Detergent Head Groups Research Articles

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.

Increase of the peroxidase activity of cytochrome c-550 by the interaction with detergents

The effect was studied of detergents on the peroxidase activity of the heme-containing electron-transport protein, cytochrome c-550 from Paracoccus versutus (cytc550). Cytc550 does not interact with non-ionic or zwitterionic detergents, but its peroxidase activity is significantly enhanced in the presence of both anionic and cationic detergents. The increase in peroxidase activity is caused by (partial) unfolding of cytc550, resulting in weakening of the bond between the axial methionine ligand and the heme-iron. With sodium dodecyl sulfate (SDS) and dodecyltrimethylammonium bromide (DTAB), the activity increases roughly 10- and 100-fold, respectively. The detergent concentration required to enhance the peroxidase activity of cytc550 consistently coincides with the critical micellisation concentration (CMC). When cytc550 carries substantial opposite charge to that of the detergent headgroup, the protein–detergent complex is formed at lower detergent concentration than the critical micellisation concentration, i.e. at acid pH for anionic detergent and at alkaline pH for cationic detergent. It is concluded that in the presence of ionic detergents, cytc550 can acquire considerable peroxidase activity. This may be exploited by applying cytc550 as an oxidative catalyst in detergent-rich environments, where regular peroxidases rapidly lose their catalytic potential.

Effect of Detergents on the Association of the Glycophorin A Transmembrane Helix

We have examined the role of the environment on the interactions between transmembrane helices using, as a model system, the dimerization of the glycophorin A transmembrane helix. In this study we have focused on micellar environments and have examined a series of detergents that include a range of alkyl chain lengths, combined with ionic, zwitterionic, and nonionic headgroups. For each we have measured how the apparent equilibrium constant depends on the detergent concentration. In two detergents we also measured the thermal sensitivity of the equilibrium constant, from which we derive the van’t Hoff enthalpy and entropy. We show that several simple models are inadequate for explaining our results; however, models that include the effect of detergent concentration on detergent binding are able to account for our measurements. Our analysis suggests that the effects of detergents on helix association are due to a pair of opposing effects: an enthalpic effect, which drives association as the detergent concentration is increased and which is sensitive to the chemical nature of the detergent headgroup, opposed by an entropic effect, which drives peptide dissociation as the detergent concentration is raised. Our results also indicate that the monomer-monomer interface is relatively hydrophilic and that association within detergent micelles is driven by the enthalpy change. The wide variations in glycophorin a dimmer, stability with the detergent used, together with the realization that this results from the balance between two opposing effects, suggests that detergents might be selected that drive association rather than dissociation of peptide dimers.

Protein Unfolding in Detergents: Effect of Micelle Structure, Ionic Strength, pH, and Temperature

The 101-residue monomeric protein S6 unfolds in the anionic detergent sodium dodecyl sulfate (SDS) above the critical micelle concentration, with unfolding rates varying according to two different modes. Our group has proposed that spherical micelles lead to saturation kinetics in unfolding (mode 1), while cylindrical micelles prevalent at higher SDS concentrations induce a power-law dependent increase in the unfolding rate (mode 2). Here I investigate in more detail how micellar properties affect protein unfolding. High NaCl concentrations, which induce cylindrical micelles, favor mode 2. This is consistent with our model, though other effects such as electrostatic screening cannot be discounted. Furthermore, unfolding does not occur in mode 2 in the cationic detergent LTAB, which is unable to form cylindrical micelles. A strong retardation of unfolding occurs at higher LTAB concentrations, possibly due to the formation of dead-end protein-detergent complexes. A similar, albeit much weaker, effect is seen in SDS in the absence of salt. Chymotrypsin inhibitor 2 exhibits the same modes of unfolding in SDS as S6, indicating that this type of protein unfolding is not specific for S6. The unfolding process in mode 1 has an activation barrier similar in magnitude to that in water, while the activation barrier in mode 2 is strongly concentration-dependent. The strong pH-dependence of unfolding in SDS and LTAB suggests that the rate of unfolding in anionic detergent is modulated by repulsion between detergent headgroups and anionic side chains, while cationic side chains modulate unfolding rates in cationic detergents.

The polar headgroup of the detergent governs the accessibility to water of tryptophan octyl ester in host micelles

