Lauryldimethylamine N-oxide Research Articles

Spectral modulation of B850 bacteriochlorophyll a in light-harvesting complex 2 from purple photosynthetic bacterium Thermochromatium tepidum by detergents and calcium ions

Spectral variations of light-harvesting (LH) proteins of purple photosynthetic bacteria provide insight into the molecular mechanisms underlying spectral tuning of circular bacteriochlorophyll (BChl) arrays, which play crucial roles in photoenergy conversion in these organisms. Here we investigate spectral changes of the Qy band of B850 BChl a in LH2 protein from purple sulfur bacterium Thermochromatium tepidum (tepidum-LH2) by detergents and Ca2+. The tepidum-LH2 solubilized with lauryl dimethylamine N-oxide and n-octyl-β-D-glucoside (LH2LDAO and LH2OG, respectively) exhibited blue-shift of the B850 Qy band with hypochromism compared with the tepidum-LH2 solubilized with n-dodecyl-β-D-maltoside (LH2DDM), resulting in the LH3-like spectral features. Resonance Raman spectroscopy indicated that this blue-shift was ascribable to the loss of hydrogen-bonding between the C3-acetyl group in B850 BChl a and the LH2 polypeptides. Ca2+ produced red-shift of the B850 Qy band in LH2LDAO by forming hydrogen-bond for the C3-acetyl group in B850 BChl a, probably due to a change in the microenvironmental structure around B850. Ca2+-induced red-shift was also observed in LH2OG although the B850 acetyl group is still free from hydrogen-bonding. Therefore, the Ca2+-induced B850 red-shift in LH2OG would originate from an electrostatic effect of Ca2+. The current results suggest that the B850 Qy band in tepidum-LH2 is primarily tuned by two mechanisms, namely the hydrogen-bonding of the B850 acetyl group and the electrostatic effect.

Characterization of 10MAG/LDAO reverse micelles: Understanding versatility for protein encapsulation

Reverse micelles (RMs) are spontaneously organizing nanobubbles composed of an organic solvent, surfactants, and an aqueous phase that can encapsulate biological macromolecules for various biophysical studies. Unlike other RM systems, the 1-decanoyl-rac-glycerol (10MAG) and lauryldimethylamine-N-oxide (LDAO) surfactant system has proven to house proteins with higher stability than other RM mixtures with little sensitivity to the water loading (W0, defined by the ratio of water to surfactant). We investigated this unique property by encapsulating three model proteins – cytochrome c, myoglobin, and flavodoxin – in 10MAG/LDAO RMs and applying a variety of experimental methods to characterize this system's behavior. We found that this surfactant system differs greatly from the traditional, spherical, monodisperse RM population model. 10MAG/LDAO RMs were discovered to be oblate ellipsoids at all conditions, and as W0 was increased, surfactants redistributed to form a greater number of increasingly spherical ellipsoidal particles with pools of more bulk-like water. Proteins distinctively influence the thermodynamics of the mixture, encapsulating at their optimal RM size and driving protein-free RM sizes to scale accordingly. These findings inform the future development of similarly malleable encapsulation systems and build a foundation for application of 10MAG/LDAO RMs to analyze biological and chemical processes under nanoscale confinement.

Spectral changes of light-harvesting complex 2 lacking B800 bacteriochlorophyll a under neutral pH conditions.

