Protein-detergent Complex Research Articles

Simulation and modelling of the detergent corona around the membrane protein MhsT based on SAXS data

For membrane proteins, ab initio modelling based on a single curve of small-angle X-ray scattering (SAXS) is precluded by the presence of detergent molecules bound to the hydrophobic region of the protein. MEMPROT was developed for the modelling of protein–detergent complexes based on the SAXS curve of the complex, and on an a priori representation of the detergent corona by an elliptical semi-torus surrounding the protein. In previous studies, MEMPROT has succeeded in modelling several membrane proteins solubilized in n-dodecyl-β-maltopyranoside (DDM). However, it has never been tested on proteins solubilized in other detergents. To understand whether the geometrical shape currently parametrized in MEMPROT could be applied to a broader catalogue of protein–detergent complexes, here, MEMPROT was used to model the detergent corona around the multi-hydrophobic substrate transporter from Bacillus halodurans solubilized in four different detergents, namely DDM, n-decyl-β-maltopyranoside (DM), 4-cyclohexyl-1-butyl-β-D-maltoside (Cymal4) and decyl-maltose-neopentyl-glycol (DMNG). The study indicates a significant variation in detergent shapes, depending on the type of detergent. The modelling results suggest that the elliptical semi-torus with a circular closure is an excellent approximation for long-tailed detergents (DDM and DM) but leads to a slightly poorer agreement with the data for DMNG and Cymal4, which have a shorter hydrophobic tail, smaller than the half-width of the protein hydrophobic region. Here, for the latter, it is hypothesized that a corona with a flatter closure would be a better shape descriptor.

Towards understanding the crystallization of photosystem II: influence of poly(ethylene glycol) of various molecular sizes on the micelle formation of alkyl maltosides.

The influence of poly(ethylene glycol) (PEG) polymers H-(O-CH2-CH2)p-OH with different average molecular sizes on the micelle formation of n-alkyl-β-D-maltoside detergents with the number of carbon atoms in the alkyl chain ranging from to is investigated with the aim to learn more about the detergent behavior under conditions suitable for the crystallization of the photosynthetic pigment-protein complex photosystem II. PEG is shown to increase the critical micelle concentration (CMC) of all three detergents in the crystallization buffer in a way that the free energy of micelle formation increases linearly with the concentration of oxyethylene units (O-CH2-CH2) irrespective of the actual molecular weight of the polymer. The CMC shift is modeled by assuming for simplicity that it is dominated by the interaction between PEG and detergent monomers and is interpreted in terms of an increase of the transfer free energy of a methylene group of the alkyl chain by 0.2kJmol-1 per 1mol L-1 increase of the concentration of oxyethylene units at 298K. Implications of this effect for the solubilization and crystallization of protein-detergent complexes as well as detergent extraction from crystals are discussed.

Advancing Kir4.2 Channel Ligand Identification through Collision-Induced Affinity Selection Mass Spectrometry.

The inwardly rectifying potassium Kir4.2 channel plays a crucial role in regulating membrane potentials and maintaining potassium homeostasis. Kir4.2 has been implicated in various physiological processes, including insulin secretion, gastric acid regulation, and the pathogenesis of central nervous system diseases. Despite its significance, the number of identified ligands for Kir4.2 remains limited. In this study, we established a method to directly observe ligands avoiding a requirement to observe the high-mass ligand-membrane protein-detergent complexes. This method used collision-induced affinity selection mass spectrometry (CIAS-MS) to identify ligands dissociated from the Kir4.2 channel-detergent complex. The CIAS-MS approach integrated all stages of affinity selection within the mass spectrometer, offering advantages in terms of time efficiency and cost-effectiveness. Additionally, we explored the effect of collisional voltage ramps on the dissociation behavior of the ligand and the ligand at different concentrations, demonstrating dose dependency.

Melamine-cored glucosides for membrane protein solubilization and stabilization: importance of water-mediated intermolecular hydrogen bonding in detergent performance.

