Protein-detergent Interactions Research Articles

Nanostructured lipopeptide-based membranomimetics for stabilizing bacteriorhodopsin.

Nanostructured 7-9-residue cyclic and unstructured lipopeptide-based facial detergents have been engineered to stabilize the model integral membrane protein, bacteriorhodopsin. Formation of a cylindrical-type micelle assembly induced by facial amphipathic lipopeptides resembles a biological membrane more effectively than conventional micelles. The hydrophobic face of this cylindrical-type micelle provides extended stability to the membrane protein and the hydrophilic surface interacts with an aqueous environment. In our present study, we have demonstrated experimentally and computationally that lipopeptide-based facial detergents having an unstructured or β-turn conformation can stabilize membrane proteins. However, constrained peptide detergents can provide enhanced stability to bacteriorhodopsin. In this study, we have computationally examined the structural stability of bacteriorhodopsin in the presence of helical, beta-strand, and cyclic unstructured peptide detergents, and conventional detergent-like peptides. Our study demonstrates that optimal membranomimetics (detergents) for stabilizing a specific membrane protein can be screened based on the following criteria: (i) hydrodynamic radii of the self-assembled peptide detergents, (ii) stability assay of detergent-encased membrane proteins, (iii) percentage covered area of detergent-encased membrane proteins obtained computationally and (iv) protein-detergent interaction energy.

Isothermal titration calorimetry of membrane protein interactions: FNR and the cytochrome b6f complex

Ferredoxin-NADP+ reductase (FNR) was previously inferred to bind to the cytochrome b6f complex in the electron transport chain of oxygenic photosynthesis. In the present study, this inference has been examined through analysis of the thermodynamics of the interaction between FNR and the b6f complex. Isothermal titration calorimetry (ITC) was used to characterize the physical interaction of FNR with b6f complex derived from two plant sources (Spinacia oleracea and Zea maize). ITC did not detect a significant interaction of FNR with the b6f complex in detergent solution nor with the complex reconstituted in liposomes. A previous inference of a small amplitude but defined FNR-b6f interaction is explained by FNR interaction with micelles of the undecyl β-D maltoside (UDM) detergent micelles used to purify b6f. Circular dichroism, employed to analyze the effect of detergent on the FNR structure, did not reveal significant changes in secondary or tertiary structures of FNR domains in the presence of UDM detergent. However, thermodynamic analysis implied a significant decrease in an interaction between the N-terminal FAD-binding and C-terminal NADP+-binding domains of FNR caused by detergent. The enthalpy, ΔHo, and the entropy, ΔSo, associated with FNR unfolding decreased four-fold in the presence of 1 mM UDM at pH 6.5. In addition to the conclusion regarding the absence of a binding interaction of significant amplitude between FNR and the b6f complex, these studies provide a precedent for consideration of significant background protein-detergent interactions in ITC analyses involving integral membrane proteins.

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.

Energetics and Kinetics of Membrane Protein-Detergent Interactions

Energetics and Kinetics of Membrane Protein-Detergent Interactions

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.

Molecular dynamics simulation strategies for protein–micelle complexes

The structure and stability of membrane proteins can vary widely in different detergents and this variability has great practical consequences for working with membrane proteins. Nevertheless, the mechanisms that operate to alter the behavior of proteins in micelles are poorly understood and not predictable. Atomic simulations could provide considerable insight into these mechanisms. Building protein–micelle complexes for simulation is fraught with uncertainty, however, in part because it is often unknown how many detergent molecules are present in the complex. Here, we describe several convenient ways to employ Micelle Builder in CHARMM-GUI to rapidly construct protein–micelle complexes and performed simulations of the isolated voltage-sensor domain of voltage-dependent potassium-selective channel and an antimicrobial peptide papiliocin with varying numbers of detergents. We found that once the detergent number exceeds a threshold, protein–detergent interactions change very little and remain very consistent with experimental observations. Our results provide a platform for future studies of the interplays between protein structure and detergent properties at the atomic level. This article is part of a Special Issue entitled: Membrane Proteins edited by J.C. Gumbart and Sergei Noskov.