Exchange of B800 bacteriochlorophyll (BChl) a in light-harvesting complex 2 (LH2) is promising for a better understanding of the mechanism on intracomplex excitation energy transfer of this protein. Structural and spectroscopic properties of LH2 lacking B800 BChl a (B800-depleted LH2), which is an important intermediate protein in the B800 exchange, will be useful to tackle the energy transfer mechanism in LH2 by the B800 exchange strategy. In this study, we report a unique spectral change of B800-depleted LH2, in which the Qy absorption band of B800 BChl a is automatically recovered under neutral pH conditions. This spectral change was facilitated by factors for destabilization of LH2, namely, a detergent, lauryl dimethylamine N-oxide, and an increase in temperature. Spectral analyses in the preparation of an LH2 variant denoted as B800-recovered LH2 indicated that most BChl a that was released by decomposition of part of B800-depleted LH2 was a source of the production of B800-recovered LH2. Characterization of purified B800-recovered LH2 demonstrated that its spectroscopic and structural features was quite similar to those of native LH2. The current results indicate that the recovery of the B800 Qy band of B800-depleted LH2 originates from the combination of decomposition of part of B800-depleted LH2 and in situ reconstitution of BChl a into the B800 binding pockets of residual B800-depleted LH2, resulting in the formation of stable B800-recovered LH2.

A simple and effective method to remove pigments from heterologous secretory proteins expressed in Pichia pastoris

Pichia pastoris is a popular yeast host for high-level heterologous expression of proteins on an industrial scale owing to its reliable expression, robust growth, high fermentation density, and easy genetic manipulation and cultivation at a relatively low cost. Of particular interest is its high secretion efficiency for small proteins including insulin, human serum albumin, vaccines, enzymes, and llama-derived heavy-chain only antibodies (nanobodies) for pharmaceutical and research applications. However, a recurring challenge in using P. pastoris heterologous secretory proteins is the co-purification of a sticky, yellow pigment which has been identified as a tetra-benzoyl disaccharide. Current methods for pigment removal involve crystallization of the heterologous secretory protein, active carbon absorption, and chromatography using cation exchange and hydrophobic interaction. Here, we present a simple and effective method to remove the yellow pigment, demonstrated with divalent nanobodies targeting SARS-CoV-2. The method entails capturing the nanobody on an affinity column and subsequent washing with the zwitterionic detergent lauryldimethylamine N-oxide (LDAO). We anticipate the method become generally useful to remove pigments from secretion proteins produced in P. pastoris, offering a practical solution to enhance the purity of heterologous proteins in various biotechnological applications.

Interaction of detergent with complex mimics of bacterial membranes

Detergents are valuable tools to extract membrane proteins for biophysical, biochemical, and structural scrutiny. The detergent-driven solubilization of bilayers made from a single lipid species is commonly described in terms of pseudo-phase diagrams and a three-stage model accounting for three ranges comprising (i) intact vesicles, (ii) vesicle/micelle co-existence, or (iii) mixed micelles. Moreover, the pseudo-phase boundaries thus determined can often be quantitatively rationalized in terms of the molecular shapes of the lipid and the detergent used. Yet, it has remained unclear to what extent this approach can be applied to multi-component lipid membranes that more closely mimic the compositional complexity of cellular membranes. Here, we studied how lipid mixtures composed of palmitoyl oleoyl phosphatidylethanolamine (POPE), palmitoyl oleoyl phosphatidylglycerol (POPG), and tetraoleoyl cardiolipin (TOCL) are solubilized by the commonly used zwitterionic detergent lauryldimethylamine N-oxide using isothermal titration calorimetry. While phase diagrams of the diverse lipid mixtures showed the typical ranges of the three-stage model, we found that POPG-rich POPE/POPG bilayers are more difficult to solubilize than POPG-poor POPE/POPG bilayers. In turn, POPE/POPG/TOCL bilayers became increasingly resistant to detergent with increasing TOCL content. Since POPG is nearly cylindrically shaped and TOCL adopts inverted cone-like shapes under current buffer conditions, our solubilization data do not align with shape-based arguments. Instead, additional electrostatic interactions between lipids and detergents lead to non-additive mixing behavior affecting the resilience of complex lipid bilayers against solubilization.

Characterization of the 10MAG/LDAO reverse micelle surfactant system, a useful tool for studying protein biophysics.

