Detergent Head Research Articles

Merging In-Solution X-ray and Neutron Scattering Data Allows Fine Structural Analysis of Membrane-Protein Detergent Complexes.

In-solution small-angle X-ray and neutron scattering (SAXS/SANS) have become popular methods to characterize the structure of membrane proteins, solubilized by either detergents or nanodiscs. SANS studies of protein-detergent complexes usually require deuterium-labeled proteins or detergents, which in turn often lead to problems in their expression or purification. Here, we report an approach whose novelty is the combined analysis of SAXS and SANS data from an unlabeled membrane protein complex in solution in two complementary ways. First, an explicit atomic analysis, including both protein and detergent molecules, using the program WAXSiS, which has been adapted to predict SANS data. Second, the use of MONSA which allows one to discriminate between detergent head- and tail-groups in an ab initio approach. Our approach is readily applicable to any detergent-solubilized protein and provides more detailed structural information on protein-detergent complexes from unlabeled samples than SAXS or SANS alone.

Gas Phase Studies of the Amyloid-β Peptide

The amyloid-β (Aβ) peptide is an amphiphilic peptide that exhibits self-aggregating properties forming the amyloid fibrils that can be found in the brains of Alzheimer patients. Today it is believed that that it is the soluble Aβ oligomers rather than the mature fibrils that are the main neurotoxic species. These small peptide-assembles are known to associate with membranes and form pores that can transport Ca2+ into the cell, which in part could be responsible for their neurotoxic properties. However, most biophysical methods that have been developed to study Aβ target either the monomer or the mature fibrils, and not the low abundance and polydisperse oligomers.We have developed a soft ionization mass spectrometry (MS) method that retains and detects these non-covalent oligomer interactions in the gas phase. And since MS is a non-averaging technique the aggregation kinetics for different oligomeric species, as well as reduced and oxidized peptides, could be followed simultaneously. The high-resolution MS was also coupled to an ion mobility spectrometer that separates ions of the same m/z according to collisional cross section, yielding information about peptide conformations in the gas phase. Additionally, peptide interactions with other molecules, such as membrane mimicking detergents can be monitored. Large complexes between Aβ(1-40) and detergent micelles was shown to survive in the MS and could be identified. Detergent head group charges seem to be essential for peptide-micelle interactions, both in the gas phase and in solution, as no structure induction is observed upon addition of non-ionic detergent. The developed methodology is promising for future detection of pore-forming oligomers in micelles, and probably also in other more sophisticated membrane models.

Invisible detergents for structure determination of membrane proteins by small-angle neutron scattering.

A novel and generally applicable method for determining structures of membrane proteins in solution via small-angle neutron scattering (SANS) is presented. Common detergents for solubilizing membrane proteins were synthesized in isotope-substituted versions for utilizing the intrinsic neutron scattering length difference between hydrogen and deuterium. Individual hydrogen/deuterium levels of the detergent head and tail groups were achieved such that the formed micelles became effectively invisible in heavy water (D2 O) when investigated by neutrons. This way, only the signal from the membrane protein remained in the SANS data. We demonstrate that the method is not only generally applicable on five very different membrane proteins but also reveals subtle structural details about the sarco/endoplasmatic reticulum Ca2+ ATPase (SERCA). In all, the synthesis of isotope-substituted detergents makes solution structure determination of membrane proteins by SANS and subsequent data analysis available to nonspecialists.

The effects of a protein osmolyte on the stability of the integral membrane protein glycerol facilitator.