All-atom molecular dynamics simulation of a photosystem i/detergent complex.

All-atom molecular dynamics (MD) simulation was used to investigate the solution structure and dynamics of the photosynthetic pigment-protein complex photosystem I (PSI) from Thermosynechococcus elongatus embedded in a toroidal belt of n-dodecyl-β-d-maltoside (DDM) detergent. Evaluation of root-mean-square deviations (RMSDs) relative to the known crystal structure show that the protein complex surrounded by DDM molecules is stable during the 200 ns simulation time, and root-mean-square fluctuation (RMSF) analysis indicates that regions of high local mobility correspond to solvent-exposed regions such as turns in the transmembrane α-helices and flexible loops on the stromal and lumenal faces. Comparing the protein-detergent complex to a pure detergent micelle, the detergent surrounding the PSI trimer is found to be less densely packed but with more ordered detergent tails, contrary to what is seen in most lipid bilayer models. We also investigated any functional implications for the observed conformational dynamics and protein-detergent interactions, discovering interesting structural changes in the psaL subunits associated with maintaining the trimeric structure of the protein. Importantly, we find that the docking of soluble electron mediators such as cytochrome c6 and ferredoxin to PSI is not significantly impacted by the solubilization of PSI in detergent.

Dependence of micelle size and shape on detergent alkyl chain length and head group.

Micelle-forming detergents provide an amphipathic environment that can mimic lipid bilayers and are important tools for solubilizing membrane proteins for functional and structural investigations in vitro. However, the formation of a soluble protein-detergent complex (PDC) currently relies on empirical screening of detergents, and a stable and functional PDC is often not obtained. To provide a foundation for systematic comparisons between the properties of the detergent micelle and the resulting PDC, a comprehensive set of detergents commonly used for membrane protein studies are systematically investigated. Using small-angle X-ray scattering (SAXS), micelle shapes and sizes are determined for phosphocholines with 10, 12, and 14 alkyl carbons, glucosides with 8, 9, and 10 alkyl carbons, maltosides with 8, 10, and 12 alkyl carbons, and lysophosphatidyl glycerols with 14 and 16 alkyl carbons. The SAXS profiles are well described by two-component ellipsoid models, with an electron rich outer shell corresponding to the detergent head groups and a less electron dense hydrophobic core composed of the alkyl chains. The minor axis of the elliptical micelle core from these models is constrained by the length of the alkyl chain, and increases by 1.2–1.5 Å per carbon addition to the alkyl chain. The major elliptical axis also increases with chain length; however, the ellipticity remains approximately constant for each detergent series. In addition, the aggregation number of these detergents increases by ∼16 monomers per micelle for each alkyl carbon added. The data provide a comprehensive view of the determinants of micelle shape and size and provide a baseline for correlating micelle properties with protein-detergent interactions.

Sequence Hydropathy Dominates Membrane Protein Response to Detergent Solubilization

The ability to predict from amino acid sequence how membrane protein structures will respond to detergent solubilization would significantly facilitate experimental characterization of these molecules. Here we have investigated and compared the response to solubilization by the "mild" n-dodecyl-β-D-maltoside (DDM) and "harsh" sodium dodecyl sulfate (SDS) of wild-type and point mutant "hairpin" (helix-loop-helix) membrane proteins derived from the third and fourth TM segments of the human cystic fibrosis transmembrane conductance regulator (CFTR) and the intervening extracellular loop. Circular dichroism spectroscopy, size-exclusion chromatography, and pyrene fluorescence spectroscopy were used to evaluate the secondary structures, hairpin-detergent complex excluded volumes, and hairpin compactness of the detergent-solubilized sequences. Sequence hydrophobicity is found to be the dominant factor dictating membrane protein response to detergent solubilization by DDM and SDS, with hairpin secondary structure exquisitely sensitive to mutation when DDM is used for solubilization. DDM and SDS differ principally in their ability to promote approach of TM segment ends, although hairpin compactness remains sensitive to point mutations. Our overall findings suggest that protein-protein and protein-detergent interactions are determined concomitantly, with the net hydropathy of residues exposed to detergent dominating the observed properties of the solubilized protein.

