Thermodynamics of sodium dodecyl sulfate partitioning into lipid membranes

The partition equilibria of sodium dodecyl sulfate (SDS) and lithium dodecyl sulfate between water and bilayer membranes were investigated with isothermal titration calorimetry and spectroscopic methods (light scattering, (31)P-nuclear magnetic resonance) in the temperature range of 28 degrees C to 56 degrees C. The partitioning of the dodecyl sulfate anion (DS(-)) into the bilayer membrane is energetically favored by an exothermic partition enthalpy of Delta H(O)(D) = -6.0 kcal/mol at 28 degrees C. This is in contrast to nonionic detergents where Delta H(O)(D) is usually positive. The partition enthalpy decreases linearly with increasing temperature and the molar heat capacity is Delta C(O)(P) = -50 +/- 3 cal mol(-1) K(-1). The partition isotherm is nonlinear if the bound detergent is plotted versus the free detergent concentration in bulk solution. This is caused by the electrostatic repulsion between the DS(-) ions inserted into the membrane and those free in solution near the membrane surface. The surface concentration of DS(-) immediately above the plane of binding was hence calculated with the Gouy-Chapman theory, and a strictly linear relationship was obtained between the surface concentration and the extent of DS(-) partitioning. The surface partition constant K describes the chemical equilibrium in the absence of electrostatic effects. For the SDS-membrane equilibrium K was found to be 1.2 x 10(4) M(-1) to 6 x 10(4) M(-1) for the various systems and conditions investigated, very similar to data available for nonionic detergents of the same chain length. The membrane-micelle phase diagram was also studied. Complete membrane solubilization requires a ratio of 2.2 mol SDS bound per mole of total lipid at 56 degrees C. The corresponding equilibrium concentration of SDS free in solution is C (sat)(D,F) approximately 1.7 mM and is slightly below the critical micelles concentration (CMC) = 2.1 mM (at 56 degrees C and 0.11 M buffer). Membrane saturation occurs at approximately 0.3 mol SDS per mol lipid and the equilibrium SDS concentration is C (sat)(D,F)approximately equal 2.2 mM +/- 0.6 mM. SDS translocation across the bilayer is slow at ambient temperature but increases at high temperatures.     

Combining precision spin-probe partitioning with time-resolved fluorescence quenching to study micelles. Application to micelles of pure lysomyristoylphosphatidylcholine (LMPC) and LMPC mixed with sodium dodecyl sulfate

Micelles of lysomyristoylphosphatidylcholine (LMPC) and mixed micelles of LMPC with anionic detergent sodium dodecyl sulfate (SDS) have been characterized by spin-probe-partitioning electron paramagnetic resonance (SPPEPR) and time-resolved fluorescence quenching (TRFQ) experiments. SPPEPR is a novel new method to study structure and dynamics in lipid assemblies successfully applied here for the first time to micelles. Several improvements to the computer program used to analyze SPPEPR spectra have been incorporated that increase the precision in the extracted parameters considerably from which micelle properties such as effective water concentration and microviscosity may be estimated. In addition, with this increased precision, it is shown that it is feasible to study the rate of transfer of a small spin probe between micelles and the surrounding aqueous phase by SPPEPR. The rate of transfer of the spin probe di-tert-butyl nitroxide (DTBN) and the activation energy of the transfer process in LMPC and LMPC-SDS micelles have been determined with high precision. The rate of transfer increases with temperature and SDS molar fraction in mixed micelles, while it remains constant with LMPC concentration in pure LMPC micelles. The activation energy of DTBN transfer in pure lysophospholipid micelles does not change with LMPC concentration while it decreases with the increasing molar fraction of SDS in mixed LMPC-SDS micelles. Both this decrease in activation energy and the increase in the rate of transfer are rationalized in terms of an increasing micelle surface area per molecule (decreasing compactness) as SDS molecules are added. This decreasing compactness as a function of SDS content is confirmed by TRFQ measurements showing an aggregation number that decreases from 122 molecules for pure LMPC micelles to 80 molecules for pure SDS micelles. The same increase in surface area per molecule is predicted to increase the effective water concentration in the polar shell of the micelles. This increase in hydration with SDS molar fraction is confirmed by measuring the effective water concentration in the polar shell of the micelles from the hyperfine spacing of DTBN. This work demonstrates the potential to design mixed lysophospholipid surfactant micelles with variable physicochemical properties. Well-defined micellar substrates, in terms of their physicochemical properties, may improve the studies of protein structure and enzyme kinetics.     

