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The present study mainly consists of a re-evaluation of the rate at which C12E8, a typical non-ionic detergent used for membrane studies, is able to dissociate from biological membranes, with sarcoplasmic reticulum membrane vesicles being used as an example. Utilizing a brominated derivative of C12E8 and now stopped-flow fluorescence instead of rapid filtration, we found that the rate of dissociation of this detergent from these membranes, merely perturbed with non-solubilizing concentrations of detergent, was significantly faster (t1/2 < 10 ms) than what had previously been determined (t1/2 ~300-400 ms) from experiments based on a rapid filtration protocol using 14C-labeled C12E8 and glass fiber filters (Binding of a non-ionic detergent to membranes: flip-flop rate and location on the bilayer, by Marc le Maire, Jesper Møller and Philippe Champeil, Biochemistry (1987) Vol 26, pages 4803-4810). We here pinpoint a methodological problem of the earlier rapid filtration experiments, and we suggest that the true overall dissociation rate of C12E8 is indeed much faster than previously thought. We also exemplify the case of brominated dodecyl-maltoside, whose kinetics for overall binding to and dissociation from membranes comprise both a rapid and a sower phase, the latter being presumably due to flip-flop between the two leaflets of the membrane. Consequently, equilibrium is reached only after a few seconds for DDM. This work thereby emphasizes the interest of using the fluorescence quenching associated with brominated detergents for studying the kinetics of detergent/membrane interactions, namely association, dissociation and flip-flop rates., Competing Interests: The authors have declared that no competing interests exist.

Most membrane proteins studies require the use of detergents, but because of the lack of a general, accurate and rapid method to quantify them, many uncertainties remain that hamper proper functional and structural data analyses. To solve this problem, we propose a method based on matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) that allows quantification of pure or mixed detergents in complex with membrane proteins. We validated the method with a wide variety of detergents and membrane proteins. We automated the process, thereby allowing routine quantification for a broad spectrum of usage. As a first illustration, we show how to obtain information of the amount of detergent in complex with a membrane protein, essential for liposome or nanodiscs reconstitutions. Thanks to the method, we also show how to reliably and easily estimate the detergent corona diameter and select the smallest size, critical for favoring protein-protein contacts and triggering/promoting membrane protein crystallization, and to visualize the detergent belt for Cryo-EM studies., Competing Interests: The authors declare no competing financial interests.

Membrane proteins are largely dependent for their function on the phospholipids present in their immediate environment, and when they are solubilized by detergent for further study, residual phospholipids are critical, too. Here, brominated phosphatidylcholine, a phospholipid which behaves as an unsaturated phosphatidylcholine, was used to reveal the kinetics of phospholipid exchange or transfer from detergent mixed micelles to the environment of a detergent-solubilized membrane protein, the paradigmatic P-type ATPase SERCA1a, in which Trp residues can experience fluorescence quenching by bromine atoms present on phospholipid alkyl chains in their immediate environment. Using dodecylmaltoside as the detergent, exchange of (brominated) phospholipid was found to be much slower than exchange of detergent under the same conditions, and also much slower than membrane solubilization, the latter being evidenced by light scattering changes. The kinetics of this exchange was strongly dependent on temperature. It was also dependent on the total concentration of the mixed micelles, revealing the major role for such exchange of the collision of detergent micelles with the detergent-solubilized protein. Back-transfer of the brominated phospholipid from the solubilized protein to the detergent micelle was much faster if lipid-free DDM micelles instead of mixed micelles were added for triggering dissociation of brominated phosphatidylcholine from the solubilized protein, or in the additional presence of C12E8 detergent during exchange, also emphasizing the role of the chemical nature of the micelle/protein interface. This protocol using brominated lipids appears to be valuable for revealing the possibly slow kinetics of phospholipid transfer to or from detergent-solubilized membrane proteins. Independently, continuous recording of the activity of the protein can also be used in some cases to correlate changes in activity with the exchange of a specific phospholipid, as shown here by using the Drs2p/Cdc50p complex, a lipid flippase with specific binding sites for lipids., Competing Interests: The authors have declared that no competing interests exist.