Synergistic effects of detergents and organic solvents on protein refolding: Control of aggregation and folding rates

This paper presents the synergistic enhancement of the refolding yield of denatured and reduced lysozyme by using detergents as aggregation inhibitors and water-miscible organic cosolvents as modulators for the detergents. Adding only cetyltrimethylammonium bromide (CTAB) led to a slight increase in the refolding yield (up to 13%). Further addition of dimethylsulfoxide (DMSO) with CTAB drastically increased the refolding yield up to 35%, a value which was higher than the simple sum of the refolding yields in the presence of only CTAB or DMSO. The synergistic enhancement was also observed in the coexistence of other detergents, such as polyethylene glycol monooleyl ether (n = 50) and N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, and cosolvents, such as N,N-dimethylformamide and N,N-dimethylacetamide. Experimental data and a kinetic analysis revealed the guideline for selecting a couple of additives; detergents which can adequately inhibit the aggregation of proteins by binding to hydrophobic surfaces of refolding intermediates should be employed as an aggregation inhibitor, and cosolvents which can properly prevent both protein-protein and protein-detergent interactions act as effective modulators for the aggregation inhibitor, resulting in a desirable balance between folding and aggregation rates.

Mixing and Matching Detergents For Membrane Protein NMR Structure Determination

One major obstacle to membrane protein structure determination is the selection of a detergent micelle that mimics the native lipid bilayer. Currently, detergents are selected by exhaustive screening because the effects of protein-detergent interactions on protein structure are poorly understood. We have investigated the structure and dynamics of integral membrane proteins in different detergents by nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy and by small-angle X-ray scattering (SAXS).

Fibrillogenic and Non-fibrillogenic Ensembles of SDS-bound Human α-Synuclein

Fibril formation of α-synuclein is associated with several neurodegenerative diseases, including Parkinson's disease in humans. The anionic detergent sodium dodecyl sulfate (SDS) can accelerate the fibril formation in vitro. However, the molecular basis of this acceleration is not clear. Our study shows that native α-synuclein exhibits relatively less fibril growth despite providing fibril seeds for nucleation. The presence of SDS promotes the seeded fibril growth in a concentration-dependent manner, with an optimal concentration of 0.5–0.75 mM. We used isothermal calorimetry, hydrophobic dye binding and circular dichroism spectroscopy to characterize the protein–detergent interactions as a function of the concentration of SDS. Interaction of SDS with α-synuclein when studied by isothermal titration calorimetry and hydrophobic dye-binding reveals a similar characteristic optimal behavior between 0.5 mM and 0.75 mM SDS. The study shows two types of ensembles of α-synuclein and SDS: the fibrillogenic ensembles formed with optimal concentration of SDS around 0.5–0.75 mM are characterized by enhanced accessible hydrophobic surfaces and extended to partially helical conformation, while the less or non-fibrillogenic ensembles formed above 2 mM SDS are characterized by less accessible hydrophobic surfaces and maximal helical content. Little or no fibrillogenicity of the ensembles observed above 2 mM SDS could be partly because of the observed intrinsic instability of the fibrils under the condition.

Membrane protein dynamics and detergent interactions within a crystal: a simulation study of OmpA.

Molecular dynamics (MD) simulations are used to explore the dynamics of a membrane protein in its crystal environment. A 50-ns-duration simulation (at a temperature of 300 K) is performed for the crystallographic unit cell of the bacterial outer membrane protein OmpA. The unit cell contains four protein molecules, plus detergent molecules and water. An excellent correlation between simulated and experimental values of crystallographic B factors is observed. Effectively, 0.2 micros of protein trajectories are obtained, allowing a critical assessment of simulation quality. Some deficiency in conformational sampling is demonstrated, but averaging over multiple trajectories improves this limitation. The previously undescribed structure and dynamics of detergent molecules in a unit cell are reported here, providing insight into the interactions important in the formation and stabilization of the crystalline environment at room temperature. In particular, we show that at room temperature the detergent molecules form a dynamic, extended micellar structure spreading over adjacent OmpA monomers within the crystal.

