Perturbations of Native Membrane Protein Structure in Alkyl Phosphocholine Detergents: A Critical Assessment of NMR and Biophysical Studies.

Membrane proteins perform a host of vital cellular functions. Deciphering the molecular mechanisms whereby they fulfill these functions requires detailed biophysical and structural investigations. Detergents have proven pivotal to extract the protein from its native surroundings. Yet, they provide a milieu that departs significantly from that of the biological membrane, to the extent that the structure, the dynamics, and the interactions of membrane proteins in detergents may considerably vary, as compared to the native environment. Understanding the impact of detergents on membrane proteins is, therefore, crucial to assess the biological relevance of results obtained in detergents. Here, we review the strengths and weaknesses of alkyl phosphocholines (or foscholines), the most widely used detergent in solution-NMR studies of membrane proteins. While this class of detergents is often successful for membrane protein solubilization, a growing list of examples points to destabilizing and denaturing properties, in particular for α-helical membrane proteins. Our comprehensive analysis stresses the importance of stringent controls when working with this class of detergents and when analyzing the structure and dynamics of membrane proteins in alkyl phosphocholine detergents.

Cell-free production, purification and characterization of human mitochondrial ADP/ATP carriers.

Mitochondrial Carriers (MCs) are responsible for fluent traffic of a variety of compounds that need to be shuttled via mitochondrial inner membranes to maintain cell metabolism. The ADP/ATP Carriers (AACs) are responsible for the import of ADP inside the mitochondria and the export of newly synthesized ATP. In human, four different AACs isoforms are described which are expressed in tissue-specific manner. They are involved in different genetic diseases and play a role in cancerogenesis. Up to now only the structures of the bovine (isoform 1) and yeast (isoforms 2 and 3) AAC have been determined in one particular conformation, obtained in complex with the CATR inhibitor. Herein, we report that full-length human ADP/ATP Carriers isoform 1 and 3 were successfully expressed in cell-free system and purified in milligram amounts in detergent-solubilized state. The proteins exhibited the expected secondary structure content. Thermostability profiles showing stabilization by the CATR inhibitor suggest that the carriers are well folded.

The substrate specificity of the human ADP/ATP carrier AAC1.

The mitochondrial ADP/ATP carrier imports ADP from the cytosol into the mitochondrial matrix for its conversion to ATP by ATP synthase and exports ATP out of the mitochondrion to replenish the eukaryotic cell with chemical energy. Here the substrate specificity of the human mitochondrial ADP/ATP carrier AAC1 was determined by two different approaches. In the first the protein was functionally expressed in Escherichia coli membranes as a fusion protein with maltose binding protein and the effect of excess of unlabeled compounds on the uptake of [(32)P]-ATP was measured. In the second approach the protein was expressed in the cytoplasmic membrane of Lactococcus lactis. The uptake of [(14)C]-ADP in whole cells was measured in the presence of excess of unlabeled compounds and in fused membrane vesicles loaded with unlabeled compounds to demonstrate their transport. A large number of nucleotides were tested, but only ADP and ATP are suitable substrates for human AAC1, demonstrating a very narrow specificity. Next we tried to understand the molecular basis of this specificity by carrying out molecular-dynamics simulations with selected nucleotides, which were placed at the entrance of the central cavity. The binding of the phosphate groups of guanine and adenine nucleotides is similar, yet there is a low probability for the base moiety to be bound, likely to be rooted in the greater polarity of guanine compared to adenine. AMP is unlikely to engage fully with all contact points of the substrate binding site, suggesting that it cannot trigger translocation.

Production of UCP1 a membrane protein from the inner mitochondrial membrane using the cell free expression system in the presence of a fluorinated surfactant.