Membrane proteins play essential roles in a number of biological processes, and their structures are important in elucidating such processes at the molecular level and also for rational drug design and development. Membrane protein structure determination is notoriously challenging compared to that of soluble proteins, due largely to the inherent instability of their structures in non-lipid environments. Micelles formed by conventional detergents have been widely used for membrane protein manipulation, but they are suboptimal for long-term stability of membrane proteins, making downstream characterization difficult. Hence, there is an unmet need for the development of new amphipathic agents with enhanced efficacy for membrane protein stabilization. In this study, we designed and synthesized a set of glucoside amphiphiles with a melamine core, denoted melamine-cored glucosides (MGs). When evaluated with four membrane proteins (two transporters and two G protein-coupled receptors), MG-C11 conferred notably enhanced stability compared to the commonly used detergents, DDM and LMNG. These promising findings are mainly attributed to a unique feature of the MGs, i.e., the ability to form dynamic water-mediated hydrogen-bond networks between detergent molecules, as supported by molecular dynamics simulations. Thus, MG-C11 is the first example of a non-peptide amphiphile capable of forming intermolecular hydrogen bonds within a protein-detergent complex environment. Detergent micelles formed via a hydrogen-bond network could represent the next generation of highly effective membrane-mimetic systems useful for membrane protein structural studies.

Dynamics of Apolipoprotein B-100 in Interaction with Detergent Probed by Incoherent Neutron Scattering.

Apolipoprotein B-100 (apo B-100) is the protein moiety of both low- and very-low-density lipoproteins, whose role is crucial to cholesterol and triglyceride transport. Aiming at the molecular dynamics' details of apo B-100, scarcely studied, we performed elastic and quasi-elastic incoherent neutron scattering (EINS, QENS) experiments combining different instruments and time scales. Similar to classical membrane proteins, the solubilization results in remaining detergent, here Nonidet P-40 (NP40). Therefore, we propose a framework for QENS studies of protein-detergent complexes, with the introduction of a combined model, including the experimental apo B-100/NP40 ratio. Relying on the simultaneous analysis of all QENS amplitudes, this approach is sensitive enough to separate both contributions. Its application identified two points: (i) apo B-100 slow dynamics and (ii) the acceleration of NP40 dynamics in the presence of apo B-100. Direct translation of the exposed methodology now makes the investigation of more membrane proteins by neutron spectroscopy achievable.

Probing Membrane Protein Assembly into Nanodiscs by In Situ Dynamic Light Scattering: A2A Receptor as a Case Study.

Simple SummaryLocated within the biological cell membranes, integral membrane proteins are responsible for a large variety of vital cellular processes. In humans, nearly a quarter of the genome codes integral membrane proteins, therefore malfunction of these proteins is associated with a variety of symptoms and diseases such as obesity, cancer and Parkinson’s disease. Clearly, knowledge of membrane proteins behaviour, in both structural and functional terms, is important not only in medicine but also in the design of better drugs with improved pharmaceutical properties. Nevertheless, much still remains unknown about these proteins, mainly because of the technical challenges associated with their production and stability in vitro once removed from their native lipidic environment. Recently, several membrane mimetic systems have been developed including nanodisc lipid particles. Nanodiscs are self-assembled lipidic structures that “trap” membrane proteins into a disc shaped phospholipid bilayer that is stabilised by a belt made of a protein know as membrane scaffold protein (MSP). Membrane proteins assembled into lipidic nanodiscs can maintain their structural and functional integrity and are compatible with most biophysical methods. Here we demonstrate the use of in situ dynamic light scattering as a high-throughput screening tool to assess the best conditions for nanodisc assembly and protein incorporation.Membrane proteins play a crucial role in cell physiology by participating in a variety of essential processes such as transport, signal transduction and cell communication. Hence, understanding their structure–function relationship is vital for the improvement of therapeutic treatments. Over the last decade, based on the development of detergents, amphipoles and styrene maleic-acid lipid particles (SMALPs), remarkable accomplishments have been made in the field of membrane protein structural biology. Nevertheless, there are still many drawbacks associated with protein–detergent complexes, depending on the protein in study or experimental application. Recently, newly developed membrane mimetic systems have become very popular for allowing a structural and functional characterisation of membrane proteins in vitro. The nanodisc technology is one such valuable tool, which provides a more native-like membrane environment than detergent micelles or liposomes. In addition, it is also compatible with many biophysical and biochemical methods. Here we describe the use of in situ dynamic light scattering to accurately and rapidly probe membrane proteins’ reconstitution into nanodiscs. The adenosine type 2A receptor (A2AR) was used as a case study.