Reverse micelles (RMs) are spontaneously organizing nanobubbles composed of an organic solvent, surfactants, and an aqueous phase that can encapsulate biological macromolecules. Reverse micelles can facilitate novel biophysical studies, especially studies of confinement and interfacial interactions on protein thermodynamics. The 1-decanoyl-rac-glycerol (10MAG) and lauryldimethylamine-N-oxide (LDAO) surfactant system provides long-term protein sample stability without precise optimization of RM size, which is determined by the ratio of water to surfactant, known as water loading, W0. In this study, we investigated this unique property of 10MAG/LDAO by encapsulating three model proteins- cytochrome c, myoglobin, and flavodoxin, to observe changes in the system using dynamic light scattering (DLS), diffusion ordered nuclear magnetic resonance spectroscopy (DOSY NMR), and NMR T1 relaxation measurements. We found that this surfactant system differs greatly from the traditional, monodisperse RM population model. At all conditions, 10MAG/LDAO RMs exist in thermodynamic equilibrium with a large, nonspherical bulk-like water reservoir. Moreover, proteins seem to drive the thermodynamics of the mixture, encapsulating at their optimal RM size. These findings are important for developing thermodynamic models to complement applications of 10MAG/LDAO RMs for studying protein stability and water dynamics in the nanoconfined space.

Addition of Lauryldimethylamine N-Oxide (LDAO) to a Copper-Free Click Chemistry Reaction Improves the Conjugation Efficiency of a Cell-Free Generated CRM197 Variant to Clinically Important Streptococcus pneumoniae Serotypes.

Strain-promoted azide–alkyne cycloaddition (SPAAC) reactions like click chemistry have the potential to be highly scalable, robust, and cost-effective methods for generating small- and large-molecule conjugates for a variety of applications. However, despite method improvements, the rates of copper-based click chemistry reactions continue to be much faster than the rates of copper-free click chemistry reactions, which makes broader deployment of click chemistry challenging from a safety and compatibility standpoint. In this study, we used a zwitterionic detergent, namely, lauryldimethylamine N-oxide (LDAO), in a copper-free click chemistry reaction to investigate its impact on the generation of conjugate vaccines (CVs). For this, we utilized an Xpress cell-free protein synthesis (CFPS) platform to generate a proprietary variant of CRM197 (eCRM) containing non-native amino acids (nnAA) with azide-containing side chains as a carrier protein for conjugation to several clinically relevant dibenzocyclooctyne (DBCO)-derivatized S. pneumoniae serotypes (types 3, 5, 18C, and 19A). For conjugation, we performed copper-free click chemistry in the presence and absence of LDAO. Our results show that the addition of LDAO significantly enhanced the reaction kinetics to generate larger conjugates, which were similarly immunogenic and equally stable to conjugates generated without LDAO. Most importantly, the addition of LDAO substantially improved the efficiency of the conjugation process. Thus, our results for the first time show that the addition of a zwitterionic surfactant to a copper-free click chemistry reaction can significantly accelerate the reaction kinetics along with improving the efficiency of the conjugation process.

Effects of Detergents on the Spectral Features of B820 Bacteriochlorophyll a in Light-Harvesting Complex 3.

Excitonic coupling of bacteriochlorophyll (BChl) a in light-harvesting (LH) proteins of purple photosynthetic bacteria is key for efficient photon capture and energy transfer. Environmental factors can affect the spectral features of these BChl a pigments and investigating these effects can provide insight into the molecular mechanisms underlying the photosynthetic spectral tuning. The present study analyzes the spectral alterations of the Qy band of B820 BChl a within the LH3 protein in relation to the type and concentration of detergents in the buffer. Changing the detergent from lauryl dimethylamine N-oxide (LDAO) to n-dodecyl-β-d-maltoside (DDM) caused a red shift in the B820 Qy band accompanied by hyperchromism; these spectral alterations were completely reversed by exchanging back from DDM to LDAO. These results reflect the different effects of harsh vs mild detergents on the perturbation of LH3. The B820 Qy band did not change when LDAO or NaCl concentration was altered, suggesting that electrostatic effects by external components have little influence on the spectral features of B820 BChl a in LH3.

Effect of Detergents on Morphology, Size Distribution, and Concentration of Copolymer-Based Polymersomes.