This report is a follow up of our previous paper (Lund, Orlowski, de Foresta, Champeil, le Maire and Møller (1989), J Biol Chem 264:4907-4915) showing that solubilization in detergent of a membrane protein may interfere with its long-term stability, and proposing a protocol to reveal the kinetics of such irreversible inactivation. We here clarify the fact that when various detergents are tested for their effects, special attention has of course to be paid to their critical micelle concentration. We also investigate the effects of a few more detergents, some of which have been recently advertised in the literature, and emphasize the role of lipids together with detergents. Among these detergents, lauryl maltose neopentyl glycol (LMNG) exerts a remarkable ability, even higher than that of β-dodecylmaltoside (DDM), to protect our test enzyme, the paradigmatic P-type ATPase SERCA1a from sarcoplasmic reticulum. Performing such experiments for one's favourite protein probably remains useful in pre-screening assays testing various detergents., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The H(+),K(+)-ATPase pumps protons or hydronium ions and is responsible for the acidification of the gastric fluid. It is made up of an α-catalytic and a β-glycosylated subunit. The relation between cation translocation and the organization of the protein in the membrane are not well understood. We describe here how pure and functionally active pig gastric H(+),K(+)-ATPase with an apparent Stokes radius of 6.3 nm can be obtained after solubilization with the non-ionic detergent C(12)E(8), followed by exchange of C(12)E(8) with Tween 20 on a Superose 6 column. Mass spectroscopy indicates that the β-subunit bears an excess mass of 9 kDa attributable to glycosylation. From chemical analysis, there are 0.25 g of phospholipids and around 0.024 g of cholesterol bound per g of protein. Analytical ultracentrifugation shows one main complex, sedimenting at s(20,)(w) = 7.2 ± 0.1 S, together with minor amounts of irreversibly aggregated material. From these data, a buoyant molecular mass is calculated, corresponding to an H(+),K(+)-ATPase α,β-protomer of 147.3 kDa. Complementary sedimentation velocity with deuterated water gives a picture of an α,β-protomer with 0.9-1.4 g/g of bound detergent and lipids and a reasonable frictional ratio of 1.5, corresponding to a Stokes radius of 7.1 nm. An α(2),β(2) dimer is rejected by the data. Light scattering coupled to gel filtration confirms the monomeric state of solubilized H(+),K(+)-ATPase. Thus, α,β H(+),K(+)-ATPase is active at least in detergent and may plausibly function as a monomer, as has been established for other P-type ATPases, Ca(2+)-ATPase and Na(+),K(+)-ATPase.

ADP/ATP carriers (AACs) are major and essential constituents of the inner mitochondrial membrane. They drive the import of ADP and the export of newly synthesized ATP. They were described as functional dimers from the 1980s until the structures of the AAC shed doubt on this consensus. We aimed to ascertain the published biophysical data claiming that AACs are dimers and to characterize the oligomeric state of the protein before crystallization. Analytical ultracentrifugation sedimentation velocity experiments clearly show that the bovine AAC is a monomer in 3-laurylamido-N,N'-dimethylpropylaminoxide (LAPAO), whereas in Triton X-100 and reduced Triton X-100, higher molecular mass species can also be identified. Neutron scattering data for monomeric bovine AAC in LAPAO does not give definite conclusions on the association state, because the large amount of detergent and lipids is imperfectly matched by contrast methods. We discuss a possible way to integrate previously published biochemical evidence in favor of assemblies, the lack of well-defined multimers that we observe, and the information from the high-resolution structures, considering supramolecular organizations of AACs within the mitochondrial membrane.