Using micellar mole fractions to assess membrane protein stability in mixed micelles

The increased focus on the structural and physical properties of membrane proteins has made it critical to develop methods that provide a reliable estimate of membrane protein stability. A simple approach is to monitor the protein's conformational changes in mixed detergent systems, typically consisting of an anionic (denaturing) and non-ionic (non-denaturing) component. Linear correlations between, e.g., the melting temperature and the bulk mole fraction of the anionic component have been observed. However, a potential complication is that the bulk mole fraction is not identical to the mole fraction in the mixed micelle, which is the local environment experienced by the membrane protein. Here, we present an extensive analysis of the thermal stability of the membrane-integrated domain of the outer membrane protein AIDA in the presence of different mixed micelles. In the micelle system SDS-octyl-polyoxyethylene, the melting temperature in the absence of SDS extrapolates to 113 °C using bulk mole fractions. However, for mixed micelles involving short-chain detergents or phospholipids, the melting temperature calculated using bulk mole fractions reaches values up to several hundred degrees higher than 113 °C and can only be obtained by extrapolation over a narrow mole fraction interval. Furthermore, there is a non-linear relationship between the melting temperature and bulk mole fractions for mixed micelle systems involving cationic detergents (also denaturing). We show that if we instead use the micellar mole fraction as a parameter for denaturing detergent strength, we obtain linear correlations which extrapolate to more or less the same value of the melting temperature. There remains some scatter in the extrapolated values of the melting temperature in different binary systems, which suggest that additional micellar interactions may play a role. Nevertheless, in general terms, the mixed micellar composition is a good parameter to describe the membrane protein's microenvironment. Note, however, that for the mixed micelle system involving SDS and dodecyl maltoside, which has been used by several research groups to determine membrane protein stability, the estimate provided by bulk mole fraction leads to similar values as that of micellar mole fractions.     

Using micellar mole fractions to assess membrane protein stability in mixed micelles

The increased focus on the structural and physical properties of membrane proteins has made it critical to develop methods that provide a reliable estimate of membrane protein stability. A simple approach is to monitor the protein's conformational changes in mixed detergent systems, typically consisting of an anionic (denaturing) and non-ionic (non-denaturing) component. Linear correlations between, e.g., the melting temperature and the bulk mole fraction of the anionic component have been observed. However, a potential complication is that the bulk mole fraction is not identical to the mole fraction in the mixed micelle, which is the local environment experienced by the membrane protein. Here, we present an extensive analysis of the thermal stability of the membrane-integrated domain of the outer membrane protein AIDA in the presence of different mixed micelles. In the micelle system SDS-octyl-polyoxyethylene, the melting temperature in the absence of SDS extrapolates to 113 degrees C using bulk mole fractions. However, for mixed micelles involving short-chain detergents or phospholipids, the melting temperature calculated using bulk mole fractions reaches values up to several hundred degrees higher than 113 degrees C and can only be obtained by extrapolation over a narrow mole fraction interval. Furthermore, there is a non-linear relationship between the melting temperature and bulk mole fractions for mixed micelle systems involving cationic detergents (also denaturing). We show that if we instead use the micellar mole fraction as a parameter for denaturing detergent strength, we obtain linear correlations which extrapolate to more or less the same value of the melting temperature. There remains some scatter in the extrapolated values of the melting temperature in different binary systems, which suggest that additional micellar interactions may play a role. Nevertheless, in general terms, the mixed micellar composition is a good parameter to describe the membrane protein's microenvironment. Note, however, that for the mixed micelle system involving SDS and dodecyl maltoside, which has been used by several research groups to determine membrane protein stability, the estimate provided by bulk mole fraction leads to similar values as that of micellar mole fractions.     

