Two-Dimensional Detergent Expansion Strategy for Membrane Protein Studies

Detergents are the most frequently applied reagents in membrane protein (MP) studies. The limited diversity of one-head-one-tailed traditional detergents, however, is far from sufficient for structurally distinct MPs. Expansion of detergent repertoire has a continuous momentum. In line with the speculation that detergent pre-assembly exerts superiority, herein we report for the first time cross-conjugation of two series of monomeric detergents for constructing a two-dimensional library of dimeric detergents. Optimum detergents stood out with unique preferences in the systematic evaluation of individual MPs. Furthermore, unprecedented hybrid detergents 14M8G and 14M9G enabled high-quality EM study of transporter MsbA and NMR study of G protein-coupled receptor A_2AAR, respectively. Given the abundance of cross-coupling chemistries, comprehensive diversity could be readily covered that would facilitate the finding of new detergents for the manipulation of thorny MPs and innovation of the functional and structural study in future.     

Molecular models of lipopeptide detergents: large coiled-coils with hydrocarbon interiors

We have constructed molecular models of octameric micelles formed by a recently developed lipopeptide detergent consisting of a single amphipathic alpha-helix coupled to two acyl chains at either end of the helix. The models explain the experimentally observed aggregation behavior of peptides with different acyl chain lengths. The octameric micelles form a unique coiled-coil structure, with the acyl chains in a nearly frozen conformation inside the cylindrical assemblies. Two extreme models with helices either all parallel or in an alternating orientation suggest that the alternating orientation is energetically more favorable. The models suggest several new directions for further diversifying this new class of detergents for the structural studies of membrane proteins.     

An accurate method for determining the residual base catalyst in commercial nonionic detergents

Summary A new, sensitive method for determining the concentration of the residual base catalyst present in nonionic detergents is given. It is based on measuring the rate constant of the color fading of malachite green in the presence of the detergent reversed micelles.With 1 figure     

Micelles,Bicelles, and Nanodiscs: Comparing the Impact of Membrane Mimetics on Membrane Protein Backbone Dynamics

Detergents are often used to investigate the structure and dynamics of membrane proteins. Whereas the structural integrity seems to be preserved in detergents for many membrane proteins, their functional activity is frequently compromised, but can be restored in a lipid environment. Herein we show with per‐residue resolution that while OmpX forms a stable β‐barrel in DPC detergent micelles, DHPC/DMPC bicelles, and DMPC nanodiscs, the pico‐ to nanosecond and micro‐ to millisecond motions differ substantially between the detergent and lipid environment. In particular for the β‐strands, there is pronounced dynamic variability in the lipid environment, which appears to be suppressed in micelles. This unexpected complex and membrane‐mimetic‐dependent dynamic behavior indicates that the frequent loss of membrane protein activity in detergents might be related to reduced internal dynamics and that membrane protein activity correlates with lipid flexibility.     

Formation and dissociation processes of gas-phase detergent micelles

Growing interest in micelles to protect membrane complexes during the transition from solution to gas phase prompts a better understanding of their properties. We have used ion mobility mass spectrometry to separate and assign detergent clusters formed from the n-trimethylammonium bromide series of detergents. We show that cluster size is independent of detergent concentration in solution, increases with charge state, but surprisingly decreases with alkyl chain length. This relationship contradicts the thermodynamics of micelle formation in solution. However, the liquid drop model, which considers both the surface energy and charge, correlates extremely well with the experimental cluster size. To explore further the properties of gas-phase micelles, we have performed collision-induced dissociation on them during tandem mass spectrometry. We observed both sequential asymmetric charge separation and neutral evaporation from the precursor ion cluster. Interestingly, however, we also found markedly different dissociation pathways for the longer alkyl chain detergents, with significantly fewer intermediate ions formed than for those with a shorter alkyl chain. These experiments provide an essential foundation for understanding the process of the gas-phase analysis of membrane protein complexes. Moreover they imply valuable mechanistic details of the protection afforded to protein complexes by detergent clusters during gas-phase activation processes.     