For structural studies of integral membrane proteins, including their 3D crystallization, the judicious use of detergent for solubilization and purification is required. Detergent binding by the solubilized protein is an important parameter to determine the hydrodynamic properties in terms of size and aggregational (monomeric/oligo(proto)meric) state of the protein. Detergent binding can be measured by gel filtration chromatography under equilibrium conditions and after separation from mixed micelles of solubilized lipid and detergent. Using sarcoplasmic reticulum Ca(2+)-ATPase as an example, we demonstrate in this protocol complete procedures for measurement of detergent binding using (i) radiolabeled n-dodecyl-beta-D-maltoside (DM) or (ii) from measurements of the increase in refractive index due to the presence of bound detergent on the protein. The latter measurement can also be performed by sedimentation velocity (SV) analysis in the analytical ultracentrifuge which in addition allows determination of the sedimentation coefficient. In combination with estimation of Stokes radius by gel filtration calibration, the molecular mass and asymmetry of the solubilized protein can be calculated. In the proposed protocols, the gel chromatographic procedures require 1 d; SV experiments are performed just after size exclusion. The whole time for these experiments is 24 h. Data analysis of analytical ultracentrifugation requires a couple of days.

BmrA from Bacillus subtilis is a half-size ABC (ATP-binding cassette) transporter involved in multidrug resistance. Although its supramolecular organization has been investigated after reconstitution in a lipid bilayer environment, and shows a dimeric and possibly a tetrameric form, the precise quaternary structure in a detergent-solubilized state has never been addressed. In the present study, BmrA was purified from Escherichia coli membranes using an optimized purification protocol and different detergents. Furthermore, the ATPase activity of BmrA and the quantity of bound lipids and detergent were determined, and the oligomeric state was analysed using SEC (size-exclusion chromatography) and analytical ultracentrifugation. The activity and the quaternary structure of BmrA appeared to be strongly influenced by the type and concentration of the detergent used. SEC data showed that BmrA could be purified in a functional form in 0.05 and 0.01% DDM (n-dodecyl-beta-D-maltoside) and was homogeneous and monodisperse with an R(s) (Stokes radius) of 5.6 nm that is compatible with a dimer structure. Sedimentation-velocity and equilibrium experiments unequivocally supported that BmrA purified in DDM is a dimer and excluded the presence of other oligomeric states. These observations, which are discussed in relation to results obtained in proteoliposomes, also constitute an important first step towards crystallographic studies of BmrA structure.

To evaluate the role of hydrophobic and electrostatic or other polar interactions for protein-ligand binding, we studied the interaction of human serum albumin (HSA) and beta-lactoglobulin with various aliphatic (C10-C14) cationic and zwitterionic detergents. We find that cationic detergents, at levels that do not cause unfolding, interact with a single site on beta-lactoglobulin and with two primary and five to six secondary sites on HSA with an affinity that is approximately the same as that with which zwitterionic (dimethylamineoxide) detergents interact, suggesting the absence of significant electrostatic interactions in the high-affinity binding of these compounds. The binding affinity for all of the groups of compounds was dependent upon hydrocarbon chain length, suggesting the predominant role of hydrophobic forces, supported by polar interactions at the protein surface. A distinct correlation between the binding energy and the propensity for micelle formation within the group of cationic or noncharged (nonionic and zwitterionic) detergents indicated that the critical micellar concentration (CMC) for each of these detergent groups, rather than the absolute length of the hydrocarbon chain, can be used to compare their hydrophobicities during their interaction with protein. Intrinsic fluorescence data suggest that the two primary binding sites on serum albumin for the zwitterionic and cationic compounds are located in the C-terminal part of the albumin molecule, possibly in the Sudlow II binding region. Comparisons with previous binding data on anionic amphiphiles emphasize the important contribution of ion bond formation and other polar interactions in the binding of fatty acids and dodecyl sulfate (SDS) by HSA but not by beta-lactoglobulin. Electrostatic interactions by cationic detergents played a significant role in destabilizing the protein structure at high binding levels, with beta-lactoglobulin being more susceptible to unfolding than HSA. Zwitterionic detergents, in contrast to the cationic detergents, had no tendency to unfold the proteins at high concentrations.