Structural studies of membrane protein are still challenging due to several severe bottlenecks, the first being the overproduction of well-folded proteins. Several expression systems are often explored in parallel to fulfil this task, or alternately prokaryotic analogues are considered. Although, mitochondrial carriers play key roles in several metabolic pathways, only the structure of the ADP/ATP carrier purified from bovine heart mitochondria was determined so far. More generally, characterisations at the molecular level are restricted to ADP/ATP carrier or the uncoupling protein UCP1, another member of the mitochondrial carrier family, which is abundant in brown adipose tissues. Indeed, mitochondrial carriers have no prokaryotic homologues and very few efficient expression systems were described so far for these proteins. We succeeded in producing UCP1 using a cell free expression system based on E. coli extracts, in quantities that are compatible with structural approaches. The protein was synthesised in the presence of a fluorinated surfactant, which maintains the protein in a soluble form. Further biochemical and biophysical analysis such as size exclusion chromatography, circular dichroism and thermal stability, of the purified protein showed that the protein is non-aggregated, monodisperse and well-folded.

Dangerous liaisons between detergents and membrane proteins. The case of mitochondrial uncoupling protein 2.

The extraction of membrane proteins from their native environment by detergents is central to their biophysical characterization. Recent studies have emphasized that detergents may perturb the structure locally and modify the dynamics of membrane proteins. However, it remains challenging to determine whether these perturbations are negligible or could be responsible for misfolded conformations, altering the protein's function. In this work, we propose an original strategy combining functional studies and molecular simulations to address the physiological relevance of membrane protein structures obtained in the presence of detergents. We apply our strategy to a structure of isoform 2 of an uncoupling protein (UCP2) binding an inhibitor recently obtained in dodecylphosphocholine detergent micelles. Although this structure shares common traits with the ADP/ATP carrier, a member of the same protein family, its functional and biological significance remains to be addressed. In the present investigation, we demonstrate how dodecylphosphocholine severely alters the structure as well as the function of UCPs. The proposed original strategy opens new vistas for probing the physiological relevance of three-dimensional structures of membrane proteins obtained in non-native environments.

The non-Newtonian behavior of detergents during concentration is increased by macromolecules, in trans, and results in their over-concentration.

Concentration of pure membrane proteins in detergent solution results in detergent concentration, albeit in unknown amounts. This phenomenon is observed in every lab working on membrane proteins, but has seldom been investigated. In this study, we explored the behavior of detergents mixed with membrane proteins during the step of sample concentration using centrifugal devices. We show that detergent over-concentrate with the presence of polymers, typically membrane or soluble proteins but also polysaccharides. The over-concentration of detergents depends on centrifugal force applied to the device. With the use of a specific dye, we observed the formation of a mesh on the concentrator device. Importantly, reducing the centrifugal speed allows to reduce the concentration of detergents when mixed to macromolecules, as tested with 3 different membrane proteins. All together, these results highlight the non-Newtonian behavior of detergents and provides a solid framework to investigators to improve drastically biochemical and structural studies of membrane proteins.

Zwitterionic fluorinated detergents: From design to membrane protein applications.

We report herein the synthesis of zwitterionic sulfobetaine (SB) and dimethylamine oxide (AO) detergents whose alkyl chain is made of either a perfluorohexyl (F6H3) or a perfluoropentyl (F5H5) group linked to a hydrogenated spacer arm. In aqueous solution, the critical micellar concentrations (CMCs) measured by surface tensiometry (SFT) and isothermal titration calorimetry (ITC) were found in the millimolar range (1.3-2.4 mM). The morphologies of the aggregates were evaluated by dynamic light scattering (DLS), analytical ultracentrifugation (AUC), small-angle X-ray scattering (SAXS), and transmission electron microscopy (TEM), demonstrating that the two perfluoropentyl derivatives formed small micelles less than 10 nm in diameter, whereas the perfluorohexyl derivatives formed larger and more heterogeneous micelles. The two SB detergents were able to solubilize synthetic lipid vesicles in a few hours; by contrast, the perfluoropentyl AO induced much faster solubilization, whereas the perfluorohexyl AO did not show any solubilization. All detergents were tested for their abilities to stabilize three membrane proteins, namely, bacteriorhodopsin (bR), the Bacillus subtilis ABC transporter BmrA, and the Streptococcus pneumoniae enzyme SpNOX. The SB detergents outperformed the AO derivatives as well as their hydrogenated analogs in stabilizing these proteins. Among the four new compounds, F5H5SB combines many desirable properties for membrane-protein study, as it is a powerful yet gentle detergent.

Expression of a chloroplast ATP/ADP transporter in E. coli membranes: behind the Mistic strategy.