Simulation studies of the interactions between membrane proteins and detergents

Interactions between membrane proteins and detergents are important in biophysical and structural studies and are also biologically relevant in the context of folding and transport. Despite a paucity of high-resolution data on protein–detergent interactions, novel methods and increased computational power enable simulations to provide a means of understanding such interactions in detail. Simulations have been used to compare the effect of lipid or detergent on the structure and dynamics of membrane proteins. Moreover, some of the longest and most complex simulations to date have been used to observe the spontaneous formation of membrane protein–detergent micelles. Common mechanistic steps in the micelle self-assembly process were identified for both α-helical and β-barrel membrane proteins, and a simple kinetic mechanism was proposed. Recently, simplified (i.e. coarse-grained) models have been utilized to follow long timescale transitions in membrane protein–detergent assemblies.

Simulation studies of the interactions between membrane proteins and detergents

Interactions between membrane proteins and detergents are important in biophysical and structural studies and are also biologically relevant in the context of folding and transport. Despite a paucity of high-resolution data on protein-detergent interactions, novel methods and increased computational power enable simulations to provide a means of understanding such interactions in detail. Simulations have been used to compare the effect of lipid or detergent on the structure and dynamics of membrane proteins. Moreover, some of the longest and most complex simulations to date have been used to observe the spontaneous formation of membrane protein-detergent micelles. Common mechanistic steps in the micelle self-assembly process were identified for both alpha-helical and beta-barrel membrane proteins, and a simple kinetic mechanism was proposed. Recently, simplified (i.e. coarse-grained) models have been utilized to follow long timescale transitions in membrane protein-detergent assemblies.

Analysis of protein–surfactant interactions—a titration calorimetric and fluorescence spectroscopic investigation of interactions between Humicola insolens cutinase and an anionic surfactant

We have studied interactions of cutinase (HiC) from Humicula insolens and sodium dodecyl sulphate (SDS) by parallel calorimetric and fluorescence investigations of systems in which the concentration of both components was changed systematically. Results from the two methods exhibit a number of synchronous characteristics, when plotted against the total SDS concentration, [SDS] tot. The molecular origin of several of these anomalies was assigned, and five intervals of [SDS] tot in which different modes of interactions dominated were identified. Going from low to high [SDS] tot, these modes were: binding of (a few) SDS to native HiC, formation of oligomeric protein aggregates, denaturation of HiC and adsorption of SDS on denatured protein. For [SDS] tot > 3–6 mM (depending on the protein concentration), the adsorption saturated, and no further protein–detergent interaction could be detected. Two particularly conspicuous anomalies in the calorimetric data were ascribed to respectively denaturation and saturation. It was found that [SDS] tot at these points depended linearly on the (total) protein concentration, [HiC]. We suggest that this reflects the balance between bound and free SDS [SDS] tot = [SDS] aq + [HiC] N b where [SDS] aq and N b are, respectively, the aqueous (“free”) concentration of SDS and the average number of SDS bound per protein. Interpretation of the results along these lines showed that at 22 °C and pH 7.0, HiC denatures with ∼14 bound surfactant molecules at [SDS] aq = 1.0 mM. Saturation is characterized by N b ∼39 and [SDS] aq = 2.2 mM. The latter value is equal to CMC in the (protein free) buffer. These results are discussed with respect to the SDS-binding capacity of HiC and the origin and location of the saturation point.

Membrane protein-lipid interactions in mixed micelles studied by NMR spectroscopy with the use of paramagnetic reagents.