Structural insights into the membrane receptor ShuA in DDM micelles and in a model of gram-negative bacteria outer membrane as seen by SAXS and MD simulations

Successful crystallization of membrane proteins in detergent micelles depends on key factors such as conformational stability of the protein in micellar assemblies, the protein-detergent complex (PDC) monodispersity and favorable protein crystal contacts by suitable shielding of the protein hydrophobic surface by the detergent belt. With the aim of studying the influence of amphiphilic environment on membrane protein structure, stability and crystallizability, we combine molecular dynamics (MD) simulations with SEC-MALLS and SEC-SAXS (Size Exclusion Chromatography in line with Multi Angle Laser Light Scattering or Small Angle X-ray Scattering) experiments to describe the protein-detergent interactions that could help to rationalize PDC crystallization. In this context, we compare the protein-detergent interactions of ShuA from Shigella dysenteriae in n-Dodecyl-β-D-Maltopyranoside (DDM) with ShuA inserted in a realistic model of gram-negative bacteria outer membrane (OM) containing a mixture of bacterial lipopolysaccharide and phospholipids. To evaluate the quality of the PDC models, we compute the corresponding SAXS curves from the MD trajectories and compare with the experimental ones. We show that computed SAXS curves obtained from the MD trajectories reproduce better the SAXS obtained from the SEC-SAXS experiments for ShuA surrounded by 268 DDM molecules. The MD results show that the DDM molecules form around ShuA a closed belt whose the hydrophobic thickness appears slightly smaller (~22 Å) than the hydrophobic transmembrane domain of the protein (24.6 Å) suggested by Orientations of Proteins in Membranes (OPM) database. The simulations also show that ShuA transmembrane domain is remarkably stable in all the systems except for the extracellular and periplasmic loops that exhibit larger movements due to specific molecular interactions with lipopolysaccharides (LPS). We finally point out that this detergent behavior may lead to the occlusion of the periplasmic hydrophilic surface and poor crystal contacts leading to difficulties in crystallization of ShuA in DDM.

Solution Structure of the Detergent-Photosystem II Core Complex Investigated by Small-Angle Scattering Techniques.

Albeit achieving the X-ray diffraction structure of dimeric photosystem II core complexes (dPSIIcc) at the atomic resolution, the nature of the detergent belt surrounding dPSIIcc remains ambiguous. Therefore, the solution structure of the whole detergent-protein complex of dPSIIcc of Thermosynechococcus elongatus (T. elongatus) solubilized in n-dodecyl-ß-d-maltoside (ßDM) was investigated by a combination of small-angle X-ray scattering (SAXS) and small-angle neutron scattering (SANS) with contrast variation. First, the structure of dPSIIcc was studied separately in SANS experiments using a contrast of 5% D2O. Guinier analysis reveals that the dPSIIcc solution is virtually free of aggregation in the studied concentration range of 2-10 mg/mL dPSIIcc, and characterized by a radius of gyration of 62 Å. A structure reconstitution shows that dPSIIcc in buffer solution widely retains the crystal structure reported by X-ray free electron laser studies at room temperature with a slight expansion of the entire protein. Additional SANS experiments on dPSIIcc samples in a buffer solution containing 75% D2O provide information about the size and shape of the whole detergent-dPSIIcc. The maximum position of P(r) function increases to 68 Å, i.e., it is about 6 Å larger than that of dPSIIcc only, thus indicating the presence of an additional structure. Thus, it can be concluded that dPSIIcc is surrounded by a monomolecular belt of detergent molecules under appropriate solubilization conditions. The homogeneity of the ßDM-dPSIIcc solutions was also verified using dynamic light scattering. Complementary SAXS experiments indicate the presence of unbound detergent micelles by a separate peak consistent with a spherical shape possessing a radius of about 40 Å. The latter structure also contributes to the SANS data but rather broadens the SANS curve artificially. Without the simultaneous inspection of SANS and SAXS data, this effect may lead to an apparent underestimation of the size of the PS II-detergent complex. The formation of larger unbound detergent aggregates in solution prior to crystallization may have a significant effect on the crystal formation or quality of the ßDM-dPSIIcc.