Synthesis and properties of dodecyl trehaloside detergents for membrane protein studies

Sugar-based detergents, mostly derived from maltose or glucose, prevail in the extraction, solubilization, stabilization, and crystallization of membrane proteins. Inspired by the broad use of trehalose for protecting biological macromolecules and lipid bilayer structures, we synthesized new trehaloside detergents for potential applications in membrane protein research. We devised an efficient synthesis of four dodecyl trehalosides, each with the 12-carbon alkyl chain attached to different hydroxyl groups of trehalose, thus presenting a structurally diverse but related family of detergents. The detergent physical properties, including solubility, hydrophobicity, critical micelle concentration (CMC), and size of micelles, were evaluated and compared with the most popular maltoside analogue, β-d-dodecyl maltoside (DDM), which varied from each other due to distinct molecular geometries and possible polar group interactions in resulting micelles. Crystals of 2-dodecyl trehaloside (2-DDTre) were also obtained in methanol, and the crystal packing revealed multiple H-bonded interactions among adjacent trehalose groups. The few trehaloside detergents were tested for the solubilization and stabilization of the nociceptin/orphanin FQ peptide receptor (ORL1) and MsbA, which belong to the G-protein coupled receptor (GPCR) and ATP-binding cassette transporter families, respectively. Our results demonstrated the utility of trehaloside detergents as membrane protein solubilization reagents with the optimal detergents being protein dependent. Continuing development and investigations of trehaloside detergents are attractive, given their interesting and unique chemical-physical properties and potential interactions with membrane lipids.     

Process of destruction of large unilamellar vesicles by a nonionic detergent,octylglucoside, and size growth factor in vesicle formation from phospholipid–detergent mixed micelles

The process of vesicle solubilization and size growth by detergents, especially by octylglucoside, was examined in detail in order to elucidate the phenomena observed in the vesicle-to-micelle transition and to clarify the size-determining factor of vesicles prepared by removing detergent from phospholipid–detergent mixed micelles. In the vesicle solubilization process, when the detergent concentration in the vesicle membrane reached a critical value, the collapse of large unilamellar vesicles (LUV) into small unilamellar vesicles (SUV) was observed. This newly appeared SUV were named SUV*. The SUV* could be produced by adding an appropriate amount of detergent to the SUV prepared by an ultrasonication method so as to increase the concentration to a little over the critical value, such as, in the case of adding octylglucoside, a molar ratio of 1.0–1.1 to phospholipid in the membrane phase. The SUV* containing octylglucoside were fusible and grow time-dependently, but those containing sodium cholate were not fusible. On the basis of the SUV* data, the following problems were solved: the variety of the size of the vesicles prepared by detergent removal from mixed micelles composed of a phospholipid and different detergents, or by different removal methods; the complex appearance of turbidity or vesicle size observed in vesicle destruction and formation; the conflict between LUV and SUV in the partition behavior of detergent and the size change with addition of detergent.     

Mesitylene‐Cored Glucoside Amphiphiles (MGAs) for Membrane Protein Studies: Importance of Alkyl Chain Density in Detergent Efficacy

Detergents serve as useful tools for membrane protein structural and functional studies. Their amphipathic nature allows detergents to associate with the hydrophobic regions of membrane proteins whilst maintaining the proteins in aqueous solution. However, widely used conventional detergents are limited in their ability to maintain the structural integrity of membrane proteins and thus there are major efforts underway to develop novel agents with improved properties. We prepared mesitylene‐cored glucoside amphiphiles (MGAs) with three alkyl chains and compared these agents with previously developed xylene‐linked maltoside agents (XMAs) with two alkyl chains and a conventional detergent (DDM). When these agents were evaluated for four membrane proteins including a G protein‐coupled receptor (GPCR), some agents such as MGA‐C13 and MGA‐C14 resulted in markedly enhanced stability of membrane proteins compared to both DDM and the XMAs. This favourable behaviour is due likely to the increased hydrophobic density provided by the extra alkyl chain. Thus, this study not only describes new glucoside agents with potential for membrane protein research, but also introduces a new detergent design principle for future development.     

Action of phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis is significantly influenced by coexisting lipids in substrate-detergent micelles.