Human plasma paraoxonase (HuPON1) is a high density lipoprotein (HDL)-bound enzyme exhibiting antiatherogenic properties. The molecular basis for the binding specificity of HuPON1 to HDL has not been established. Isolation of HuPON1 from HDL requires the use of detergents. We have determined the activity, dispersity, and oligomeric states of HuPON1 in solutions containing mild detergents using nondenaturing electrophoresis, size exclusion chromatography, and cross-linking. HuPON1 was active whatever its oligomeric state. In nonmicellar solutions, HuPON1 was polydisperse. In contrast, HuPON1 exhibited apparent homogeneity in micellar solutions, except with CHAPS. The enzyme apparent hydrodynamic radius varied with the type of detergent and protein concentration. In C(12)E(8) micellar solutions, from sedimentation velocity, equilibrium analytical ultracentrifugation, and radioactive detergent binding, HuPON1 was described as monomers and dimers in equilibrium. A decrease of the detergent concentration shifted this equilibrium toward the formation of dimers. About 100 detergent molecules were associated per monomer and dimer. The assembly of amphiphilic molecules, phospholipids in vivo, in sufficiently large aggregates could be a prerequisite for anchoring of HuPON1 and then allowing stabilization of the enzyme activity. Changes of HDL size and shape could strongly affect the binding affinity and stability of HuPON1 and result in reduced antioxidative capacity of the lipoprotein.

As an extension of our studies on the interaction of detergents with membranes and membrane proteins, we have investigated their binding to water-soluble proteins. Anionic aliphatic compounds (dodecanoate and dodecylsulfate) were bound to serum albumin with high affinity at nine sites; related nonionic detergents (C12E8 and dodecylmaltoside) were bound at seven to eight sites, many in common with those of dodecanoate. The compounds were also bound in the hydrophobic cavity of beta-lactoglobulin, but not to ovalbumin. In addition to the generally recognized role of the Sudlow binding region II of serum albumin (localized at the IIIA subdomain) in fatty acid binding, quenching of the fluorescence intensity of tryptophan-214 by 7,8-dibromododecylmaltoside and 12-bromododecanoate also implicate the Sudlow binding region I (subdomain IIA) as a locus for binding of aliphatic compounds. Our data document the usefulness of dodecyl amphipathic compounds as probes of hydrophobic cavities in water-soluble proteins. In conjunction with recent x-ray diffraction analyses of fatty acid binding as the starting point we propose a new symmetrical binding model for the location of nine high-affinity sites on serum albumin for aliphatic compounds.

Detergents are indispensable in the isolation of integral membrane proteins from biological membranes to study their intrinsic structural and functional properties. Solubilization involves a number of intermediary states that can be studied by a variety of physicochemical and kinetic methods; it usually starts by destabilization of the lipid component of the membranes, a process that is accompanied by a transition of detergent binding by the membrane from a noncooperative to a cooperative interaction already below the critical micellar concentration (CMC). This leads to the formation of membrane fragments of proteins and lipids with detergent-shielded edges. In the final stage of solubilization membrane proteins are present as protomers, with the membrane inserted sectors covered by detergent. We consider in detail the nature of this interaction and conclude that in general binding as a monolayer ring, rather than as a micelle, is the most probable mechanism. This mode of interaction is supported by neutron diffraction investigations on the disposition of detergent in 3-D crystals of membrane proteins. Finally, we briefly discuss the use of techniques such as analytical ultracentrifugation, size exclusion chromatography, and mass spectrometry relevant for the structural investigation of detergent solubilized membrane proteins.

