDS? (4) How reversible is unfolding in SDS? (5) How do solvent conditions affect the way in which surfactants denature proteins? The last three issues compare SDS with "proper" membranes. (6) Do anionic surfactants such as SDS mimic biological membranes? (7) How do mixed micelles interact with globular proteins? (8) How can mixed micelles be used to measure the stability of membrane proteins? The growing efforts to understand the unique features of membrane proteins have encouraged the development of mixed micelles to study the equilibria and kinetics of this class of proteins, and traits which unite globular and membrane proteins have also emerged. These issues emphasise the amazing power of surfactants to both extend the protein conformational landscape and at the same time provide convenient and reversible short-cuts between the native and denatured state for otherwise obdurate membrane proteins.     

Molecular dissection of the large mechanosensitive ion channel (MscL) of E. coli: Mutants with altered channel gating and pressure sensitivity

In the search for the essential functional domains of the large mechanosensitive ion channel (MscL) of E. coli, we have cloned several mutants of the mscL gene into a glutathione S-transferase fusion protein expression system. The resulting mutated MscL proteins had either amino acid additions, substitutions or deletions in the amphipathic N-terminal region, and/or deletions in the amphipathic central or hydrophilic C-terminal regions. Proteolytic digestion of the isolated fusion proteins by thrombin yielded virtually pure recombinant MscL proteins that were reconstituted into artificial liposomes and examined for function by the patch-clamp technique. The addition of amino acid residues to the N-terminus of the MscL did not affect channel activity, whereas N-terminal deletions or changes to the N-terminal amino acid sequence were poorly tolerated and resulted in channels exhibiting altered pressure sensitivity and gating. Deletion of 27 amino acids from the C-terminus resulted in MscL protein that formed channels similar to the wild-type, while deletion of 33 C-terminal amino acids extinguished channel activity. Similarly, deletion of the internal amphipathic region of the MscL abolished activity. In accordance with a recently proposed spatial model of the MscL, our results suggest that (i) the N-terminal portion participates in the channel activation by pressure, and (ii) the essential channel functions are associated with both, the putative central amphipathic α-helical portion of the protein and the six C-terminal residues RKKEEP forming a charge cluster following the putative M2 membrane spanning α-helix. Received: 25 September 1996/Revised: 21 November 1996     

Fluorine: a new element in protein design

Fluorocarbons are quintessentially man-made molecules, fluorine being all but absent from biology. Perfluorinated molecules exhibit novel physicochemical properties that include extreme chemical inertness, thermal stability, and an unusual propensity for phase segregation. The question we and others have sought to answer is to what extent can these properties be engineered into proteins? Here, we review recent studies in which proteins have been designed that incorporate highly fluorinated analogs of hydrophobic amino acids with the aim of creating proteins with novel chemical and biological properties. Fluorination seems to be a general and effective strategy to enhance the stability of proteins, both soluble and membrane bound, against chemical and thermal denaturation, although retaining structure and biological activity. Most studies have focused on small proteins that can be produced by peptide synthesis as synthesis of large proteins containing specifically fluorinated residues remains challenging. However, the development of various biosynthetic methods for introducing noncanonical amino acids into proteins promises to expand the utility of fluorinated amino acids in protein design.     

The mechanosensitive channel protein MscL is targeted by the SRP to the novel YidC membrane insertion pathway of Escherichia coli

The mechanosensitive channel MscL in the inner membrane of Escherichia coli is a homopentameric complex involved in homeostasis when cells are exposed to hypo-osmotic conditions. The E. coli MscL protein is synthesized as a polypeptide of 136 amino acid residues and uses the bacterial signal recognition particle (SRP) for membrane targeting. The protein is inserted into the membrane independently of the Sec translocon. Mutants affected in the Sec-components are competent for MscL assembly. Translocation of the periplasmic domain was detected using a membrane-impermeant, sulfhydryl-specific gel-shift reagent. The modification of a single cysteine residue at position 68 indicated its translocation across the inner membrane. From these in vivo experiments, it is concluded that the electrical chemical membrane potential is not necessary for membrane insertion of MscL. However, depletion of the membrane insertase YidC inhibits translocation of the protein across the membrane. We show here that YidC is essential for efficient membrane insertion of the MscL protein. YidC is a component of a recently identified membrane insertion pathway that is evolutionarily conserved in bacteria, mitochondria and chloroplasts.     