The effects of phosphatidylcholine (PC), phosphatidylethanolamine (PE), sphingomyelin (SM), and cholesterol on the activity of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis were studied in detail in phosphatidylinositol (PI)/detergent mixed micelles. By addition of PC, the enzymatic hydrolysis of PI was significantly stimulated in PI/Triton X-100 as well as PI/sodium deoxycholate (SDC) mixed micelles. SM stimulated enzyme activity toward PI/Triton X-100 micelles at a lower molar ratio of SM to PI, but was rather inhibitory at a ratio higher than 2.0. The enzyme activity became significantly lower with an increase of PE or cholesterol in PI/Triton X-100 micelles. Actually, both PE and cholesterol were intensively inhibitory when added at a higher molar ratio to PI in Triton X-100-containing micelles. In the system of PI/SDC mixed micelles, not only PC but also SM, PE and cholesterol enhanced the enzymatic hydrolysis of PI. The difference between PI/Triton X-100 and PI/SDC micelles regarding the effects of these lipids on PI-PLC action, must be dependent on the physical state of micelles formed by these detergents and lipids.     

Glycosyl‐Substituted Dicarboxylates as Detergents for the Extraction,Overstabilization, and Crystallization of Membrane Proteins

To tackle the problems associated with membrane protein (MP) instability in detergent solutions, we designed a series of glycosyl‐substituted dicarboxylate detergents (DCODs) in which we optimized the polar head to clamp the membrane domain by including, on one side, two carboxyl groups that form salt bridges with basic residues abundant at the membrane–cytoplasm interface of MPs and, on the other side, a sugar to form hydrogen bonds. Upon extraction, the DCODs 8 b , 8 c , and 9 b preserved the ATPase function of BmrA, an ATP‐binding cassette pump, much more efficiently than reference or recently designed detergents. The DCODs 8 a , 8 b , 8 f , 9 a , and 9 b induced thermal shifts of 20 to 29 °C for BmrA and of 13 to 21 °C for the native version of the G‐protein‐coupled adenosine receptor A_2AR. Compounds 8 f and 8 g improved the diffraction resolution of BmrA crystals from 6 to 4 Å. DCODs are therefore considered to be promising and powerful tools for the structural biology of MPs.     

Glycosyl‐Substituted Dicarboxylates as Detergents for the Extraction,Overstabilization, and Crystallization of Membrane Proteins

To tackle the problems associated with membrane protein (MP) instability in detergent solutions, we designed a series of glycosyl‐substituted dicarboxylate detergents (DCODs) in which we optimized the polar head to clamp the membrane domain by including, on one side, two carboxyl groups that form salt bridges with basic residues abundant at the membrane–cytoplasm interface of MPs and, on the other side, a sugar to form hydrogen bonds. Upon extraction, the DCODs 8 b , 8 c , and 9 b preserved the ATPase function of BmrA, an ATP‐binding cassette pump, much more efficiently than reference or recently designed detergents. The DCODs 8 a , 8 b , 8 f , 9 a , and 9 b induced thermal shifts of 20 to 29 °C for BmrA and of 13 to 21 °C for the native version of the G‐protein‐coupled adenosine receptor A_2AR. Compounds 8 f and 8 g improved the diffraction resolution of BmrA crystals from 6 to 4 Å. DCODs are therefore considered to be promising and powerful tools for the structural biology of MPs.     

Self-Assembly Behavior and Application of Terphenyl-Cored Trimaltosides for Membrane-Protein Studies: Impact of Detergent Hydrophobic Group Geometry on Protein Stability

Amphipathic agents are widely used in various fields including biomedical sciences. Micelle-forming detergents are particularly useful for in vitro membrane-protein characterization. As many conventional detergents are limited in their ability to stabilize membrane proteins, it is necessary to develop novel detergents to facilitate membrane-protein research. In the current study, we developed novel trimaltoside detergents with an alkyl pendant-bearing terphenyl unit as a hydrophobic group, designated terphenyl-cored maltosides (TPMs). We found that the geometry of the detergent hydrophobic group substantially impacts detergent self-assembly behavior, as well as detergent efficacy for membrane-protein stabilization. TPM-Vs, with a bent terphenyl group, were superior to the linear counterparts (TPM-Ls) at stabilizing multiple membrane proteins. The favorable protein stabilization efficacy of these bent TPMs is likely associated with a binding mode with membrane proteins distinct from conventional detergents and facial amphiphiles. When compared to n-dodecyl-β-d -maltoside (DDM), most TPMs were superior or comparable to this gold standard detergent at stabilizing membrane proteins. Notably, TPM-L3 was particularly effective at stabilizing the human β₂ adrenergic receptor (β₂AR), a G-protein coupled receptor, and its complex with G_s protein. Thus, the current study not only provides novel detergent tools that are useful for membrane-protein study, but also suggests a critical role for detergent hydrophobic group geometry in governing detergent efficacy.     