For solution NMR studies of the structure and function of membrane proteins, these macromolecules have to be reconstituted and solubilized in detergent micelles. Detailed characterization of the mixed detergent/protein micelles is then of key importance to validate the results from such studies, and to evaluate how faithfully the natural environment of the protein in the biological membrane is mimicked by the micelle. In this paper, a selection of paramagnetic probes with different physicochemical properties are used to characterize the 60 kDa mixed micelles consisting of about 90 molecules of the detergent dihexanoylphosphatidylcholine (DHPC) and one molecule of the Escherichia coli outer-membrane protein X (OmpX), which had previously been extensively studied by solution NMR techniques. The observation of highly selective relaxation effects on the NMR spectra of OmpX and DHPC from a water-soluble relaxation agent and from nitroxide spin labels attached to lipophilic molecules, confirmed data obtained previously with more complex NMR studies of the diamagnetic OmpX/DHPC system, and yielded additional novel insights into the protein-detergent interactions in the mixed micelles. The application of paramagnetic probes to the well-characterized OmpX/DHPC system indicates that such probes should be widely applicable as an efficient support of NMR studies of the topology of mixed membrane protein-detergent micelles.

Direct analysis of protein sedimentation equilibrium in detergent solutions without density matching

Characterizing membrane proteins by sedimentation equilibrium is challenging because detergents and/or lipid molecules, usually required for solubilization, form a complex with the protein. The most common way to overcome this problem is Tanford and Reynolds’ density matching method, which eliminates the buoyant mass contributions of detergents/lipids by adjusting the solvent density with D 2O/H 2O mixtures to render either detergent or lipid molecules neutrally buoyant. Unfortunately, the method is practical only for detergent densities between 1.0 (H 2O) and 1.1 (D 2O) g ml −1, excluding many of the more commonly used detergents for membrane protein studies. Here, we present a modern variant of Tanford and Reynolds’ method that (1) is applicable to any detergent regardless of its specific density, (2) does not compromise accuracy and precision, and (3) provides additional information about the number of detergent molecules that are bound to each protein. The new method was applied successfully to Δ(1-43)A-I, an amino-terminal deletion mutant of human apolipoprotein A-I. Interestingly, we observed a significantly lower Δ(1-43)A-I/octyl-glucoside complex partial specific volume than that expected from volume additivity rules, indicative of specific protein–detergent interactions.

Lipid-protein interactions in DHPC micelles containing the integral membrane protein OmpX investigated by NMR spectroscopy.

Intermolecular nuclear Overhauser effects (NOEs) between the integral outer membrane protein OmpX from Escherichia coli and dihexanoylphosphatidylcholine (DHPC) provided a detailed description of protein-detergent interactions. The NOEs were measured in 3D (15)N- and (13)C-resolved [(1)H,(1)H]-NOESY spectra recorded with selectively methyl-protonated and otherwise uniformly (2)H,(13)C,(15)N-labeled OmpX in micelles of DHPC at natural isotope abundance. In these mixed micelles the NMR structure of OmpX consists of an eight-stranded antiparallel beta-barrel. The OmpX surface area covered with intermolecular NOEs to the DHPC hydrophobic tails forms a continuous cylinder jacket of approximately 28 A in height, which is centered about the middle of the long axis through the beta-barrel. In addition, some intermolecular NOEs with methyl groups of the DHPC polar head were identified along both boundaries of this cylinder jacket. The experimental data suggest that the hydrophobic surface areas of OmpX are covered with a monolayer of DHPC molecules, which appears to mimic quite faithfully the embedding of the beta-barrel in a double-layer lipid membrane.

The reaction order of the dissociation reaction of the B820 subunit of Rhodospirillum rubrum light-harvesting I complex

We have studied the equilibrium between the dissociated B777 form (absorbing at 777 nm) of the light-harvesting complex of Rhodospirillum rubrum and the oligomeric B820 form. Analysis of the reaction order for the B820 dissociation reaction to form B777 shows that this reaction depends on the concentration of octylglucoside detergent ( n-octyl-β- D-glucopyranoside (βOG)) present in the sample. At low βOG concentrations (less than 1.2%) this reaction requires two components, presumably one α-B777 and one β-B777, implying that the B820 subunit is a dimer. At higher βOG concentrations this reaction requires four components, implying that B820 is a tetramer. These results partly explain the discrepancies in the literature about the stoichiometry of B820 and open an original way for studying protein–detergent interactions.