Cooperative gating and spatial organization of membrane proteins through elastic interactions

Biological membranes are elastic media in which the presence of a transmembrane protein leads to local bilayer deformation. The energetics of deformation allow two membrane proteins in close proximity to influence each other's equilibrium conformation via their local deformations, and spatially organize the proteins based on their geometry. We use the mechanosensitive channel of large conductance (MscL) as a case study to examine the implications of bilayer-mediated elastic interactions on protein conformational statistics and clustering. The deformations around MscL cost energy on the order of 10 k_BT and extend ~3 nm from the protein edge, as such elastic forces induce cooperative gating, and we propose experiments to measure these effects. Additionally, since elastic interactions are coupled to protein conformation, we find that conformational changes can severely alter the average separation between two proteins. This has important implications for how conformational changes organize membrane proteins into functional groups within membranes.     

OCAM: a new tool for studying the oligomeric diversity of MscL channels

We have developed a new technique to study the oligomeric state of proteins in solution. OCAM or Oligomer Characterization by Addition of Mass counts protein subunits by selectively shaving a protein mass tag added to a protein subunit via a short peptide linker. Cleavage of each mass tag reduces the total mass of the protein complex by a fixed amount. By performing limited proteolysis and separating the reaction products by size on a blue native PAGE gel, a ladder of reaction products corresponding to the number of subunits can be resolved. The pattern of bands may be used to distinguish the presence of a single homo-oligomer from a mixture of oligomeric states. We have applied OCAM to study the mechanosensitive channel of large conductance (MscL) and find that these proteins can exist in multiple oligomeric states ranging from tetramers up to possible hexamers. Our results demonstrate the existence of oligomeric forms of MscL not yet observed by X-ray crystallography or other techniques and that in some cases a single type of MscL subunit can assemble as a mixture of oligomeric states.     

Chaperoning of insertion of membrane proteins into lipid bilayers by hemifluorinated surfactants: application to diphtheria toxin

Hemifluorinated compounds, such as HF-TAC, make up a novel class of nondetergent surfactants designed to keep membrane proteins soluble under nondissociating conditions [Breyton, C., et al. (2004) FEBS Lett. 564, 312]. Because fluorinated and hydrogenated chains do not mix well, supramicellar concentrations of these surfactants can coexist with intact lipid vesicles. To test the ability of HF-TAC to assist proper membrane insertion of proteins, we examined its effect on the pH-triggered insertion of the diphtheria toxin T-domain. The function of the T-domain is to translocate the catalytic domain across the lipid bilayer in response to acidification of the endosome. This translocation is accompanied by the formation of a pore, which we used as a measure of activity in a vesicle leakage assay. We have also used F?rster resonance energy transfer to follow the effect of HF-TAC on aggregation of aqueous and membrane-bound T-domain. Our data indicate that the pore-forming activity of the T-domain is affected by the dynamic interplay of two principal processes: productive pH-triggered membrane insertion and nonproductive aggregation of the aqueous T-domain at low pH. The presence of HF-TAC in the buffer is demonstrated to suppress aggregation in solution and ensure correct insertion and folding of the T-domain into the lipid vesicles, without solubilizing the latter. Thus, hemifluorinated surfactants stabilize the low-pH conformation of the T-domain as a water-soluble monomer while acting as low-molecular weight chaperones for its insertion into preformed lipid bilayers.     

Correlating a protein structure with function of a bacterial mechanosensitive channel

MscL, a mechanosensitive channel found in many bacteria, protects cells from hypotonic shock by reducing intracellular pressure through release of cytoplasmic osmolytes. First isolated from Escherichia coli, this protein has served as a model for how a protein senses and responds to membrane tension. Recently the structure of a functionally uncharacterized MscL homologue from Mycobacterium tuberculosis was solved by x-ray diffraction to a resolution of 3.5 A. Here we demonstrate that the protein forms a functional MscL-like mechanosensitive channel in E. coli membranes and azolectin proteoliposomes. Furthermore, we show that M. tuberculosis MscL crystals, when re-solubilized and reconstituted, yield wild-type channel currents in patch clamp, demonstrating that the protein does not irreversibly change conformation upon crystallization. Finally, we apply functional clues acquired from the E. coli MscL to the M. tuberculosis channel and show a mechanistic correlation between these channels. However, the inability of the M. tuberculosis channel to gate at physiological membrane tensions, demonstrated by in vivo E. coli expression and in vitro reconstitution, suggests that the membrane environment or other additional factors influence the gating of this channel.     