Microstructures formed in aqueous solutions of a hydrophobically modified nonionic cellulose derivative and sodium dodecyl sulfate: a fluorescence probe investigation

The association behavior of hydrophobically modified ethyl hydroxyethyl cellulose (HM-EHEC) and its interaction with the anionic surfactant sodium dodecyl sulfate (SDS) has been studied in the dilute concentration regime. Steady-state fluorescence probe techniques have been utilized to obtain microstructural information of the system properties and combined with macroscopic bulk information from equilibrium dialysis experiments in order to determine binding isotherms of SDS to HM-EHEC. HM-EHEC was found to self-associate and form polymeric micelles in semi-dilute aqueous solutions. c^* for the self-association process was determined to be approximately 0.4%. The microviscosity of the polymeric micelles is much higher, and the micropolarity slightly higher, than that of ordinary SDS micelles. The onset of interaction between HM-EHEC and SDS was evidenced by a simultaneous strong increase in microviscosity and decrease in micropolarity upon successive addition of SDS. There is a minor, noncooperative SDS binding to the HM-EHEC starting from low concentrations of SDS (<5 mM) followed by a highly cooperative binding region at SDS concentrations ≥5 mM. The polymer–surfactant aggregates are rigid and hydrophobic with a maximum in microviscosity in the noncooperative binding region at a very low degree of SDS-adsorption.     

Functional reconstitution of carrier proteins by removal of detergent with a hydrophobic ion exchange column

A method has been developed for the functional reconstitution of membrane proteins in phospholipid vesicles. This method is an extension of a previously published procedure (Ueno, M., Tanford, C. and Reynolds, A. (1984) Biochemistry 23, 3070-3076) for the formation of unilamellar vesicles from mixed micelles of egg phosphatidylcholine and dodecyl octaoxyethylene ether. Mixed micelles are formed from detergent-solubilized protein and egg-yolk phospholipid vesicles. These micelles are subjected to repeated passage through small columns filled with Amberlite XAD-2 beads. Several carrier proteins from the inner mitochondrial membrane have been reconstituted in this way; experimental data are shown for the aspartate/glutamate carrier and the ADP/ATP carrier. Certain parameters proved to be important for optimal efficiency of reconstitution: the ratio of detergent/phospholipid in the mixed micelles, the concentration of phospholipid during the hydrophobic chromatography, the ratio of phospholipid/protein, (d) the ratio of detergent/Amberlite XAD 2 beads, the number of column passages, and the type of detergent. After optimization of these parameters, phospholipid vesicles with a diameter of about 150 nm were obtained. The main advantage of this procedure, however, lies in the fact that high amounts of membrane protein can be incorporated into the phospholipid vesicles, i.e. up to 15% (w/w).     

Microviscosity in dilute aqueous solutions of SDS and non-ionic cellulose derivatives of different hydrophobicity: fluorescence probe investigations

The microviscosity in mixed micelles formed in dilute aqueous solutions of sodium dodecyl sulphate (SDS) and a set of non-ionic cellulose ethers of different hydrophobicity has been determined by means of steady-state fluorescence probe techniques. Two hydrophobic probes have been applied in this investigation: 1,3-di(1-pyrenyl)propane (P3P) and perylene. Reference measurements of microviscosity have also been performed on SDS solutions including the uncharged polymers poly(ethyleneoxide) (PEO) or poly(vinylpyrrolidone) (PVP). All compositions investigated showed qualitatively the same general behaviour with an abrupt increase in microviscosity at the critical surfactant concentration where the polymer-surfactant interaction starts (c₁) followed by a maximum and an asymptotically declining region as the surfactant concentration was increased further. Comparison with a recent investigation of a specific ethyl(hydroxyethyl)cellulose (EHEC fraction CST-103)/ SDS/water system (Evertsson & Nilsson (1997) Macromolecules, 30, 2377) revealed that the maximum in microviscosity generally corresponds to a low degree of SDS adsorption (≈ 0.5 mmol of SDS per gram of polymer) and consequently to a high polymer content of the mixed micelles formed in the type of systems studied herein. The hydrophobicity of the cellulose derivatives was found to correlate to the amplitude of the overall microviscosity pattern for the mixed micelles, i.e. an increased polymer hydrophobicity gave an increased rigidity of the polymersurfactant aggregates. An approximately exponential relation was demonstrated between the maxima in microviscosity of the different mixed micelles and the surface activities of the corresponding cellulose derivatives. All polymer/surfactant combinations investigated gave aggregates with a higher rigidity than ordinary SDS micelles. The microviscosity of the mixed micelles of the cellulose derivatives and SDS formed close to c₁ increased as the temperature rose from 20 to 50 °C. This effect was attributed to an increased hydrophobicity of the cellulose ethers upon temperature elevation, hence giving rise to further close-packing of the aggregate structures.     