Promoting crystallization of intrinsic membrane proteins with conjugated micelles

A new technique for promoting nucleation and growth of membrane protein (MP) crystals from micellar environments is reported. It relies on the conjugation of micelles that sequester MPs in protein detergent complexes (PDCs). Conjugation via amphiphilic [metal:chelator] complexes presumably takes place at the micelle/water interface, thereby bringing the PDCs into proximity, promoting crystal nucleation and growth. We have successfully applied this approach to two light-driven proton pumps: bacteriorhodopsin (bR) and the recently discovered King Sejong 1–2 (KS1–2), using the amphiphilic 4,4′-dinonyl-2,2′-dipyridyl (Dinonyl) (0.7 mM) chelator in combination with Zn2+, Fe2+, or Ni2+ (0.1 mM). Crystal growth in the presence of the [metal–chelator] complexes leads to purple, hexagonal crystals (50–75 µm in size) of bR or pink, rectangular/square crystals (5–15 µm) of KS1–2. The effects of divalent cation identity and concentration, chelator structure and concentration, ionic strength and pH on crystal size, morphology and process kinetics, are described.

Pendant-bearing glucose-neopentyl glycol (P-GNG) amphiphiles for membrane protein manipulation: Importance of detergent pendant chain for protein stabilization

Glucoside detergents are successfully used for membrane protein crystallization mainly because of their ability to form small protein-detergent complexes. In a previous study, we introduced glucose neopentyl glycol (GNG) amphiphiles with a branched diglucoside structure that has facilitated high resolution crystallographic structure determination of several membrane proteins. Like other glucoside detergents, however, these GNGs were less successful than DDM in stabilizing membrane proteins, limiting their wide use in protein structural study. As a strategy to improve GNG efficacy for protein stabilization, we introduced two different alkyl chains (i.e., main and pendant chains) into the GNG scaffold while maintaining the branched diglucoside head group. Of these pendant-bearing GNGs (P-GNGs), three detergents (GNG-2,14, GNG-3,13 and GNG-3,14) were not only notably better than both DDM (a gold standard detergent) and the previously described GNGs at stabilizing all six membrane proteins tested here, but were also as efficient as DDM at membrane protein extraction. The results suggest that the C14 main chain of the P-GNGs is highly compatible with the hydrophobic widths of membrane proteins, while the C2/C3 pendant chain is effective at strengthening detergent hydrophobic interactions. Based on the marked effect on protein stability and solubility, these glucoside detergents hold significant potential for membrane protein structural study. Furthermore, the independent roles of the detergent two alkyl chains first introduced in this study have shed light on new amphiphile design for membrane protein study. Statement of significanceDetergent efficacy for protein stabilization tends to be protein-specific, thus it is challenging to find a detergent that is effective at stabilizing multiple membrane proteins. By incorporating a pendant chain into our previous GNG scaffold, we prepared pendant chain-bearing GNGs (P-GNGs) and identified three P-GNGs that were highly effective at stabilizing all membrane proteins tested here including two GPCRs. In addition, the new detergents were as efficient as DDM at extracting membrane proteins, enabling use of these detergents over the multiple steps of protein isolation. The key difference between the P-GNGs and other glucoside detergents, the presence of a pendant chain, is likely to be responsible for their markedly enhanced protein stabilization behavior.

An automated data processing and analysis pipeline for transmembrane proteins in detergent solutions

The application of small angle X-ray scattering (SAXS) to the structural characterization of transmembrane proteins (MPs) in detergent solutions has become a routine procedure at synchrotron BioSAXS beamlines around the world. SAXS provides overall parameters and low resolution shapes of solubilized MPs, but is also meaningfully employed in hybrid modeling procedures that combine scattering data with information provided by high-resolution techniques (eg. macromolecular crystallography, nuclear magnetic resonance and cryo-electron microscopy). Structural modeling of MPs from SAXS data is non-trivial, and the necessary computational procedures require further formalization and facilitation. We propose an automated pipeline integrated with the laboratory-information management system ISPyB, aimed at preliminary SAXS analysis and the first-step reconstruction of MPs in detergent solutions, in order to streamline high-throughput studies, especially at synchrotron beamlines. The pipeline queries an ISPyB database for available a priori information via dedicated services, estimates model-free SAXS parameters and generates preliminary models utilizing either ab initio, high-resolution-based, or mixed/hybrid methods. The results of the automated analysis can be inspected online using the standard ISPyB interface and the estimated modeling parameters may be utilized for further in-depth modeling beyond the pipeline. Examples of the pipeline results for the modelling of the tetrameric alpha-helical membrane channel Aquaporin0 and mechanosensitive channel T2, solubilized by n-Dodecyl β-D-maltoside are presented. We demonstrate how increasing the amount of a priori information improves model resolution and enables deeper insights into the molecular structure of protein-detergent complexes.