Force Transduction and Lipid Binding in MscL: A Continuum-Molecular Approach

The bacterial mechanosensitive channel MscL, a small protein mainly activated by membrane tension, is a central model system to study the transduction of mechanical stimuli into chemical signals. Mutagenic studies suggest that MscL gating strongly depends on both intra-protein and interfacial lipid-protein interactions. However, there is a gap between this detailed chemical information and current mechanical models of MscL gating. Here, we investigate the MscL bilayer-protein interface through molecular dynamics simulations, and take a combined continuum-molecular approach to connect chemistry and mechanics. We quantify the effect of membrane tension on the forces acting on the surface of the channel, and identify interactions that may be critical in the force transduction between the membrane and MscL. We find that the local stress distribution on the protein surface is largely asymmetric, particularly under tension, with the cytoplasmic side showing significantly larger and more localized forces, which pull the protein radially outward. The molecular interactions that mediate this behavior arise from hydrogen bonds between the electronegative oxygens in the lipid headgroup and a cluster of positively charged lysine residues on the amphipathic S1 domain and the C-terminal end of the second trans-membrane helix. We take advantage of this strong interaction (estimated to be 10–13 kT per lipid) to actuate the channel (by applying forces on protein-bound lipids) and explore its sensitivity to the pulling magnitude and direction. We conclude by highlighting the simple motif that confers MscL with strong anchoring to the bilayer, and its presence in various integral membrane proteins including the human mechanosensitive channel K2P1 and bovine rhodopsin.     

Loss-of-function mutations at the rim of the funnel of mechanosensitive channel MscL

MscL is a bacterial mechanosensitive channel that is activated directly by membrane stretch. Although the gene has been cloned and the crystal structure of the closed channel has been defined, how membrane tension causes conformational changes in MscL remains largely unknown. To identify the site where MscL senses membrane tension, we examined the function of the mutants generated by random and scanning mutagenesis. In vitro (patch-clamp) and in vivo (hypoosmotic-shock) experiments showed that when a hydrophilic amino acid replaces one of the hydrophobic residues that are thought to make contact with the membrane lipid near the periplasmic end of the M1 or M2 transmembrane domain, MscL loses the ability to open in response to membrane tension. Hydrophilic (asparagine) substitution of the other residues in the lipid-protein interface did not impair the channel's mechanosensitivity. These observations suggest that the disturbance of the hydrophobic interaction between the membrane lipid and the periplasmic rim of the channel's funnel impairs the function of MscL.     

Dual-color fluorescence-burst analysis to probe protein efflux through the mechanosensitive channel MscL

The mechanosensitive channel protein of large conductance, MscL, from Escherichia coli has been implicated in protein efflux, but the passage of proteins through the channel has never been demonstrated. We used dual-color fluorescence-burst analysis to evaluate the efflux of fluorescent labeled compounds through MscL. The method correlates the fluctuations in intensity of fluorescent labeled membranes and encapsulated (macro)molecules (labeled with second fluorophore) for each liposome diffusing through the observation volume. The analysis provides quantitative information on the concentration of macromolecules inside the liposomes and the fraction of functional channel proteins. For MscL, reconstituted in large unilamellar vesicles, we show that insulin, bovine pancreas trypsin inhibitor, and other compounds smaller than 6.5 kDa can pass through MscL, whereas larger macromolecules cannot.     

Fluorinated and hemifluorinated surfactants derived from maltose: synthesis and application to handling membrane proteins in aqueous solution

Two novel fluorinated surfactants have been obtained by grafting by radical reaction either a fluorocarbon or an ethyl end-capped fluorocarbon chain onto the double bond of beta-D-allyl maltose. The two compounds thus obtained form polydisperse aggregates in water. They can keep membrane proteins water-soluble, but the protein/surfactant complexes are polydisperse, which affects neither the native state nor the stability of the proteins.