Spontaneous formation of DPPC monolayers at aqueous/vapor interfaces and the impact of charged surfactants

Surface tensiometry and vibrational sum-frequency spectroscopy were used to examine the structure and organization in phospholipid monolayers at the aqueous/vapor interface in the absence and in the presence of simple, charged surfactants. 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was the phospholipid employed in these studies and surfactants included sodium dodecyl sulfate (SDS) and dodecyl trimethyl ammonium bromide (DTAB). DPPC spontaneously spreads on a pure water (pH = 5.5) surface to form monolayers as evidenced by an equilibrium spreading pressure (ESP) of 7.9 ± 2.3 mN/m and a clearly resolved vibrational spectrum. Low concentrations of surfactants inhibit the spreading of DPPC and result in significantly lower ESP values. Anionic and cationic surfactants at higher concentrations have opposite effects on monolayer organization; SDS creates well-organized monolayers while DTAB leads to poor organization of lipid molecules. Surface-specific vibrational spectra showed that high concentrations of charged surfactants (≥ 100 µM) lead to accumulation of net surface charges as evidenced by destructive and constructive interferences. Selectively deuterating surfactants results in changes in vibrational band intensities and phases enabling assignment of relative orientations of equivalent functional groups belonging to the lipid and surfactant.     

Assay of detergents by rocket electrophoresis in agarose gels containing red blood cells: "rocket hemolysis"

A method is described for quantitation of charged detergents using their hemolytic property in an electrophoresis assay in agarose gels containing red blood cells. After electrophoresis the zone of hemolysis is directly proportional to the concentration of detergent in the sample. Using this technique we have determined the smallest detectable concentration for the negatively charged detergents, sodium dodecyl sulfate (SDS) and Quil A to about 10 micrograms/ml and 25 micrograms/ml, respectively and the positively charged cetyltrimethylammonium bromide (CTAB) to about 10 micrograms/ml.     

Effect of detergents on the enzyme activities of the rat liver plasma membrane

The anionic detergents sodium dodecyl sulfate (SDS) and Alipal CO-433 and the non-ionic detergent Trition X-100 at concentrations of 0.02–0.10% cause a more rapid solubilization of phospholipid than proteins in isolated rat liver plasma membranes. All three detergents cause an increase in membrane turbidity at low detergent concentration (0.01–0.04%) but then decrease the turbidity at higher detergent concentration (0.04–0.10%). Each detergent gives a characteristic turbidity-detergent concentration profile which is pH dependent.The activities of the membrane-bound enzymes Mg²⁺ ATPase, 5′-nucleotidase and acid and aklaline phosphatase were influenced by each detergent to a different extent. Each enzyme gave a characteristic activity-detergent concentration profile. Mg²⁺ ATPase was inhibited by all detergents. 5′-Nucleotidase was stimulated by Triton and Alipal but inhibited by SDS. Alkaline phosphatase was stimulated by Alipal and SDS and not influenced by Triton. Acid phosphatase was stimulated by Triton and inhibited by Alipal and SDS. 56% of the total membrane-bound alkaline phosphatase and 23% of the total membrane-bound 5′-nucleotidase was solubilized in an active form by 0.06% and 0.05% SDS respectively.     