Osmolytes are naturally occurring molecules used by a wide variety of organisms to stabilize proteins under extreme conditions of temperature, salinity, hydrostatic pressure, denaturant concentration, and desiccation. The effects of the osmolyte trimethylamine N-oxide (TMAO) as well as the influence of detergent head group and acyl chain length on the stability of the Escherichia coli integral membrane protein glycerol facilitator (GF) tetramer to thermal and chemical denaturation by sodium dodecyl sulphate (SDS) are reported. TMAO promotes the association of the normally tetrameric α-helical protein into higher order oligomers in dodecyl-maltoside (DDM), but not in tetradecyl-maltoside (TDM), lyso-lauroylphosphatidyl choline (LLPC), or lyso-myristoylphosphatidyl choline (LMPC), as determined by dynamic light scattering (DLS); an octameric complex is particularly stable as indicated by SDS polyacrylamide gel electrophoresis. TMAO increases the heat stability of the GF tetramer an average of 10 °C in the 4 detergents and also protects the protein from denaturation by SDS. However, it did not promote re-association to the tetramer when added to SDS-dissociated protein. TMAO also promotes the formation of rod-like detergent micelles, and DLS was found to be useful for monitoring the structure of the protein and the redistribution of detergent during thermal dissociation of the protein. The protein is more thermally stable in detergents with the phosphatidylcholine head group (LLPC and LMPC) than in the maltoside detergents. The implications of the results for osmolyte mechanism, membrane protein stability, and protein-protein interactions are discussed.

Polar Interactions Trump Hydrophobicity in Stabilizing a Membrane-Interacting Protein

The vast majority of membrane-bound proteins consist of bundles of hydrophobic α-helical transmembrane domains. How these proteins adopt their native, biologically active structures in the apolar milieu of a membrane is increasingly well understood. However, it is still under debate to what extent polar interactions contribute to membrane-protein stability. For example, the membrane-interacting protein Mistic exhibits a net charge of −12 at pH 7 but, in NMR experiments, displays a concentric ring of interactions with apolar detergent tails typical of more hydrophobic membrane proteins. Moreover, as a fusion tag, Mistic supports the production and membrane insertion of integral membrane proteins in Escherichia coli. Thus, in spite of its unusually polar surface, Mistic in many aspects behaves like a conventional membrane protein.Here, we demonstrate that, upon addition of urea, Mistic unfolds reversibly form detergent micelles following a two-state equilibrium and exhibits the same unfolded reference state irrespective of the detergent used. Unfolding titrations from alkyl maltoside detergents revealed that alkyl tails of 12 carbon atoms preserve the conformational stability of the protein best. However, on top of such hydrophobic interactions, we discovered that zwitter-ionic and charged detergent head groups render Mistic extraordinarily resistant against unfolding, so that the protein largely retains its secondary structure even at urea concentrations higher than 7 M. Notably, for the application of Mistic as a fusion tag, polar or charged detergents have been found to be very successful in solubilizing the protein, which is in excellent agreement with our in vitro stability data. Thus, Mistic represents a unique model system to quantitatively assess the contribution of polar interactions to membrane-protein folding and extends the established view that hydrophobic interactions are the single most important contributors to the stability of membrane-interacting proteins.

Conformational Analysis of the Frog Skin Peptide, Plasticin-L1 and its Effects on the Production of Proinflammatory Cytokines by Macrophages

Plasticin-L1 (GLVNGLLSSVLGGGQGGGGLLGGIL) is a conformationally flexible glycine/leucine-rich peptide originally isolated from norepinephrine-stimulated skin secretions of the South-American Santa Fe frog Leptodactylus laticeps (Leptodactylidae). A nuclear magnetic resonance (NMR)/molecular dynamics (MD) characterization of plasticin-L1 in the presence of dodecylphosphocholine (DPC) and DPC/sodium dodecylsulphate (SDS) micelles as membrane-mimetic models showed that the peptide has affinity for both neutral and anionic membranes. The peptide adopts a stable helical conformation at the N-terminal region and a more disordered helix at the C-terminal region, separated by an unstructured loop wherein the highest number of glycines is localized. In both micelle environments, plasticin-L1 slowly inserts between the detergent head groups, but always remains localized at the micelle/water interface. Plasticin-L1 lacks direct antimicrobial activity but stimulates cytokine production by macrophages. Incubation with plasticin-L1 (20 µg/mL) significantly (P < 0.05) increased the production of the proinflammatory cytokines IL-1β, IL-12 IL-23 and TNF-α from unstimulated peritoneal macrophages from both C57BL/6 and BALB/C mice. The peptide also increased IL-6 production by unstimulated (P < 0.01) and lipopolysaccharide-stimulated (P < 0.01) macrophages, while the effects on production of the anti-inflammatory cytokine IL-10 were not significant. These findings suggest that plasticin-L1 may play an immunomodulatory role in vivo by stimulating cytokine production from frog skin macrophages in response to microbial pathogens. This peptide may represent a template for the design of peptides with therapeutic applications as immunostimulatory agents.