Polymersomes made of amphiphilic diblock copolymers are generally regarded as having higher physical and chemical stability than liposomes composed of phospholipids. This enhanced stability arises from the higher molecular weight of polymer constituents. Despite their increased stability, polymer bilayers are solubilized by detergents in a similar manner to lipid bilayers. In this work, we evaluated the stability of poly(ethylene glycol)-block-poly(ε-caprolactone) (PEG-PCL)-based polymersomes exposed to three different detergents: N-octyl-β-d-glucopyranoside (OG), lauryldimethylamine N-oxide (LDAO), and Triton X-100 (TX-100). Changes in morphology, particle size distribution, and concentrations of the polymersomes were evaluated during the titration of the detergents into the polymersome solutions. Furthermore, we discussed the effect of detergent features on the solubilization of the polymeric bilayer and compared it to the results reported in the literature for liposomes and polymersomes. This information can be used for tuning the properties of PEG-PCL polymersomes for use in applications such as drug delivery or protein reconstitution studies.

Fast and Robust Quantification of Detergent Micellization Thermodynamics from Isothermal Titration Calorimetry.

Detergents are widely used in modern in vitro biochemistry and biophysics, in particular to aid the characterization of integral membrane proteins. An important characteristic of these chemicals in aqueous solutions is the concentration above which their molecular monomers self-associate to form micelles, termed the critical micellar concentration (CMC). Micelles are supramolecular assemblies arranged with the hydrophobic portions oriented inward and the hydrophilic head groups positioned outward to interact with the aqueous solvent. Knowledge of the CMC is not only of practical relevance but also of theoretical interest because it provides thermodynamic insights. Isothermal titration calorimetry (ITC) is a powerful method to determine CMCs, as it furnishes additional information on the enthalpy and entropy of micellization. Here we describe our extension of previous methods to determine CMCs and other thermodynamic parameters from ITC demicellization curves. The new algorithm, incorporated into the stand-alone software package D/STAIN, analyzes ITC demicellization curves by taking advantage of state-of-the-art thermogram-integration techniques and automatically providing rigorous confidence intervals on the refined parameters. As a demonstration of the software's capabilities, we undertook ITC experiments to determine the respective CMCs of n-octyl β-d-glucopyranoside (OG), n-dodecyl β-d-maltopyranoside (DDM), and lauryldimethylamine N-oxide (LDAO). Motivated by the fact that in vitro membrane protein studies often require additives such as precipitants (e.g., polyethylene glycol (PEG)), we also carried out ITC demicellization studies in the presence of PEG3350, finding in all cases that PEG had significant effects on the thermodynamics of detergent micellization.

Reverse Micelle Surfactant System Comprising the 1-decanoyl-rac-glycerol and the Lauryldimethylamine-N-oxide: Structure and Dynamics of Confined Water

A mixture of two surfactants 1-decanoyl-rac-glycerol (MAG) and lauryldimethylamine-N-oxide (LDA) in pentane solvent was investigated by molecular dynamics simulation. The simulation was carried out in the presence and absence of flavodoxin protein. In each of the simulations, hexanol was used as a co-solvent. Water molecules were added to the mixtures to adapt to experimental conditions. The ratio of surfactants, solvent and water were selected according to experimental data. The moments of inertia and the radius of gyration of micelles were calculated and the results indicated that the protein caused the micelles dimensions to be smaller. Also, the protein has reduced the number of hydrogen bonds between the water molecules confined in the reverse micelles core, while the strength of these bonds increases. Calculating the diffusion coefficient also indicates that the flavodoxin reduces the mobility of water molecules. In the presence of 5 hexanol molecules, micelle retains its structure in the presence of flavodoxin and is oblate ellipsoid. Increasing the number of hexanol to 10, has caused the geometric shape of the reverse micelle to change in the presence and absence of flavodoxin.