Solubilization of Myelin Membranes by Detergents

The major proteins of myelin have classically been extracted in organic solvents. Here we investigated some of the characteristics of brain myelin solubilization in aqueous detergent solutions. At comparable molar concentrations, two nonionic detergents, i.e., octyl glucoside and Lubrol PX, proved relatively better myelin solubilizers than the detergents related to the bile salts, i.e., cholate and CHAPS. The two former detergents solubilized more protein than lipid and the two latter ones more lipid than protein from myelin membranes. All four detergents solubilized the phospholipid more efficiently than the cholesterol component of myelin. The detergent concentrations required for myelin solubilization were reduced substantially if the temperature and the salt concentration of the media were increased. As much as 3 mg of lyophilized myelin (about 1 mg of protein) were solubilized readily per milliliter of a solution containing 30 mM octyl glucoside and 0.1 M sodium sulfate in 0.1 M sodium phosphate buffer, pH 6.7. Each of the detergents studied, including the above four, sodium dodecyl sulfate (SDS). Triton X-100, and Zwittergent 3-14, had its own advantages and drawbacks as myelin protein extractors. The nonionic amphiphiles and CHAPS left a small residue mainly composed of proteins of the Wolfgram fraction, as revealed by SDS-polyacrylamide gel electrophoresis. Octyl glucoside was preferred, given its versatility as solubilizer, ultraviolet transparency, and high critical micellar concentration. Observations on possible difficulties that may be encountered are also included.     

Bilayer interactions of ether- and ester-linked phospholipids: dihexadecyl- and dipalmitoylphosphatidylcholines

Mixed phospholipid systems of ether-linked 1,2-dihexadecylphosphatidylcholine (DHPC) and ester-linked 1,2-dipalmitoylphosphatidylcholine (DPPC) have been studied by differential scanning calorimetry and X-ray diffraction. At maximum hydration (60 wt % water), DHPC shows three reversible transitions: a main (chain melting) transition, TM = 44.2 degrees C; a pretransition, TP = 36.2 degrees C; and a subtransition, TS = 5.5 degrees C. DPPC shows two reversible transitions: TM = 41.3 degrees C and TP = 36.5 degrees C. TM decreases linearly from 44.2 to 41.3 degrees C as DPPC is incorporated into DHPC bilayers; TP exhibits eutectic behavior, decreasing sharply to reach 23.3 degrees C at 40.4 mol % DPPC and then increasing over the range 40-100 mol % DPPC; TS remains constant at 4-5 degrees C and is not observed at greater than 20 mol % DPPC. At 50 degrees C, X-ray diffraction shows a liquid-crystalline bilayer L alpha phase at all DHPC:DPPC mole ratios. At 22 degrees C, DHPC shows an interdigitated bilayer gel L beta phase (bilayer periodicity d = 47.0 A) into which approximately 30 mol % DPPC can be incorporated. Above 30 mol % DPPC, a noninterdigitated gel L beta' phase (d = 64-66 A) is observed. Thus, at T greater than TM, DHPC and DPPC are miscible in all proportions in an L alpha bilayer phase. In contrast, a composition-dependent gel----gel transition between interdigitated and noninterdigitated bilayers is observed at T less than TP, and this leads to eutectic behavior of the DHPC/DPPC system.     

Energetic and binding properties of DNA upon interaction with dodecyl trimethylammonium bromide.

The interaction of dodecyl trimethylammonium bromide (DTAB), a cationic surfactant, with calf thymus DNA has been studied by various methods, including potentiometric technique using DTAB-selective plastic membrane electrode at 27 and 37 degreesC, isothermal titration microcalorimetry and UV spectrophotometry at 27 degreesC using 0.05 M Tris buffer and 0.01 M NaCl at pH 7.4. The free energy is calculated from binding isotherms on the basis of Wyman binding potential theory and the enthalpy of binding according to van't Hoff relation. The enthalpy of unfolding has been determined by subtraction of the enthalpy of binding from the microcalorimetric enthalpy. The results show that, after the interaction of first DTAB molecule to DNA (base molarity) through the electrostatic interaction, the second DTAB molecule also binds to DNA through electrostatic interaction. At this stage, the predom-inant DNA conformational change occurs. Afterwards up to 20 DTAB molecules, below the critical micelle concentration of DTAB, bind through hydrophobic interactions.     