A Fluorinated Detergent for Membrane‐Protein Applications

Surfactants carrying fluorocarbon chains hold great promise as gentle alternatives to conventional hydrocarbon‐based detergents for the solubilization and handling of integral membrane proteins. However, their inertness towards lipid bilayer membranes has limited the usefulness of fluorinated surfactants in situations where detergent‐like activity is required. We demonstrate that fluorination does not necessarily preclude detergency, as exemplified by a fluorinated octyl maltoside derivative termed F₆OM. This nonionic compound readily interacts with and completely solubilizes phospholipid vesicles in a manner reminiscent of conventional detergents without, however, compromising membrane order at subsolubilizing concentrations. Owing to this mild and unusual mode of detergency, F₆OM outperforms a lipophobic fluorinated surfactant in chaperoning the functional refolding of an integral membrane enzyme by promoting bilayer insertion in the absence of micelles.     

STUDIES OF SODIUM TAUROCHOLATE MICELLES USING FLUORESCENT PROBE MOLECULES

Abstract Micelles formed by sodium taurocholate (NaTC) and mixed micelles formed by NaTC with detergents (SDS, reduced Triton X-100 and CTAC) were studied with fluorescent probes. Pyrene was used as an indicator of the polarity of the micellar binding site by comparison of the fluorescence spectra and vibronic band intensity ratios of pyrene in the different systems. Perylene was used as a fluorescence polarization probe to study the rigidity of the NaTC and mixed micelles. The fluorescence lifetime of perylene in the different systems was also measured. Results of the studies were compared with measurements of the probes in cyclohexane, ethanol and aqueous beta-cyclodextrin. Perylene was found to be more rigidly bound in the NaTC micelles than in the detergent micelles. Insertion of small amounts of reduced Triton X-100 into the NaTC micelles appears to increase the rigidity. The binding sites of NaTC and CTAC have similar polarities, and are more polar than those of SDS and reduced Triton X-100. Insertion of any of the detergents into the NaTC micelle decreases the polarity of the binding site, possibly by reducing the penetration of water into the micelle.     

A new azobenzene-based design strategy for detergents in membrane protein research

Mass spectrometry enables the in-depth structural elucidation of membrane protein complexes, which is of great interest in structural biology and drug discovery. Recent breakthroughs in this field revealed the need for design rules that allow fine-tuning the properties of detergents in solution and gas phase. Desirable features include protein charge reduction, because it helps to preserve native features of protein complexes during transfer from solution into the vacuum of a mass spectrometer. Addressing this challenge, we here present the first systematic gas-phase study of azobenzene detergents. The utility of gas-phase techniques for monitoring light-driven changes of isomer ratios and molecular properties are investigated in detail. This leads to the first azobenzene detergent that enables the native mass spectrometry analysis of membrane proteins and whose charge-reducing properties can be tuned by irradiation with light. More broadly, the presented work outlines new avenues for the high-throughput characterization of supramolecular systems and opens a new design strategy for detergents in membrane protein research.

Here, L. H. Urner and co-workers identify a new detergent design strategy for the non-denaturing structural analysis of membrane proteins by studying the gas-phase properties of azobenzene-based oligoglycerol detergents.     