Conformational Analysis of the Frog Skin Peptide, Plasticin-L1, and Its Effects on Production of Proinflammatory Cytokines by Macrophages

Plasticin-L1 (GLVNGLLSSVLGGGQGGGGLLGGIL) is a conformationally flexible glycine/leucine-rich peptide originally isolated from norepinephrine-stimulated skin secretions of the South-American Santa Fe frog Leptodactylus laticeps (Leptodactylidae). A nuclear magnetic resonance/molecular dynamics characterization of plasticin-L1 in the presence of dodecylphosphocholine (DPC) and DPC/sodium dodecylsulphate micelles as membrane-mimetic models showed that the peptide has affinity for both neutral and anionic membranes. The peptide adopts a stable helical conformation at the N-terminal region and a more disordered helix at the C-terminal region, separated by an unstructured loop wherein the highest number of glycines is localized. In both micelle environments, plasticin-L1 slowly inserts between the detergent head groups but always remains localized at the micelle/water interface. Plasticin-L1 lacks direct antimicrobial activity but stimulates cytokine production by macrophages. Incubation with plasticin-L1 (20 μg/mL) significantly (P < 0.05) increased the production of the proinflammatory cytokines IL-1β, IL-12, IL-23, and TNF-α from unstimulated peritoneal macrophages from both C57BL/6 and BALB/C mice. The peptide also increased IL-6 production by unstimulated (P < 0.01) and lipopolysaccharide-stimulated (P < 0.01) macrophages, whereas the effects on production of the anti-inflammatory cytokine IL-10 were not significant. These findings suggest that plasticin-L1 may play an immunomodulatory role in vivo by stimulating cytokine production from frog skin macrophages in response to microbial pathogens. This peptide may represent a template for the design of peptides with therapeutic applications as immunostimulatory agents.

Dependence of micelle size and shape on detergent alkyl chain length and head group.

Micelle-forming detergents provide an amphipathic environment that can mimic lipid bilayers and are important tools for solubilizing membrane proteins for functional and structural investigations in vitro. However, the formation of a soluble protein-detergent complex (PDC) currently relies on empirical screening of detergents, and a stable and functional PDC is often not obtained. To provide a foundation for systematic comparisons between the properties of the detergent micelle and the resulting PDC, a comprehensive set of detergents commonly used for membrane protein studies are systematically investigated. Using small-angle X-ray scattering (SAXS), micelle shapes and sizes are determined for phosphocholines with 10, 12, and 14 alkyl carbons, glucosides with 8, 9, and 10 alkyl carbons, maltosides with 8, 10, and 12 alkyl carbons, and lysophosphatidyl glycerols with 14 and 16 alkyl carbons. The SAXS profiles are well described by two-component ellipsoid models, with an electron rich outer shell corresponding to the detergent head groups and a less electron dense hydrophobic core composed of the alkyl chains. The minor axis of the elliptical micelle core from these models is constrained by the length of the alkyl chain, and increases by 1.2–1.5 Å per carbon addition to the alkyl chain. The major elliptical axis also increases with chain length; however, the ellipticity remains approximately constant for each detergent series. In addition, the aggregation number of these detergents increases by ∼16 monomers per micelle for each alkyl carbon added. The data provide a comprehensive view of the determinants of micelle shape and size and provide a baseline for correlating micelle properties with protein-detergent interactions.