Eukaryotic membrane protein expression is still a major bottleneck for structural studies. Production in E. coli often leads to low expression level and/or aggregated proteins. In the last decade, strategies relying on new fusion protein expression revealed promising results. Fusion with the amphipatic Mistic protein has been described to favor expression in E. coli membranes. Although, this approach has already been reported for a few membrane proteins, little is known about the activity of the fused proteins. We used this strategy and obtained high expression levels of a chloroplast ATP/ADP transporter from A. thaliana (NTT1) and characterized its transport properties. NTT1 fused to Mistic has a very low transport activity which can be recovered after in vivo Mistic fusion cleavage. Moreover, detailed molecular characterization of purified NTT1 mature form, NTT1 fused to Mistic or NTT1 cleaved-off from this fusion highlights the correct fold of the latter one. Therefore, considering the higher quantity of purified NTT1 mature form obtained via the Mistic fusion approach, this is a valuable strategy for obtaining quantities of pure and active proteins that are adequate for structural studies.

Contamination from an affinity column: an encounter with a new villain in the world of membrane-protein crystallization.

Attempts to crystallize AtNTT1, a chloroplast ATP/ADP transporter from Arabidopsis thaliana, revealed an unexpected contaminant, Strep-Tactin, a variant of streptavidin that was used during purification of the protein. Although it was present in very small amounts, crystals of Strep-Tactin were reproducibly grown from the AtNTT1 solution. AtNTT1 was overexpressed in Escherichia coli and purified from detergent-solubilized membrane fractions using Strep-Tactin affinity chromatography based on an engineered streptavidin. The contamination of protein solutions purified on Strep-Tactin columns has never been described previously and seems to be specific to membrane proteins solubilized in detergents. Trace amounts of Strep-Tactin were observed to be eluted from a Strep-Tactin column using several routinely used detergents, illustrating their possible role in the contamination. This finding raises an alarm and suggests caution in membrane-protein purification using Strep-Tactin affinity columns, where detergents are essential components. The small crystals of contaminant protein led to the structure at 1.9 Å resolution of Strep-Tactin in complex with desthiobiotin.

Assaying the proton transport and regulation of UCP1 using solid supported membranes.

The uncoupling protein 1 (UCP1) is a mitochondrial protein that carries protons across the inner mitochondrial membrane. It has an important role in non-shivering thermogenesis, and recent evidence suggests its role in human adult metabolism. Using rapid solution exchange on solid supported membranes, we succeeded in measuring electrical currents generated by the transport activity of UCP1. The protein was purified from mouse brown adipose tissue, reconstituted in liposomes and absorbed on solid supported membranes. A fast pH jump activated the ion transport, and electrical signals could be recorded. The currents were characterized by a fast rise and a slow decay, were stable over time, inhibited by purine nucleotides and activated by fatty acids. This new assay permits direct observation of UCP1 activity in controlled cell-free conditions, and opens up new possibilities for UCP1 functional characterization and drug screening because of its robustness and its potential for automation.

Determining membrane protein structures: still a challenge!

Determination of structures and dynamics events of transmembrane proteins is important for the understanding of their function. Analysis of such events requires high-resolution 3D structures of the different conformations coupled with molecular dynamics analyses describing the conformational pathways. However, the solution of 3D structures of transmembrane proteins at atomic level remains a particular challenge for structural biochemists--the need for purified and functional transmembrane proteins causes a 'bottleneck'. There are various ways to obtain 3D structures: X-ray diffraction, electron microscopy, NMR and modelling; these methods are not used exclusively of each other, and the chosen combination depends on several criteria. Progress in this field will improve knowledge of ligand-induced activation and inhibition of membrane proteins in addition to aiding the design of membrane-protein-targeted drugs.

Conformational transitions and ligand-binding to a muscle-type nicotinic acetylcholine receptor.