Phospholipid distributions in purple phototrophic bacteria and LH1-RC core complexes

In contrast to plants, algae and cyanobacteria that contain glycolipids as the major lipid components in their photosynthetic membranes, phospholipids are the dominant lipids in the membranes of anoxygenic purple phototrophic bacteria. Although the phospholipid compositions in whole cells or membranes are known for a limited number of the purple bacteria, little is known about the phospholipids associated with individual photosynthetic complexes. In this study, we investigated the phospholipid distributions in both membranes and the light-harvesting 1-reaction center (LH1-RC) complexes purified from several purple sulfur and nonsulfur bacteria. 31P NMR was used for determining the phospholipid compositions and inductively coupled plasma atomic emission spectroscopy was used for measuring the total phosphorous contents. Combining these two techniques, we could determine the numbers of specific phospholipids in the purified LH1-RC complexes. A total of approximate 20–30 phospholipids per LH1-RC were detected as the tightly bound lipids in all species. The results revealed that while cardiolipin (CL) exists as a minor component in the membranes, it became the most abundant phospholipid in the purified core complexes and the sum of CL and phosphatidylglycerol accounted for more than two thirds of the total phospholipids for most species. Preferential association of these anionic phospholipids with the LH1-RC is discussed in the context of the recent high-resolution structure of this complex from Thermochromatium (Tch.) tepidum. The detergent lauryldimethylamine N-oxide was demonstrated to selectively remove phosphatidylethanolamine from the membrane of Tch. tepidum.

Characterization of Biocompatible Nanoparticles for Biophysical and Biomedical Applications

Reverse micelles are soft nanoparticles created when amphiphilic molecules surround small volumes of water in a bulk nonpolar solvent. Typically these mixtures spontaneously organize into roughly spherical complexes. RM particles are quite stable, and can be used to encapsulate small hydrophilic molecules. Under optimal conditions, they can also be used to encapsulate macromolecules without perturbing their structures. Recently, a mixture composed of decylmonoacyl glycerol (10MAG) and lauryldimethylamine-N-oxide (LDAO) has been shown to encapsulated a wide array of proteins and nucleic acids. In addition to its versatility, this mixture also has the benefit of biocompatibility. The Nucci lab is exploring the utility of this mixture for a variety of biomedical and nanotechnology applications. The purpose of the present study is to thoroughly characterize the shape and dispersity of 10MAG/LDAO RMs in the presence and absence of macromolecular cargo. Dynamic light scattering is a primary tool for this endeavor and is augmented by electron microscopy and nuclear magnetic resonance. This characterization informs further application of this mixture as a biotechnological development platform.

Evaluation of eco-friendly zwitterionic detergents for enveloped virus inactivation.

Inclusion of a detergent in protein biotherapeutic purification processes is a simple and very robust method for inactivating enveloped viruses. The detergent Triton X-100 has been used for many years and is part of the production process of several commercial therapeutic proteins. However, recent ecological studies have suggested that Triton X-100 and its break-down products can potentially behave as endocrine disrupters in aquatic organisms, raising concerns from an environmental impact perspective. As such, discharge of Triton X-100 into the waste water treatment plants is regulated in some jurisdictions, and alternative detergents for viral inactivation are required. In this work, we report on the identification and evaluation of more eco-friendly detergents as viable replacements for Triton X-100. Five detergent candidates with low to moderate environmental impact were initially identified and evaluated with respect to protein stability, followed by proof-of-concept virus inactivation studies using a model enveloped virus. From the set of candidates lauryldimethylamine N-oxide (LDAO) was identified as the most promising detergent due to its low ecotoxicity, robust anti-viral activity (LRV >4 at validation set-point conditions with X-MuLX), and absence of any negative impact on protein function. This detergent exhibited effective and robust virus inactivation in a broad range of protein concentrations, solution conductivities, pHs, and in several different cell culture fluid matrices. The only process parameter which correlated with reduced virus inactivation potency was LDAO concentration, and then only when the concentration was reduced to below the detergent's critical micelle concentration (CMC). Additionally, this work also demonstrated that LDAO was cleared to below detectable levels after Protein A affinity chromatography, making it suitable for use in a platform process that utilizes this chromatographic mode for protein capture. All these findings suggest that LDAO may be a practical alternative to Triton X-100 for use in protein therapeutic production processes for inactivating enveloped viruses. Biotechnol. Bioeng. 2017;114: 813-820. © 2016 Wiley Periodicals, Inc.