Functional Detergents Containing an Imidazole Ring and Typical Fragments of α-Nucleophiles Underlying Micellar Systems for Cleavage of Esters of Prosphorus Acids

1-Cetyl-3-(2-oximinopropyl)imidazolium, 1-cetyl-3-(2-oxaminoethyl-2-one)imidazolium, and 1-cetyl-3-(2-amino-2-oximinoethyl)imidazolium halides were synthesized.These compounds form in water solutions functional zwitter-ionic micelles as surfactants. The cleavage kinetics of 4-nitrophenyl diethyl phosphate, 4-nitrophenyl ethyl ethylphosphonate, and 4-nitrophenyl tosylate in micelles of the functional detergents and combined micelles of the functional detergents with cetyltrimethylammonium chloride are adequately described in a framework of a simple pseudophase distribution model, and the micellar systems of the detergents are typical -nucleophilic reagents. An equation was suggested for quantitative estimation of the micellar effect of the surfactants that took into account the change in the nucleophilic and basic characteristics of the -nucleophilic center of the detergent and also the influence of the reagents concentrating on transition of the reaction from the water into micelle phase. The maximum acceleration of the S N 2-reaction in the micelles of the functional zwitter-ionic detergents for the cleavage of 4-nitrophenyl diethyl phosphate and 4-nitrophenyl tosylate reached 3500 and 75 000 (oximate surfactant), 3300 and 66 000 (amidoximate surfactant ), and 4800 and 12200 (hydroxamate surfactant) times respectively. New functional detergents underlie unique supernucleophilic micellar system affording extremely high cleavage rates of organophosphorus substrates-ecotoxicants.     

Comparative investigations of the molecular properties of detergents and protein–detergent complexes

Investigations of monomeric and micellar detergents, protein–detergent complexes, as well as native and denatured proteins by means of various physicochemical techniques yield a wide range of molecular characteristics of the components under analysis. Varying the experimental conditions (e.g., the concentration of solutes or the ionic strength of the medium) allows the mass, gross structure, and structural details of the macromolecular components to be determined. However, several modifications of the conventional techniques and evaluation procedures have to be applied in order to analyze multicomponent systems consisting of several low-molecular, micellar, and macromolecular components in an appropriate way. In the case of weakly absorbing detergents, labeling of the detergent micelles by specific dyes is required. Evidently, impurities and lack of homogeneity of many detergents may severely disturb the precise evaluation of the experiments; both necessitate a series of precautions in order to avoid misinterpretations. Analytical ultracentrifugation, size-exclusion chromatography, together with viscometry and densimetry, yield molar masses, mass distributions, and the overall structure of micellar and macromolecular molecules. In contrast, spectroscopic methods (UV absorption, fluorescence emission and excitation, far- and near-UV circular dichroism) monitor only local details of detergent-induced changes in the environment of aromatic residues. The technique of sodium dodecyl sulfate–polyacrylamide gel electrophoresis is routinely applied in biochemical work in order to establish molar masses of simple and conjugated proteins. To study the binding behavior of detergents to proteins in quantitative terms, however, techniques (e.g., equilibrium centrifugation, electrophoresis and chromatography) involving detergent concentrations have to be used. Received: 9 August 1999/Accepted: 19 October 1999     

Infrared Multiphoton Dissociation Enables Top-Down Characterization of Membrane Protein Complexes and G Protein-Coupled Receptors

Membrane proteins are challenging to analyze by native mass spectrometry (MS) as their hydrophobic nature typically requires stabilization in detergent micelles that are removed prior to analysis via collisional activation. There is however a practical limit to the amount of energy which can be applied, which often precludes subsequent characterization by top-down MS. To overcome this barrier, we have applied a modified Orbitrap Eclipse Tribrid mass spectrometer coupled to an infrared laser within a high-pressure linear ion trap. We show how tuning the intensity and time of incident photons enables liberation of membrane proteins from detergent micelles. Specifically, we relate the ease of micelle removal to the infrared absorption of detergents in both condensed and gas phases. Top-down MS via infrared multiphoton dissociation (IRMPD), results in good sequence coverage enabling unambiguous identification of membrane proteins and their complexes. By contrasting and comparing the fragmentation patterns of the ammonia channel with two class A GPCRs, we identify successive cleavage of adjacent amino acids within transmembrane domains. Using gas-phase molecular dynamics simulations, we show that areas prone to fragmentation maintain aspects of protein structure at increasing temperatures. Altogether, we propose a rationale to explain why and where in the protein fragment ions are generated.     