Fast synaptic communication requires receptors that respond to the presence of neurotransmitter by opening an ion channel across the post-synaptic membrane. The muscle-type nicotinic acetylcholine receptor from the electric fish, Torpedo, is the prototypic ligand-gated ion channel, yet the structural changes underlying channel activation remain undefined. Here we use cryo-EM to solve apo and agonist-bound structures of the Torpedo nicotinic receptor embedded in a lipid nanodisc. Using both a direct biochemical assay to define the conformational landscape and molecular dynamics simulations to assay flux through the pore, we correlate structures with functional states and elucidate the motions that lead to pore activation of a heteromeric nicotinic receptor. We highlight an underappreciated role for the complementary subunit in channel gating, establish the structural basis for the differential agonist affinities of α/δ versus α /γ sites, and explain why nicotine is less potent at muscle nicotinic receptors compared to neuronal ones.

Glucose-Based Fluorinated Surfactants as Additives for the Crystallization of Membrane Proteins: Synthesis and Preliminary Physical-Chemical and Biochemical Characterization.

We report herein the synthesis of a series of fluorinated surfactants with a glucose moiety as a polar head group and whose alkyl chain was varied in length and in fluorine/hydrogen ratio. They were synthesized in two or four steps in 20 to 50% overall yields allowing gram-scale synthesis. Their solubility in water is between 0.2 and 13.8 g/L, which indicates low water solubility. Two derivatives of the series were found to form micelles in water at ∼11 mM. Their hydrophilic-lipophilic balance was determined both by Griffin's and Davies' methods; they may exhibit a "harsh" character toward membrane proteins. This, combined with their low water solubility, suggest that they could advantageously be used in detergent mixtures containing a "mild" detergent. Finally, the potency of one of the derivatives, F3H5-ß-Glu, to act as an additive for the crystallization of AcrB was evaluated in detergent mixtures with n-dodecyl-ß-d-maltopyranoside (DDM). Among the six crystallization conditions investigated, adding F3H5-ß-Glu improved the crystallization for three of them, as compared to control drops without additives. Moreover, preliminary tests with other compounds of the series showed that none of them hampered crystallization and suggested improvement for three of them. These novel glucose-based fluorinated detergents should be regarded as potential additives that could be included in screening kits used in crystallization.

NADPH oxidase activator p67(phox) behaves in solution as a multidomain protein with semi-flexible linkers.

The NADPH oxidase complex is involved in the destruction of phagocytosed pathogens through the production of reactive oxygen species. This activatable complex consists of a membranous heterodimeric flavocytochrome b, a small G-protein Rac1/Rac2 and cytosolic factors, p47(phox), p67(phox) and p40(phox). p67(phox), due to its modular structure, is the NADPH oxidase component for which global structure information is most scarce despite its mandatory role in activation and its central position in the whole complex organization. Indeed, p67(phox) is the only factor establishing interaction with all others. In this study, we report the SAXS analysis of p67(phox). Our data reveals that p67(phox) behaves as a multidomain protein with semi-flexible linkers. On the one hand, it appears to be a very elongated molecule with its various domains organized as beads on a string. Linkers are predicted to be partially or mainly unstructured and features of our experimental data do point towards inter-domain flexibility. On the other hand, our work also suggests that the protein is not as extended as unstructured linkers could allow, thereby implying the existence of intra-molecular interactions within p67(phox). We suggest that the dual character of p67(phox) conformation in solution is central to ensure the numerous interactions to be accommodated.

Structure of mitochondrial ADP/ATP carrier in complex with carboxyatractyloside.

ATP, the principal energy currency of the cell, fuels most biosynthetic reactions in the cytoplasm by its hydrolysis into ADP and inorganic phosphate. Because resynthesis of ATP occurs in the mitochondrial matrix, ATP is exported into the cytoplasm while ADP is imported into the matrix. The exchange is accomplished by a single protein, the ADP/ATP carrier. Here we have solved the bovine carrier structure at a resolution of 2.2 A by X-ray crystallography in complex with an inhibitor, carboxyatractyloside. Six alpha-helices form a compact transmembrane domain, which, at the surface towards the space between inner and outer mitochondrial membranes, reveals a deep depression. At its bottom, a hexapeptide carrying the signature of nucleotide carriers (RRRMMM) is located. Our structure, together with earlier biochemical results, suggests that transport substrates bind to the bottom of the cavity and that translocation results from a transient transition from a 'pit' to a 'channel' conformation.