Size and Shape of Detergent Micelles Determined by Small-Angle X-ray Scattering

We present a systematic analysis of the aggregation number and shape of micelles formed by nine detergents commonly used in the study of membrane proteins. Small-angle X-ray scattering measurements are reported for glucosides with 8 and 9 alkyl carbons (OG/NG), maltosides and phosphocholines with 10 and 12 alkyl carbons (DM/DDM and FC-10/FC-12), 1,2-dihexanoyl-sn-glycero-phosphocholine (DHPC), 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] (LPPG), and 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS). The SAXS intensities are well described by two-component ellipsoid models, with a dense outer shell corresponding to the detergent head groups and a less electron dense hydrophobic core. These models provide an intermediate resolution view of micelle size and shape. In addition, we show that Guinier analysis of the forward scattering intensity can be used to obtain an independent and model-free measurement of the micelle aggregation number and radius of gyration. This approach has the advantage of being easily generalizable to protein-detergent complexes, where simple geometric models are inapplicable. Furthermore, we have discovered that the position of the second maximum in the scattering intensity provides a direct measurement of the characteristic head group-head group spacing across the micelle core. Our results for the micellar aggregation numbers and dimensions agree favorably with literature values as far as they are available. We de novo determine the shape of FC-10, FC-12, DM, LPPG, and CHAPS micelles and the aggregation numbers of FC-10 and OG to be ca. 50 and 250, respectively. Combined, these data provide a comprehensive view of the determinants of micelle formation and serve as a starting point to correlate detergent properties with detergent-protein interactions.

Identification of conserved lipid/detergent-binding sites in a high-resolution structure of the membrane protein cytochrome c oxidase

Well ordered reproducible crystals of cytochrome c oxidase (CcO) from Rhodobacter sphaeroides yield a previously unreported structure at 2.0 A resolution that contains the two catalytic subunits and a number of alkyl chains of lipids and detergents. Comparison with crystal structures of other bacterial and mammalian CcOs reveals that the positions occupied by native membrane lipids and detergent substitutes are highly conserved, along with amino acid residues in their vicinity, suggesting a more prevalent and specific role of lipid in membrane protein structure than often envisioned. Well defined detergent head groups (maltose) are found associated with aromatic residues in a manner similar to phospholipid head groups, likely contributing to the success of alkyl glycoside detergents in supporting membrane protein activity and crystallizability. Other significant features of this structure include the following: finding of a previously unreported crystal contact mediated by cadmium and an engineered histidine tag; documentation of the unique His-Tyr covalent linkage close to the active site; remarkable conservation of a chain of waters in one proton pathway (D-path); and discovery of an inhibitory cadmium-binding site at the entrance to another proton path (K-path). These observations provide important insight into CcO structure and mechanism, as well as the significance of bound lipid in membrane proteins.

Structure of the monomeric outer-membrane porin OmpG in the open and closed conformation

OmpG, a monomeric pore-forming protein from Escherichia coli outer membranes, was refolded from inclusion bodies and crystallized in two different conformations. The OmpG channel is a 14-stranded beta-barrel, with short periplasmic turns and seven extracellular loops. Crystals grown at neutral pH show the channel in the open state at 2.3 A resolution. In the 2.7 A structure of crystals grown at pH 5.6, the pore is blocked by loop 6, which folds across the channel. The rearrangement of loop 6 appears to be triggered by a pair of histidine residues, which repel one another at acidic pH, resulting in the breakage of neighbouring H-bonds and a lengthening of loop 6 from 10 to 17 residues. A total of 151 ordered LDAO detergent molecules were found in the 2.3 A structure, mostly on the hydrophobic outer surface of OmpG, mimicking the outer membrane lipid bilayer, with three LDAO molecules in the open pore. In the 2.7 A structure, OmpG binds one OG and one glucose molecule as sugar substrates in the closed pore.