Up to now, removal of sodium dodecyl sulfate (SDS) from proteins in terms of restoration of their activity was an unsolved problem. A general procedure using ceramic hydroxyapatite (HAP) chromatography was developed for the complete removal of SDS bound to soluble or membrane proteins. This procedure involves (i) the binding of the SDS-protein complexes onto the ceramic hydroxyapatite column, (ii) extensive washing of bound proteins with phosphate buffer containing a mild detergent to exchange SDS, (iii) elution of the retained protein by increasing the phosphate concentration. Using this approach, complete exchange of [35S]SDS into a nonionic detergent such as dodecyl maltoside was achieved with a 90-100% protein recovery. The efficiency of protein-bound SDS removal is very likely due to the combined effect of phosphate ions and the hydrophobic tail of nonionic detergent: acting together, they are able to displace SDS molecules from their protein-binding sites. The advantages of this HAP-mediated SDS removal method include high efficiency, rapidity, simplicity and general applicability to a wide variety of detergents and soluble or membrane proteins. Of utmost importance, SDS-treated P-glycoprotein, glutamate dehydrogenase, and lysozyme fully recovered their enzymatic activities after HAP chromatography, including lysozyme electroeluted from SDS-polyacrylamide gel electrophoresis. This demonstrates that reactivation of SDS-treated protein can be achieved, provided that SDS is completely removed under mild conditions.

In order to study protein-detergent short-range interactions, we analyzed the quenching by brominated detergents of reticulum sarcoplasmic (SR) Ca(2+)-ATPase intrinsic fluorescence. For this purpose, 7,8-dibromododecyl beta-maltoside and 2-O-(10,11-dibromoundecanoyl)sucrose, brominated analogs of two non-ionic detergents, the frequently used dodecylmaltoside and the newly synthesized 2-O-lauroylsucrose respectively, were prepared. Rayleigh scattering measurements showed that the brominated detergents efficiently and rapidly solubilized SR vesicles like their non-brominated analogs although at slightly higher concentrations. Similarly, each analog had a slightly higher critical micellar concentration than its parent detergent. The partition coefficient K (expressed as the ratio of the molar fraction of detergent in the SR lipid phase to that in the aqueous phase, at pH 7.5 and 20 degrees C) was similar for brominated and non-brominated dodecyl maltoside (3.5-4 x 10(5)) and slightly lower for dibromoundecanoylsucrose (approximately 10(5)) than for lauroylsucrose (approximately 2 x 10(5)). At detergent concentrations too low to solubilize the membrane, the brominated detergents rapidly inserted (within seconds) into SR vesicles. In this concentration range, Ca(2+)-ATPase fluorescence quenching steadily increased with detergent concentration. When the membrane was saturated with detergent, the residual fluorescence was about half of its initial value, indicating significant protein-detergent, contacts, possibly due to a slightly higher affinity of Ca(2+)-ATPase for these detergents than for phospholipids. For higher detergent concentrations, solubilizing the membrane, the fluorescence continued to decrease with detergent concentration, with no evidence for a dramatic change in the average hydrophobic environment of the protein during the transition from bilayers to a soluble state. For still higher detergent concentrations, above that necessary for membrane solubilization, the fluorescence was further quenched to a residual relative value of about 20%, corresponding to further delipidation of the protein surface, in agreement with previous results [de Foresta, B., le Maire, M., Orlowski, S., Champeil, P., Lund, S., Møller, J.V., Michelangeli, F. & Lee, A.G. (1989) Biochemistry 28, 2558-2567]. Fluorescence quenching for solubilized Ca(2+)-ATPase was quickly reversed upon addition of excess non-brominated detergent. The effects of the four detergents on the Ca(2+)-ATPase hydrolysis of p-nitrophenyl phosphate were similar and correlated with the protein-detergent contacts evidenced above. In conclusion, both these brominated detergents appear to be promising tools to study protein-detergent interactions at the hydrophobic surface of a membrane protein, either in a membrane or in solubilized complexes.

Nonionic polyoxyethylene type detergents (CxEy) are widely used to solubilize and purify membrane proteins. The detergent hydrophobic moiety (Cx) replaces phospholipids at exposed hydrophobic regions of the membrane proteins. During chromatography on an immobilized anti-Kell antibody to purify Kell protein (an integral erythrocyte protein), it was observed that the size of the polar head of an non ionic detergent added to the mobile phase appeared to influence the interaction of the detergent-protein complex with the immobilized antibody. Further studies were performed using another erythrocyte membrane protein, Glycophorin C and three anti-GPC monoclonal antibodies directed against three epitopes of the extracytoplasmic domain of the protein. The interaction of GPC with the three Protein A-coupled monoclonal antibodies was studied in the presence of three detergents C12E<9>, C13E<15> and C12E<23>. It was observed in batch mode and in column chromatography experiments that the adsorption of GPC to the immunoaffinity supports decreased as the size of the detergent polar head increased. Thus, the polyoxyethylene chain of a detergent might prevent the interaction of the detergent-protein complex with the immobilized antibody.