High-chloride concentrations abolish the binding of adenine nucleotides in the mitochondrial ADP/ATP carrier family.

The ADP/ATP carrier (AAC) is a very effective membrane protein that mediates the exchange of ADP and ATP across the mitochondrial membrane. In vivo transport measurements on the AAC overexpressed in Escherichia coli demonstrate that this process can be severely inhibited by high-chloride concentrations. Molecular-dynamics simulations reveal a strong modification of the topology of the local electric field related to the number of chloride ions inside the cavity. Halide ions are shown to shield the positive charges lining the internal cavity of the carrier by accurate targeting of key basic residues. These specific amino acids are highly conserved as highlighted by the analysis of multiple AAC sequences. These results strongly suggest that the chloride concentration acts as an electrostatic lock for the mitochondrial AAC family, thereby preventing adenine nucleotides from reaching their dedicated binding sites.

A first low-resolution difference Fourier map of phosphorus in a membrane protein from near-edge anomalous diffraction.

Crystal diffraction of three membrane proteins (cytochrome bc(1) complex, sarcoplasmic reticulum Ca(2+) ATPase, ADP-ATP carrier) and of one nucleoprotein complex (leucyl tRNA synthetase bound to tRNAleu, leuRS:tRNAleu) was tested at wavelengths near the X-ray K-absorption edge of phosphorus using a new set-up for soft X-ray diffraction at the beamline ID01 of the ESRF. The best result was obtained from crystals of Ca(2+) ATPase [adenosin-5'-(beta,gamma-methylene) triphosphate complex] which diffracted out to 7 A resolution. Data were recorded at a wavelength at which the real resonant scattering factor of phosphorus reaches the extreme value of -20 electron units. The positions of the four triphosphates of the monoclinic unit cell of the ATPase have been obtained from a difference Fourier synthesis based on a limited set of anomalous diffraction data.

Mitochondrial bovine ADP/ATP carrier in detergent is predominantly monomeric but also forms multimeric species.

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

Binding of ADP in the mitochondrial ADP/ATP carrier is driven by an electrostatic funnel.

The ADP/ATP carrier (AAC) is a membrane protein of paramount importance for the energy-fueling function of the mitochondria, transporting ADP from the intermembrane space to the matrix and ATP in the opposite direction. On the basis of the high-resolution, 2.2-A structure of the bovine carrier, a total of 0.53 micros of classical molecular dynamics simulations were conducted in a realistic membrane environment to decipher the early events of ADP (3-) translocation across the inner membrane of the mitochondria. Examination of apo-AAC underscores the impermeable nature of the carrier, impeding passive transport of permeants toward the matrix. The electrostatic funnel illuminated from three-dimensional mapping of the electrostatic potential forms a privileged passageway anticipated to drive the diphosphate nucleotide rapidly toward the bottom of the internal cavity. This conjecture is verified in the light of repeated, independent numerical experiments, whereby the permeant is dropped near the mouth of the mitochondrial carrier. Systematic association of ADP (3-) to the crevice of the AAC, an early event in its transport across the inner membrane, is accompanied by the formation of an intricate network of noncovalent bonds. Simulations relying on the use of an adaptive biasing force reveal for the first time that the proposed binding site corresponds to a minimum of the free energy landscape delineating the translocation of ADP (3-) in the carrier. The present work paves the way to the design of novel nucleotides and new experiments aimed at unveiling key structural features in the chronology of ADP/ATP transport across the mitochondrial membrane.

Nucleotide exchange in mitochondria: insight at a molecular level.

Mitochondrial carrier proteins are embedded in the inner mitochondrial membrane and ensure the transport of many important metabolites. The ADP/ATP carrier imports ADP into the mitochondrial matrix in exchange for ATP after synthesis. It is the most studied mitochondrial carrier and its structure was the first to be unraveled at high resolution. The structure reveals six transmembrane helices forming a tightly closed bundle toward the matrix and a funnel-shaped cavity opening toward the intermembrane space. The cavity ends in a narrow pit 10A from the matrix. The analysis of residues located in the cavity hints at the mechanism of binding of adenine nucleotides. Additionally, the presence of conserved proline residues in three sharply kinked helices suggests a translocation mechanism.