OmpT在Tween-20胶束中折叠的单分子研究

The cell envelope of gram-negative bacteria consists of the outer membrane (OM), inner membrane (IM), and periplasm. The β-barrel outer membrane proteins (OMPs) embedded in the OM perform diverse and significant functions such as signaling, transporting, and proteolysis. The OMPs of gram-negative bacteria share similar folding pathways with that of mitochondria and chloroplasts. Therefore, the study of the OMP folding mechanism not only provides insights into antimicrobial drug design but also helps elucidate mitochondrial and chloroplast biogenesis. Most knowledge about OMP folding was obtained from ensemble experiments where OMPs were usually at micromolar concentrations and prone to aggregate, which is different from the physiological environment in the cells. Unlike ensemble techniques, single-molecule detection (SMD) can measure OMPs from nano- to picomolar concentrations and prevent aggregation. In this work, we investigated the folding of OmpT, one of the OMPs, in Tween-20 and n-dodecyl β-d-maltopyranoside (DDM) micelles by SMD. We prepared monodisperse OmpT and observed both unfolded and folded OmpT in Tween-20 and DDM micelles under different urea concentrations by single-molecule fluorescence resonance energy transfer (FRET). The folded OmpT in Tween-20 is structurally similar to the native OmpT folded in DDM but exhibits weaker resistance to urea. In contrast, OmpA barely folds and OmpC hardly folds in Tween-20 micelles. We confirmed that folded OmpT forms complexes with detergent micelles and estimated the number of bound Tween-20 and DDM molecules per OmpT by fluorescence correlation spectroscopy. We compared the enzymatic activity of OmpT folded in two detergents with a fluorescent peptide as substrate, and found that the folded form of OmpT in Tween-20 possesses weaker enzymatic activity than that in DDM. We also investigated the folding properties of OmpT, OmpA, and OmpC in the presence of the β-barrel assembly machine (BAM) complex. OmpT folds efficiently in liposome even without the BAM complex; OmpA only folds with the help of the BAM complex; and OmpC does not fold with or without the BAM complex. Based on the comparison of the folding of OmpT, OmpA, and OmpC in detergent micelles and in the presence of the BAM complex, we propose that OmpT has stronger folding tendency than OmpA and OmpC, which supports the idea that the exact role of the BAM complex is dependent on the distinct folding properties of individual OMPs. Since Tween-20 is a widely used reagent to block nonspecific adsorption in SMD experiments, our results also remind people to exercise caution to prevent possible wrong interpretations caused by the interaction between proteins and Tween-20.     

Development of 1,3-acetonedicarboxylate-derived glucoside amphiphiles (ACAs) for membrane protein study

Detergents are extensively used for membrane protein manipulation. Membrane proteins solubilized in conventional detergents are prone to denaturation and aggregation, rendering downstream characterization of these bio-macromolecules difficult. Although many amphiphiles have been developed to overcome the limited efficacy of conventional detergents for protein stabilization, only a handful of novel detergents have so far proved useful for membrane protein structural studies. Here, we introduce 1,3-acetonedicarboxylate-derived amphiphiles (ACAs) containing three glucose units and two alkyl chains as head and tail groups, respectively. The ACAs incorporate two different patterns of alkyl chain attachment to the core detergent unit, generating two sets of amphiphiles: ACA-As (asymmetrically alkylated) and ACA-Ss (symmetrically alkylated). The difference in the attachment pattern of the detergent alkyl chains resulted in minor variation in detergent properties such as micelle size, critical micelle concentration, and detergent behaviors toward membrane protein extraction and stabilization. In contrast, the impact of the detergent alkyl chain length on protein stability was marked. The two C11 variants (ACA-AC11 and ACA-SC11) were most effective at stabilizing the tested membrane proteins. The current study not only introduces new glucosides as tools for membrane protein study, but also provides detergent structure–property relationships important for future design of novel amphiphiles.

Newly developed amphiphiles, designated ACAs, are not only efficient at extracting G protein-coupled receptors from the membranes, but also conferred enhanced stability to the receptors compared to the gold standards (DDM and LMNG).