The mode by which four nonionic detergents interact with integral membrane proteins was studied by relating detergent binding to characteristic detergent monolayer and micellar dimensions and to the surface area of the hydrophobic sector of the proteins. The detergents used (C12E8, Triton X-100, dodecyl-beta-D-maltoside, and dodecyldimethylamineoxide) are efficient membrane solubilizers, which, according to our hydrodynamic analysis, form small approximately spherical micelles. Their binding by protomeric forms of bacteriorhodopsin, photosynthetic reaction center, sarcoplasmic reticulum Ca(2+)-ATPase, and cytochrome oxidase was measured after equilibration and delipidation, both by the use of successive chromatographies on silica gel and the use of agarose gel columns in combination with DEAE-cellulose chromatography. It was found that, despite detergent binding by silica gel, both chromatographic procedures gave equivalent and consistent results. The data obtained show systematic trends; thus dodecyldimethylamineoxide exhibited the highest binding levels, followed by dodecylmaltoside, whereas the polyethylene glycol detergents C12E8 and Triton X-100 had the lowest degree of binding. This ranking order is inversely related to the cross-sectional areas of these detergents in monolayers at an air-water interface and in micelles. Binding was reduced by aggregation of protomers, whereas the presence of strongly bound residual lipid slightly increased it. Among the membrane proteins with a high molecular mass (reaction center, Ca(2+)-ATPase, and cytochrome oxidase), relative binding could be related to the size of their hydrophobic sectors. On the other hand, bacteriorhodopsin bound somewhat more detergent per transmembrane helix, probably because of less steric hindrance, caused by the absence of a sizable hydrophilic domain in this protein. Theoretical calculations indicated monolayer binding of detergent to the hydrophobic surface of membrane proteins to be a better model for interaction with membrane proteins than binding of the detergents in micellar form.

Membrane solubilization by dodecyl maltoside was studied, using Ca(2+)-ATPase membranes and liposome preparations as prototypes of biological membranes. In equilibrium dialysis experiments, transition from saturable incorporation of monomeric detergent into the membrane to cooperative binding already occurred at a free detergent concentration about 50% of the cmc. This transition was discontinuous for unilamellar liposomes of dioleoylphosphatidylcholine, but gradual for Ca(2+)-ATPase membranes and multilayered liposomes of sarcoplasmic reticulum lipid. Equilibrium detergent binding by Ca(2+)-ATPase membranes (expressed on the basis of lipid content) was the same as for detergent binding by multilamellar liposomes of sarcoplasmic reticulum lipid. Equilibration involving cooperative binding was considerably delayed (for many days) if detergent was presented gradually to the membranous preparations in nonmicellar form by diffusion across the dialysis membrane, while equilibration of detergent occurred rapidly if detergent in micellar form was added directly to the membrane preparations. In contrast, equilibration was rapid in both directions if detergent was added at levels below that required to initiate cooperative binding. Detergent interaction resulted in a biphasic decrease in light scattering of Ca(2+)-ATPase membranes. The first of these decreases coincided with the onset of cooperative binding, while the second one was associated with a decreased sedimentability during ultracentrifugation, i.e., with usual criteria of solubilization. The concentration at which this occurred corresponded to the level of free detergent at which lipid, after detergent solubilization, segregated from detergent after gel chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)