Revisiting the Interaction between the Chaperone Skp and Lipopolysaccharide

The bacterial outer membrane comprises two main classes of components, lipids and membrane proteins. These nonsoluble compounds are conveyed across the aqueous periplasm along specific molecular transport routes: the lipid lipopolysaccharide (LPS) is shuttled by the Lpt system, whereas outer membrane proteins (Omps) are transported by chaperones, including the periplasmic Skp. In this study, we revisit the specificity of the chaperone-lipid interaction of Skp and LPS. High-resolution NMR spectroscopy measurements indicate that LPS interacts with Skp nonspecifically, accompanied by destabilization of the Skp trimer and similar to denaturation by the nonnatural detergent lauryldimethylamine-N-oxide (LDAO). Bioinformatic analysis of amino acid conservation, structural analysis of LPS-binding proteins, and MD simulations further confirm the absence of a specific LPS binding site on Skp, making a biological relevance of the interaction unlikely. Instead, our analysis reveals a highly conserved salt-bridge network, which likely has a role for Skp function.

Calorimetric quantification of linked equilibria in cyclodextrin/lipid/detergent mixtures for membrane-protein reconstitution

Reconstitution from detergent micelles into lipid bilayer membranes is a prerequisite for many in vitro studies on purified membrane proteins. Complexation by cyclodextrins offers an efficient and tightly controllable way of removing detergents for membrane-protein reconstitution, since cyclodextrins sequester detergents at defined stoichiometries and with tuneable affinities. To fully exploit the potential advantages of cyclodextrin for membrane-protein reconstitution, we establish a quantitative model for predicting the supramolecular transition from mixed micelles to vesicles during cyclodextrin-mediated detergent extraction. The model is based on a set of linked equilibria among all pseudophases present in the course of the reconstitution process. Various isothermal titration-calorimetric protocols are used for quantifying a detergent’s self-association as well as its colloidal and stoichiometric interactions with lipid and cyclodextrin, respectively. The detergent’s critical micellar concentration, the phase boundaries in the lipid/detergent phase diagram, and the dissociation constant of the cyclodextrin/detergent complex thus obtained provide all thermodynamic parameters necessary for a quantitative prediction of the transition from micelles to bilayer membranes during cyclodextrin-driven reconstitution. This is exemplified and validated by stepwise complexation of the detergent lauryldimethylamine N-oxide in mixtures with the phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine upon titration with 2-hydroxypropyl-β-cyclodextrin, both in the presence and in the absence of the membrane protein Mistic. The calorimetric approach presented herein quantitatively predicts the onset and completion of the reconstitution process, thus obviating cumbersome trial-and-error efforts and facilitating the rational optimisation of reconstitution protocols, and can be adapted to different cyclodextrin/lipid/detergent combinations.

Stubborn contaminants: influence of detergents on the purity of the multidrug ABC transporter BmrA.

Despite the growing interest in membrane proteins, their crystallization remains a major challenge. In the course of a crystallographic study on the multidrug ATP-binding cassette transporter BmrA, mass spectral analyses on samples purified with six selected detergents revealed unexpected protein contamination visible for the most part on overloaded SDS-PAGE. A major contamination from the outer membrane protein OmpF was detected in purifications with Foscholine 12 (FC12) but not with Lauryldimethylamine-N-oxide (LDAO) or any of the maltose-based detergents. Consequently, in the FC12 purified BmrA, OmpF easily crystallized over BmrA in a new space group, and whose structure is reported here. We therefore devised an optimized protocol to eliminate OmpF during the FC12 purification of BmrA. On the other hand, an additional band visible at ∼110 kDa was detected in all samples purified with the maltose-based detergents. It contained AcrB that crystallized over BmrA despite its trace amounts. Highly pure BmrA preparations could be obtained using either a ΔacrAB E. coli strain and n-dodecyl-β-D-maltopyranoside, or a classical E. coli strain and lauryl maltose neopentyl glycol for the overexpression and purification, respectively. Overall our results urge to incorporate a proteomics-based purity analysis into quality control checks prior to commencing crystallization assays of membrane proteins that are notoriously arduous to crystallize. Moreover, the strategies developed here to selectively eliminate obstinate contaminants should be applicable to the purification of other membrane proteins overexpressed in E. coli.