High-Throughput Screening of Protein-Detergent Complexes Using Fluorescence Polarization Spectroscopy.

This article provides detailed protocols for a high-throughput fluorescence polarization (FP) spectroscopy approach to disentangle the interactions of membrane proteins with solubilizing detergents. Existing techniques for examining the membrane protein-detergent complex (PDC) interactions are low throughput and require high amounts of proteins. Here, we describe a 96-well analytical approach, which facilitates a scalable analysis of the PDC interactions at low-nanomolar concentrations of membrane proteins in native solutions. At detergent concentrations much greater than the equilibrium dissociation constant of the PDC, Kd , the FP anisotropy reaches a saturated value, so it is independent of the detergent concentration. On the contrary, at detergent concentrations comparable with or lower than the Kd , the FP anisotropy readout undergoes a time-dependent decrease, exhibiting a sensitive and specific detergent-dissociation signature. Our approach can also be used for determining the kinetic rate constants of association and dissociation. With further development, these protocols might be used in various arenas of membrane protein research that pertain to extraction, solubilization, and stabilization. © 2019 by John Wiley & Sons, Inc.

Contrast-Matching Detergent in Small-Angle Neutron Scattering Experiments for Membrane Protein Structural Analysis and Ab Initio Modeling.

The biological small-angle neutron scattering instrument at the High-Flux Isotope Reactor of Oak Ridge National Laboratory is dedicated to the investigation of biological materials, biofuel processing, and bio-inspired materials covering nanometer to micrometer length scales. The methods presented here for investigating physical properties (i.e., size and shape) of membrane proteins (here, MmIAP, an intramembrane aspartyl protease from Methanoculleus marisnigri) in solutions of micelle-forming detergents are well-suited for this small-angle neutron scattering instrument, among others. Other biophysical characterization techniques are hindered by their inability to address the detergent contributions in a protein-detergent complex structure. Additionally, access to the Bio-Deuteration Lab provides unique capabilities for preparing large-scale cultivations and expressing deuterium-labeled proteins for enhanced scattering signal from the protein. While this technique does not provide structural details at high-resolution, the structural knowledge gap for membrane proteins contains many addressable areas of research without requiring near-atomic resolution. For example, these areas include determination of oligomeric states, complex formation, conformational changes during perturbation, and folding/unfolding events. These investigations can be readily accomplished through applications of this method.

Exploring the Potential of Dendritic Oligoglycerol Detergents for Protein Mass Spectrometry

The ability to design detergents that are suitable for protein analysis by mass spectrometry (MS) represents an on-going challenge in the field of native MS. Desirable detergent characteristics include charge-reducing properties and low gas-phase stabilities of complexes formed with proteins. In this work, the gas-phase properties of oligoglycerol detergents (OGDs) are optimized by fine tuning of their molecular structure. Furthermore, a tandem mass spectrometry (MS/MS) approach is presented that estimates the gas-phase properties of detergents simply by studying the dissociation behaviour of protein-detergent complexes (PDCs) formed with the soluble protein β-lactoglobulin (BLG).Graphical ᅟ

Kinetics of Membrane Protein-Detergent Interactions Depend on Protein Electrostatics.

Interactions of a membrane protein with a detergent micelle represent a fundamental process with practical implications in structural and chemical biology. Quantitative assessment of the kinetics of protein-detergent complex (PDC) interactions has always been challenged by complicated behavior of both membrane proteins and solubilizing detergents in aqueous phase. Here, we show the kinetic reads of the desorption of maltoside-containing detergents from β-barrel membrane proteins. Using steady-state fluorescence polarization (FP) anisotropy measurements, we recorded real-time, specific signatures of the PDC interactions. The results of these measurements were used to infer the model-dependent rate constants of association and dissociation of the proteomicelles. Remarkably, the kinetics of the PDC interactions depend on the overall protein charge despite the nonionic nature of the detergent monomers. In the future, this approach might be employed for high-throughput screening of kinetic fingerprints of different membrane proteins stabilized in micelles that contain mixtures of various detergents.

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

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

Detergent Desorption of Membrane Proteins Exhibits Two Kinetic Phases.