Cytosolic delivery of macromolecules: 4. Head group-dependent membrane permeabilization by pH-sensitive detergents

Three tertiary amine-based detergents with zero, one, or two hydroxyl groups at various positions in their head group were characterized for their ability to promote the cytosolic delivery of macromolecules. Critical micellar concentrations (CMC) and membrane-bound pKa values of the lipid constructs increased with increasing head group polarity, ranging from 1–5 μM and 5.9 to 6.3, respectively. Fluorescence resonance energy transfer (FRET) and calcein leakage experiments revealed that when the amine group is protonated introduction of –OH moieties to detergent head groups enhanced their ability to interact with and permeabilize anionic, endosome-mimicking vesicles. Different formulations of a diethanolamine-based lipid (DEL) were further evaluated for pH-dependent hemolytic activity and ability to promote cytosolic delivery of macromolecules in vitro. Intact liposomes containing DEL at its maximum limit of incorporation were less efficient than DEL-containing micelles in promoting hemoglobin leakage from human erythrocytes at acidic pH. In HeLa cells, DEL-containing detergent micelles facilitated efficient cytosolic release of endocytosed macromolecules such as fluorescein-labeled dextran of MW 10 kDa. This observation was further corroborated by a functional assay based on antisense-mediated up-regulation of enhanced green fluorescent protein (EGFP). Taken together, our findings emphasize the key role of polar head groups and micellar architecture of pH-sensitive detergents in mediating endosomal permeabilization and the efficient cytosolic delivery of macromolecules.

Spin-labeled extracellular loop from a seven-transmembrane helix receptor: Studies in solution and interaction with model membranes

A spin-labeled pentadecapeptide was synthesized containing 2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid (TOAC) as the N-terminal amino acid and residues 253-266 (EYWSTFGNLHHISL) of the mass oncogene receptor, a membrane-bound protein from the G-protein coupled receptors family. According to predictions, this protein folds into seven transmembrane helices connected by three extra- and three intracellular loops, and the peptide encompasses part of the third extracellular loop and part of the seventh helix. Electron paramagnetic resonance (EPR) spectra of the spin-labeled peptide (TOAC-14) were obtained in aqueous solution as a function of pH and temperature, in a secondary structure-inducing solvent [trifluoroethanol (TFE)], and in the presence of detergent micelles and phospholipid bilayers. The charged and uncharged amino groups of TOAC and TOAC-14 yielded spectra with different isotropic hyperfine splittings (aN). The slow exchange between protonated and unprotonated forms in the EPR time scale gave rise to composite spectra weighted by the Henderson-Hasselbalch equation. Plots of aN vs pH allowed the determination of the amino group pK values (8.4 and 4.5, for TOAC and TOAC-14, respectively). A small change in aN centered at pH 6.5 was ascribed to the titration of the histidines. Values of calculated rotational correlation times were indicative of a pH-induced conformational change. A conformational change was also observed in TFE. TOAC-14 bound to micelles irrespective of peptide and detergent head group charge. In contrast, the peptide bound to phospholipid bilayers only when both carried opposite charges. The slow exchange (in the EPR time scale) between membrane-bound and free TOAC-14 allowed the calculation of the peptide's partition coefficient. The spectral line shapes were affected by aggregate size and degree of packing of the constituent molecules. It is proposed that pH, polarity, and lipid environment can affect the conformation of water-exposed regions of membrane-bound receptors, thereby playing a role in the mechanism of signal transduction.