The mechanism of detergent solubilization of liposomes and protein-containing membranes.

The present study explores intermediate stages in detergent solubilization of liposomes and Ca2+-ATPase membranes by sodium dodecyl sulfate (SDS) and medium-sized ( approximately C12) nonionic detergents. In all cases detergent partitioning in the membranes precedes cooperative binding and solubilization, which is facilitated by exposure to detergent micelles. Nonionic detergents predominantly interact with the lipid component of Ca2+-ATPase membranes below the CMC (critical micellar concentration), whereas SDS extracts Ca2+-ATPase before solubilization of lipid. At the transition to cooperative binding, n-dodecyl octaethylene glycol monoether (C12E8), Triton X-100, and dodecyldimethylamine oxide induce fusion of small unilamellar liposomes to larger vesicles before solubilization. Solubilization of Ca2+-ATPase membranes is accompanied by membrane fragmentation and aggregation rather than vesicle fusion. Detergents with strongly hydrophilic heads (SDS and beta-D-dodecylmaltoside) only very slowly solubilize liposomal membranes and do not cause liposome fusion. These properties are correlated with a slow bilayer flip-flop. Our data suggest that detergent solubilization proceeds by a combination of 1) a transbilayer attack, following flip-flop of detergent molecules across the lipid bilayer, and 2) extraction of membrane components directly by detergent micelles. The present study should help in the design of efficient solubilization protocols, accomplishing the often delicate balance between preserving functional properties of detergent sensitive membrane proteins and minimizing secondary aggregation and lipid content.     

The interaction between hemoglobin and two surfactants with different charges

The interactions of hemoglobin (Hb) with sodium dodecyl sulfate (SDS) and dodecyl trimethylammonium bromide (DTAB) are investigated by several methods. We observed the formation of hemichrome below the critical micelle concentration (cmc) of surfactant and the release of heme from Hb above the cmc. When pH value of Hb/surfactant system is lower than isoelectric point (pI) of Hb, the interaction of SDS with Hb is both electrostatic and hydrophobic, while the interaction of DTAB with Hb is hydrophobic mainly. On the contrary, when pH > pI, the interaction of SDS with Hb is hydrophobic mainly, while the interaction of DTAB with Hb is both electrostatic and hydrophobic. In the case where both the electrostatic interaction and hydrophobic interaction exist, the electrostatic interaction plays a more important role. Thus, SDS tends to interact with Hb more obviously than DTAB does when pH < pI and the interaction between DTAB and Hb is stronger when pH > pI.     

Characterization of Phospholipid Mixed Micelles by Translational Diffusion

The concentration dependence of the translational self diffusion rate, D (s), has been measured for a range of micelle and mixed micelle systems. Use of bipolar gradient pulse pairs in the longitudinal eddy current delay experiment minimizes NOE attenuation and is found critical for optimizing sensitivity of the translational diffusion measurement of macromolecules and aggregates. For low volume fractions Phi (Phi\\ le 15% v/v) of the micelles, experimental measurement of the concentration dependence, combined with use of the D (s)= D (o)(1-3.2lambdaPhi) relationship, yields the hydrodynamic volume. For proteins, the hydrodynamic volume, derived from D (s) at infinitely dilute concentration, is found to be about 2.6 times the unhydrated molecular volume. Using the data collected for hen egg white lysozyme as a reference, diffusion data for dihexanoyl phosphatidylcholine (DHPC) micelles indicate approximately 27 molecules per micelle, and a critical micelle concentration of 14 mM. Differences in translational diffusion rates for detergent and long chain phospholipids in mixed micelles are attributed to rapid exchange between free and micelle-bound detergent. This difference permits determination of the free detergent concentration, which, for a high detergent to long chain phospholipid molar ratio, is found to depend strongly on this ratio. The hydrodynamic volume of DHPC/POPC bicelles, loaded with an M2 channel peptide homolog, derived from translational diffusion, predicts a rotational correlation time that slightly exceeds the value obtained from peptide (15)N relaxation data.     