Gradual dissociation of detergent molecules from water-insoluble membrane proteins culminates in protein aggregation. However, the time-dependent trajectory of this process remains ambiguous because the signal-to-noise ratio of most spectroscopic and calorimetric techniques is drastically declined by the presence of protein aggregates in solution. We show that by using steady-state fluorescence polarization (FP) spectroscopy the dissociation of the protein-detergent complex (PDC) can be inspected in real time at detergent concentrations below the critical micelle concentration. This article provides experimental evidence of the coexistence of two distinct phases of the dissociations of detergent monomers from membrane proteins. We first noted a slow detergent predesolvation process, which was accompanied by a relatively modest change in the FP anisotropy, suggesting a small number of dissociated detergent monomers from the proteomicelles. This predesolvation phase was followed by a fast detergent desolvation process, which was highlighted by a major alteration in the FP anisotropy. The durations and rates of these phases were dependent on both the detergent concentration and the interfacial PDC interactions. Further development of this approach might lead to the creation of a new semiquantitative method for the assessment of the kinetics of association and dissociation of proteomicelles.

Small-angle Scattering of X-rays and Neutrons as Structural Tools inNanoscience: Block Copolymer Micelles, Protein-detergent Complexes,Microgels and other Nanoparticles

Small-angle Scattering of X-rays and Neutrons as Structural Tools inNanoscience: Block Copolymer Micelles, Protein-detergent Complexes,Microgels and other Nanoparticles

Quantification of Membrane Protein-Detergent Complex Interactions.

Although fundamentally significant in structural, chemical, and membrane biology, the interfacial protein-detergent complex (PDC) interactions have been modestly examined because of the complicated behavior of both detergents and membrane proteins in aqueous phase. Membrane proteins are prone to unproductive aggregation resulting from poor detergent solvation, but the participating forces in this phenomenon remain ambiguous. Here, we show that using rational membrane protein design, targeted chemical modification, and steady-state fluorescence polarization spectroscopy, the detergent desolvation of membrane proteins can be quantitatively evaluated. We demonstrate that depleting the detergent in the sample well produced a two-state transition of membrane proteins between a fully detergent-solvated state and a detergent-desolvated state, the nature of which depended on the interfacial PDC interactions. Using a panel of six membrane proteins of varying hydrophobic topography, structural fingerprint, and charge distribution on the solvent-accessible surface, we provide direct experimental evidence for the contributions of the electrostatic and hydrophobic interactions to the protein solvation properties. Moreover, all-atom molecular dynamics simulations report the major contribution of the hydrophobic forces exerted at the PDC interface. This semiquantitative approach might be extended in the future to include studies of the interfacial PDC interactions of other challenging membrane protein systems of unknown structure. This would have practical importance in protein extraction, solubilization, stabilization, and crystallization.

Purification and characterization of the colicin A immunity protein in detergent micelles

The immunity proteins against pore-forming colicins represent a family of integral membrane proteins that reside in the inner membrane of producing cells. Cai, the colicin A immunity protein, was characterized here in detergent micelles by circular dichroism (CD), size exclusion chromatography, chemical cross-linking, nuclear magnetic resonance (NMR) spectroscopy, cysteine accessibility, and colicin A binding in detergent micelles. Bile-salt derivatives induced extensive protein polymerization that precluded further investigation. The physical characterization of detergent-solubilized protein indicates that phosphate-containing detergents are more efficient in extracting, solubilizing and maintaining Cai in a monomeric state. Yet, their capacity to ensure protein activity, reconstitution, helix packing, and high-quality NMR spectra was inferior to that of milder detergents. Solvent ionic strength and composition greatly modified the solubilizing capacity of milder detergents. Most importantly, binding to the colicin A pore-forming domain (pf-ColA) occurred almost exclusively in sugar-derived detergents. The relative performance of the different detergents in each experiment depends on their impact not only on Cai structure, solubility and oligomerization state, but also on other reaction components and technical aspects. Thus, proteoliposomes were best obtained from protein in LDAO micelles, possibly also due to indirect effects on the lipidic bilayer. The compatibility of a detergent with Cai/pf-ColA complex formation is influenced by its effect on the conformational landscape of each protein, where detergent-mediated pf-ColA denaturation could also lead to negative results. The NMR spectra were greatly affected by the solubility, monodispersity, fold and dynamics of the protein-detergent complexes, and none of those tested here provided NMR spectra of sufficient quality to allow for peak assignment. Cai function could be proven in alkyl glycosides and not in those detergents that afforded the best solubility, reconstitution efficiency or spectral quality indicating that these criteria cannot be taken as unambiguous proof of nativeness without the support of direct activity measurements.