The kinetic aspects of amphiphile interaction with intact membranes (unilamellar and multilamellar liposomes, sarcoplasmic reticulum vesicles) were studied, with the nonionic detergent octa(ethylene glycol) dodecyl monoether (C12E8) as a prototype. C12E8 was bound to these membranes noncooperatively and with a maximum of 0.6-0.8 mol per mole of phospholipid, before the onset of solubilization. Binding was not affected by ultrasonication to expose internal binding sites on the inner leaflet. All detergent could be removed from the membranes by treatment with hydrophobic beads. Furthermore, bound detergent, also from the inside of multilayered liposomes, comprising 10-20 bilayers, was quickly released by dilution of the membranes, followed by gel filtration. The time course of these processes was investigated with a rapid-filtration apparatus, using glass fiber filters to deposit membrane material. Both detergent binding and removal could be described by a monoexponential process with a half-time of approximately 350 ms for all types of membranes. Binding of detergent enhanced the intrinsic fluorescence of sarcoplasmic reticulum vesicles. This occurred in less than 100 ms, probably as the result of direct interaction of C12E8 with Ca2+-ATPase at a few binding sites. The data show that flip-flop of C12E8 across lipid membranes is a rapid process that cannot account for incomplete detergent removal in reconstitution experiments [Ueno, M., Tanford, C., & Reynolds, J. A. (1984) Biochemistry 23, 3070-3076]. It is also suggested that other nonionized amphiphiles, including those with an anesthetic action, rapidly gain access to membrane proteins on the inside of the cell, even when used at low, clinical doses.

The properties of detergents required to substitute the lipid environment of sarcoplasmic reticulum Ca2+-ATPase with retention of good functional properties were determined by the use of a large number of diverse detergents and delipidated enzyme. Detergents having an intermediate chain length (approximately equal to C12) and a polyoxyethylene glycol or carbohydrate polar group were optimal for Ca2+-ATPase function and stabilization, while detergents with short alkyl chain (C8) or bulky head groups and many zwitterionic detergents led to rapid inactivation. Under optimal conditions (including solubilization in the E1 state), stability of delipidated Ca2+-ATPase approximated that obtained by solubilization of Ca2+-ATPase with a layer of bound lipid. Some detergents (in particular long chain members of the Tween family) were characterized by an inadequate interaction with delipidated Ca2+-ATPase, resulting in biphasic inactivation. According to analytical ultracentrifugation and high performance liquid chromatography experiments, the rapid and slow components of biphasic inactivation were due to the formation of monomeric and oligomeric Ca2+-ATPase, respectively. It is concluded that both hydrophobic and polar interactions are important for the detergent effect and that solubilizing detergents of intermediate and short chain length may be bound as a monolayer, differently than the membrane lipid. Long chain detergents cause protein aggregation and, despite their resemblance to natural lipids, are inferior in their activity-retaining properties. The previous use of such detergents to prepare oligomeric Ca2+-ATPase with long term retention of activity (cf. Møller, J. V., Anderson, J. P., and le Maire, M. (1988) Methods Enzymol. 157, 261-270) is shown to depend on the presence of residual lipid in these preparations.