NMR solution structure of the peptide fragment 1-30, derived from unprocessed mouse Doppel protein, in DHPC micelles

The downstream prion-like Doppel (Dpl) protein is a homologue related to the prion protein (PrP). Dpl is expressed in the brains of mice that do not express PrP, and Dpl is known to be toxic to neurons. One mode of toxicity has been suggested to involve direct membrane interactions. PrP under certain conditions of cell trafficking retains an uncleaved signal peptide, which may also hold for the much less studied Dpl. For a peptide with a sequence derived from the N-terminal part (1-30) of mouse Dpl (mDpl(1-30)) CD spectroscopy shows about 40% alpha-helical structure in DHPC and SDS micelles. In aqueous solution it is mostly a random coil. The three-dimensional solution structure was determined by NMR for mDpl(1-30) associated with DHPC micelles. 2D 1H NMR spectra of the peptide in q = 0.25 DMPC/DHPC bicelles only showed signals from the unstructured termini, indicating that the structured part of the peptide resides within the lipid bilayer. Together with 2H2O exchange data in the DHPC micelle solvent, these results show an alpha-helix protected from solvent exchange between residues 7 and 19, and suggest that the alpha-helical segment can adopt a transmembrane localization also in a membrane. Leakage studies with entrapped calcein in large unilamellar phospholipid vesicles showed that the peptide is almost as membrane perturbing as melittin, known to form pores in membranes. The results suggest a possible channel formation mechanism for the unprocessed Dpl protein, which may be related to toxicity through direct cell membrane interaction and damage.     

Solubilization of DMPC and DPPC vesicles by detergents below their critical micellization concentration: high-sensitivity differential scanning calorimetry, Fourier transform infrared spectroscopy and freeze-fracture electron microscopy reveal two interaction sites of detergents in vesicles

The interaction of sodium deoxycholate, sodium cholate and octyl glucoside with sonicated vesicles of L alpha-dimyristoyl-phosphatidylcholine (DMPC) and L alpha-dipalmitoylphosphatidylcholine (DPPC) at concentrations below the critical micellization concentration (cmc) of the detergents was studied by high-sensitivity DSC (hs-DSC), Fourier transform infrared spectroscopy (FT-IR) and freeze-fracture electron microscopy. The two phospholipids exhibited a striking different thermotropic behaviour in the presence of these detergents. For DPPC vesicles, the detergents were found to interact exclusively in the aqueous interface region of the bilayer below the membrane saturation concentration Rsat while in DMPC vesicles two coexisting interaction sites below this concentration persist. These are detergents which interact at the aqueous interface region (site 1) and in the acyl chain region (site 2) of the DMPC vesicles. The partition coefficients K of the detergents between DPPC vesicles and the water phase were calculated from the hs-DSC results at two detergent/phospholipid molar ratios Rtot less than or equal to Rsat as 0.35, 0.049 and 0.040 mol-1 for sodium deoxycholate, sodium cholate and octyl glucoside, respectively. In contrast, for DMPC the K values for Rtot less than or equal to Rsat were found to be dependent on Rtot due to the occupation of site 2 by the detergents above a certain Rtot. The model is discussed on the basis of the detergents free energies of transfer from the water phase to site 1 and site 2 of the vesicles, respectively. The solubilization behaviour of DPPC vesicles, dependent on whether the total detergent concentration is above or below the cmc at Rsat, differed significantly as revealed by hs-DSC. This suggests that in the latter case an additional hydrophobic effect could facilitate the formation of disc shaped mixed micelles. Moreover, this different behaviour was employed to measure the cmc values of the detergents studied in the presence of the vesicles by hs-DSC.     

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.