Many attempts have been made to rationalize the use of detergents for membrane protein studies [J. Biol. Chem. 264 (1989) 4907]. The barrier properties of the detergent headgroup may be one parameter critically involved in protein protection. In this paper, we analyzed these properties using a model system, by comparing the accessibility of tryptophan octyl ester (TOE) to water-soluble collisional quenchers (iodide and acrylamide) in three detergent micelles. The detergents used differed only in the chemical nature of their polar headgroups, zwitterionic for dodecylphosphocholine (DPC) and nonionic for octa(ethylene glycol) dodecyl monoether (C 12E 8) and dodecylmaltoside (DM). In all cases, in phosphate buffer at pH 7.5, the binding of 5 μM TOE was complete in the presence of a slight excess of detergent micelles over TOE molecules, resulting in a significant blue shift and greater intensity of TOE fluorescence emission. The resulting quantum yield of bound TOE was between 0.08 (in DPC) and 0.12 (in DM) with an emission maximum ( λ max) of ∼335 nm whatever the detergent micelle. Time-resolved fluorescence intensity decays of TOE at λ max were heterogeneous in all micelles (3–4 lifetime populations), with mean lifetimes of 1.7 ns in DPC, and 2 ns in both C 12E 8 and DM. TOE fluorescence quenching by iodide, in detergent micelles, yielded linear Stern–Volmer plots characteristic of a dynamic quenching process. The accessibility of TOE to this ion was the greatest with C 12E 8, followed by DPC and finally DM (Stern–Volmer quenching constants K sv of 2 to 5.5 M −1). In contrast, the accessibility of TOE to acrylamide was greatest with DPC, followed by C 12E 8 and finally DM ( K sv=2.7–7.1 M −1). TOE also presents less rotational mobility in DM than in the other two detergents, as shown from anisotropy decay measurements. These results, together with previous TOE quenching measurements with brominated detergents [Biophys. J. 77 (1999) 3071] provide reference data for analyzing Trp characteristics in peptide (and more indirectly protein)–detergent complexes. The main finding of this study was that TOE was less accessible (to soluble quenchers) in DM than in DPC and C 12E 8, the cohesion of DM headgroup region being suggested to play a role in the ability of this detergent to protect function and stability of solubilized membrane proteins.

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.

Stages of the bilayer-micelle transition in the system phosphatidylcholine-C12E8 as studied by deuterium- and phosphorous-NMR, light scattering, and calorimetry

The perturbation of phospholipid bilayer membranes by a nonionic detergent, octaethyleneglycol mono-n-dodecylether (C12E8), was investigated by 2H- and 31P-NMR, static and dynamic light scattering, and differential scanning calorimetry. Preequilibrated mixtures of the saturated phospholipids 1,2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC), and 1,2-dilauroyl-sn-glycero-3-phosphorylcholine (DLPC) with the detergent were studied over a broad temperature range including the temperature of the main thermotropic phase transition of the pure phospholipids. Above this temperature, at a phospholipid/detergent molar ratio 2:1, the membranes were oriented in the magnetic field. Cooling of the mixtures below the thermotropic phase transition temperatures of the pure phospholipids led to micelle formation. In mixtures of DPPC and DMPC with C12E8, a narrow calorimetric signal at the onset temperature of the solubilization suggested that micelle formation was related to the disorder-order transition in the phospholipid acyl chains. The particle size changed from 150 nm to approximately 7 nm over the temperature range of the bilayer-micelle transition. The spontaneous orientation of the membranes at high temperatures enabled the direct determination of segmental order parameters from the deuterium spectra. The order parameter profiles of the phospholipid acyl chains could be attributed to slow fluctuations of the whole membrane and to detergent-induced local perturbations of the bilayer order. The packing constraints in the mixed bilayers that eventually lead to bilayer solubilization were reflected by the order parameters of the interfacial phospholipid acyl chain segments and of the phospholipid headgroup. These results are interpreted in terms of the changing average shape of the component molecules. Considering the decreasing cross sectional areas in the acyl chain region and the increasing hydration of the detergent headgroups, the bilayer-micelle transition is the result of an imbalance in the chain and headgroup repulsion. A neutral or pivotal plane can be defined on the basis of the temperature dependence of the interfacial quadrupolar splittings.

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.

A 31P- and 2H-NMR study on the structural perturbations induced by charged detergents in the headgroup region of phosphatidylcholine bilayers

The effect of cetyltrimethylammonium bromide (CTAB) and dodecyltrimethylammonium bromide (DTAB) on the structure and mobility of phosphatidylcholine membranes was studied by 31P- and 2H-nuclear magnetic resonance. CTAB and DTAB induced a strong increase in the effective phosphorus chemical shift anisotropy of the phospholipid. In phosphatidylcholine which had been specifically labeled by deuterium in the headgroup segments, the detergents caused an increase in the quadrupole splittings from the N- methyl deuterons whereas the splittings from both methylene segments collapsed to a singlet signal at a ratio of about 2 mol of phosphatidylcholine per mol of detergent. The effect of DTAB on the headgroup spectral parameters was largely compensated by the negatively charged detergent sodium dodecylsulfate (SDS). In contrast to the phospholipid headgroup the hydrophobic region of the membrane was almost unaffected by CTAB or DTAB. The trimethylammonium headgroups of membrane-bound detergent molecules were found to be motionally much more restricted than the corresponding headgroup segments of the host phospholipid. From these observations it is concluded that the detergent headgroups are located close to the water/hydrocarbon interface of the phospholipid membrane thereby tilting the phospholipid headgroup dipole from the membrane parallel into a more upright orientation.

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.