The solubilization and delipidation of sarcoplasmic reticulum Ca2+-ATPase by different nonionic detergents were measured from changes in turbidity and recovery of intrinsic fluorescence of reconstituted ATPase in which tryptophan residues had been quenched by replacement of endogenous phospholipids with brominated phospholipids. It was found that incorporation of C12E8 or dodecyl maltoside (DM) at low concentrations in the membrane, resulting in membrane "perturbation" without solubilization, displaced a few of the phospholipids in contact with the protein; perturbation was evidenced by a parallel drop in ATPase activity. As a result of further detergent addition leading to solubilization, the tendency toward delipidation of the immediate environment of the protein was stopped, and recovery of enzyme activity was observed, suggesting reorganization of phospholipid and detergent molecules in the solubilized ternary complex, as compared to the perturbed membrane. After further additions of C12E8 or DM to the already solubilized membrane, the protein again experienced progressive delipidation which was only completed at a detergent concentration about 100-fold higher than that necessary for solubilization. Delipidation was correlated with a decrease in enzyme activity toward a level similar to that observed during perturbation. On the other hand, Tween 80, Tween 20, and Lubrol WX failed to solubilize SR membranes and to induce further ATPase delipidation when added after preliminary SR solubilization by C12E8 or dodecyl maltoside. For Tween 80, this can be related to an inability to solubilize pure lipid membrane; in contrast, Tween 20 and Lubrol WX were able to solubilize liposomes but not efficiently to solubilize SR membranes. In all three cases, insertion of the detergent in SR membranes is, however, demonstrated by perturbation of enzyme activity. Correlation between detergent structure and ability to solubilize and delipidate the ATPase suggests that one parameter impeding ATPase solubilization might be the presence of a bulky detergent polar headgroup, which could not fit close to the protein surface. We also conclude that in the active protein/detergent/lipid ternary complexes, solubilized by C12E8 or dodecyl maltoside, most phospholipids remain closely associated with the ATPase hydrophobic surface as in the membranous form. Binding of only a few detergent molecules on this hydrophobic surface may be sufficient for inhibition of ATPase activity observed at high ATP concentration, both during perturbation and in the completely delipidated, solubilized protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Binding of dodecyloctaethyleneglycol monoether (C12E3) and purified Triton X-100 to various integral membrane proteins was studied by chromatographic procedures. Binding capacity decreased in the following order: bovine rhodopsin greater than photochemical reaction center greater than sarcoplasmic reticulum Ca2+-ATPase. The detergents were bound in different amounts to the proteins and less than corresponding to the aggregation number of the pure micelles. Appreciable binding of C12E8 to Ca2+-ATPase was observed far below the critical micelle concentration, consistent with interaction of the membrane protein with non-micellar detergent. Model calculations indicate that the detergents cannot combine with the membrane proteins, forming an oblate ring similar to that of pure detergent micelles, such as has been previously proposed for e.g. cytochrome b5 [Robinson and Tanford (1975) Biochemistry, 14, 365-378]. Other arrangements (prolate and monolayer rings), in which all detergent molecules are in contact with the protein, are considered as alternatives for covering the hydrophobic surface of the membrane protein with a continuous layer of detergent.

We previously characterized the structural features of the interaction of sarcoplasmic reticulum membranes with nonsolubilizing concentrations of C12E8, the non-ionic detergent octaethylene glycol monododecyl ether (Andersen, J.P., le Maire, M., Kragh-Hansen, V., Champeil, P., and Møller, J. V. (1983) Eur. J. Biochem. 134, 205-214). The present study characterizes especially the functional aspects and implications of the detergent-induced perturbation for an understanding of ATPase function. Perturbing detergent decreased Vmax, but left Ca2+ transport intact. Detergent incorporation affected neither the calcium-dependent phosphorylation from ATP, as judged from multimixer quenching experiments, nor the calcium-releasing transition between the two phosphoenzyme forms (Ca2E1P to E2P), as judged from kinetically resolved dual-wavelength measurements with the calcium-sensitive dye antipyrylazo III. However, the decrease in Vmax was accounted for by a decrease in the rate of enzyme dephosphorylation by a factor of 3-4, whereas the Ca2+-dependent transition between the nonphosphorylated enzyme forms (E2 to Ca2E1) was enhanced almost 10-fold. Evidence of a conformational change of E2 by C12E8 toward that of the E1 state to account for the perturbed reactions was obtained from experiments on vanadate reactivity and tryptic degradation pattern. Both direct and steady-state evidence was obtained for an acceleration by ATP of the Ca2E1P to E2P transition which may account for the low affinity modulatory effect of the nucleotide on enzyme turnover. The kinetic data indicated that reduction of ATP hydrolysis by C12E8 coincided with conditions where E2P dephosphorylation becomes rate-limiting (high ATP concentration, low pH, absence of potassium). Otherwise, the Ca2E1P to E2P transition is deduced to be a rate-limiting step for the ATPase cycle, whereas the potential for rate control of the cycle by modulation of the E2 to Ca2E1 transition is very small. Only in special circumstances (absence of potassium, high temperature, and using ITP as a substrate) did this transition become a rate-limiting step, subject to rate enhancement of the whole cycle by detergent perturbation.