Nanostructures from Self‐Assembling Triazine Tertiary Amine N‐Oxide Amphiphiles

A new set of amphiphilic tertiary amine N-oxides has been prepared and their self-assembly properties observed in aqueous solution by tensiometry, dynamic and static light scattering. X-ray crystallographic analysis of parent amines and sulfoxide congeners indicates the formation of hydrogen-bonded dimers as the primary assembly unit for formation of vesicles in preference to the compact micelles typical of lauryl dimethylamine N-oxide (LDAO). 6-Benzyloxy-N,N'-bis(5-diethylaminopentylamine oxide)[1,3,5]triazine-2,4-diamine forms a 1 μm vesicle observed to entrap fluorescein. The [1,3,5]triazine core thus allows variation of the new self-assembled structures from nano- to micrometre length scales.

Single-Molecule Spectroscopy on RC-LH1 Complexes ofRhodopseudomonas acidophilaStrain 10050

We have revisited the RC-LH1 complex from Rhodopseudomonas (Rps.) acidophila for single-molecule spectroscopy. For the current study the pigment-protein complexes were stabilized in the detergent buffer solution using a relatively mild detergent (dodecyl-β-D-maltoside (DDM) instead of lauryldimethylamine N-oxide (LDAO)). This leads to a significant reduction of the fraction of broken/dissociated RC-LH1 complexes with respect to previous studies and has allowed us to investigate a sufficiently large sample of individual RC-LH1 complexes. For most of the complexes the fluorescence-excitation spectra exhibit a narrow spectral feature at the red end of the spectrum. Analysis of the statistics of the spectral properties yields a close resemblance with the results obtained on RC-LH1 complexes from Rps. palustris for which a low-resolution X-ray structure is available. Based on this comparison we come to the conclusion that for both species the RC-LH1 complex can be described by the same structural model, that is, an overall elliptical assembly of pigments that features a gap.

Transmembrane Gate Movements in the Type II ATP-binding Cassette (ABC) Importer BtuCD-F during Nucleotide Cycle

ATP-binding cassette (ABC) transporters are ubiquitous integral membrane proteins that translocate substrates across cell membranes. The alternating access of their transmembrane domains to opposite sides of the membrane powered by the closure and reopening of the nucleotide binding domains is proposed to drive the translocation events. Despite clear structural similarities, evidence for considerable mechanistic diversity starts to accumulate within the importers subfamily. We present here a detailed study of the gating mechanism of a type II ABC importer, the BtuCD-F vitamin B(12) importer from Escherichia coli, elucidated by EPR spectroscopy. Distance changes at key positions in the translocation gates in the nucleotide-free, ATP- and ADP-bound conformations of the transporter were measured in detergent micelles and liposomes. The translocation gates of the BtuCD-F complex undergo conformational changes in line with a "two-state" alternating access model. We provide the first direct evidence that binding of ATP drives the gates to an inward-facing conformation, in contrast to type I importers specific for maltose, molybdate, or methionine. Following ATP hydrolysis, the translocation gates restore to an apo-like conformation. In the presence of ATP, an excess of vitamin B(12) promotes the reopening of the gates toward the periplasm and the dislodgment of BtuF from the transporter. The EPR data allow a productive translocation cycle of the vitamin B(12) transporter to be modeled.