Effects of zwitterionic detergents on the primary donor of bacterial reaction centers

AbstractThe effects of zwitterionic detergents on the spectroscopic properties of the primary electron donor P of photosynthetic reaction centers (RCs) from the purple nonsulfur bacterium Rhodobacter sphaeroides are investigated. When RCs are solubilized with N‐(n‐alkyl)‐N,N‐dimethyl‐3‐ammonio‐1‐propanesulfonates (sulfobetaines) the maximum of the characteristic near‐infrared absorption of P, λmax, is shifted to lower wavelengths at room temperature as compared with nonionic detergents. While the range of the blueshift (λmax = 850 – 862 nm) is identical for different sulfobetaines, the dependence of the shift on the detergent/RC ratio varies systematically with the number of carbon atoms in the n‐alkyl‐chain. N‐lauryl‐N,N‐dimethyl‐amine‐N‐oxide (LDAO), a zwitterionic detergent with a less polar head group, is only able to induce blueshifts of λmax down to 859 nm at much higher detergent/RC ratios. ENDOR experiments performed on the primary donor radical cation P+• reveal that two distinct states of P exist in such samples. Thus the optical bandshifts are caused by transitions between two spectroscopic species, called P866 and P850. We interpret the detergent effect as an electrostatic influence of the detergent head groups on the equilibrium between two protein conformations which in turn affect the structure of the primary donor.

Membrane protein crystallization: observations and use of short chain phospholipids as amphiphiles

An integral membrane protein, porin OmpF located in E. coli outer membrane, has been crystallized in the sole presence of short chain phospholipids. Although the amphiphilic characteristics of these molecules are comparable to those of conventional detergents, they appear to be unique probes for the study of the phospholipid-protein interactions by crystallographic methods. Here we also discuss the influence of the detergent head group on the crystal packing, and the role of additives. The stabilization of crystals after termination of growth in protein-free buffer is described.

The crystallization of outer membrane proteins from Escherichia coli. Studies on lamB and ompA gene products.

The outer membrane protein LambB from Escherichia coli has been crystallized from detergent-containing solutions. Several different crystal habits can be obtained under the same ionic and precipitant conditions by altering the detergent head group composition of the protein-detergent mixed micelle or by adding polar organic compounds. Two crystal forms have been partially characterized as P1 and C2221, the former diffracting to beyond 4 A resolution and the latter to 6 A. The detergents used were beta-octyl glucoside, octyl tetraoxyethylene, and octyl polyoxyethylene (polydisperse) either alone or as mixtures. In some experiments, the addition of small nonionic amphiphiles having n-butyl alkyl tails significantly influenced crystallization. The experiments suggest that the detergent region of the mixed micelle plays a critical role in crystal formation. Using the methods developed here for LamB and also for matrix porin (Garavito, R. M., Jenkins, J. A., Jansonius, J. N., Karlsson, R., and Rosenbusch, J. P. (1983) J. Mol. Biol. 164, 313-327), an additional protein from the outer membrane, OmpA, has been obtained as a microcrystalline preparation.

The importance of coulombic interactions for the induction of beta structure in lysine oligomers by sodium dodecyl sulfate.

AbstractCircular dichroism spectra have been obtained for tri(L‐lysine), tetra(L‐lysine), and penta(L‐lysine) in aqueous sodium dodecyl sulfate at 25°C. None of the oligomers are affected significantly by sodium dodecyl sulfate at detergent concentrations exceeding 0.01 M. Literature results show that the high‐molecular‐weight polymer forms a β strucure under these conditions. At detergent concentrations near 3.5 × 10−4 M the penta(L‐lysine), but not the smaller oligomers, undergoes a conformational change. Its circular dichroism under these conditions is essentially identical to that observed with poly(L‐lysine) when it forms a β structure in sodium dodecyl sulfate. Solutions of the penta(L‐lysine), which exhibit this modified circular dichroism, are also turbid, leading to the conclusion that the oligomer has formed an intermolecular β structure. When these experiments are conducted in the presence of 0.1 M sodium hydroxide, the sodium dodecyl sulfate produces neither turbidity nor a modified circular dichroism spectrum. These observations provide compelling evidence that Coulombic interaction between the anionic detergent head and the cationic lysyl amino groups is essential for the conformational change induced in penta(L‐lysine) by sodium dodecyl sulfate.