[Show abstract][Hide abstract] ABSTRACT: Plant cell-to-cell communication is mediated by nanopores called plasmodesmata (PDs) which are complex structures comprising plasma membrane (PM), highly packed endoplasmic reticulum and numerous membrane proteins. Although recent advances on proteomics have led to insights into mechanisms of transport, there is still an inadequate characterization of the lipidic composition of the PM where membrane proteins are inserted. It has been postulated that PDs could be formed by lipid rafts, however no structural evidence has shown to visualize and analyse their lipid components. In this perspective article, we discuss proposed experiments to characterize lipid rafts and proteins in the PDs. By using atomic force microscopy (AFM) and mass spectrometry (MS) of purified PD vesicles it is possible to determine the presence of lipid rafts, specific bound proteins and the lipidomic profile of the PD under physiological conditions and after changing transport permeability. In addition, MS can determine the stoichiometry of intact membrane proteins inserted in lipid rafts. This will give novel insights into the role of membrane proteins and lipid rafts on the PD structure.
Frontiers in Plant Science 01/2014; 5:234. · 3.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The vas deferens is a simple bioassay widely used to study the physiology of sympathetic neurotransmission and the pharmacodynamics of adrenergic drugs. The role of ATP as a sympathetic co-transmitter has gained increasing attention and furthered our understanding of its role in sympathetic reflexes. In addition, new information has emerged on the mechanisms underlying the storage and release of ATP. Both noradrenaline and ATP concur to elicit the tissue smooth muscle contractions following sympathetic reflexes or electrical field stimulation of the sympathetic nerve terminals. ATP and adenosine (its metabolic byproduct) are powerful presynaptic regulators of co-transmitter actions. In addition, neuropeptide Y, the third member of the sympathetic triad, is an endogenous modulator. The peptide plus ATP and/or adenosine play a significant role as sympathetic modulators of transmitter’s release. This review focuses on the physiological principles that govern sympathetic co-transmitter activity, with special interest in defining the motor role of ATP. In addition, we intended to review the recent structural biology findings related to the topology of the P2X1R based on the crystallized P2X4 receptor from Danio rerio, or the crystallized adenosine A2A receptor as a member of the G protein coupled family of receptors as prototype neuro modulators. This review also covers structural elements of ectonucleotidases, since some members are found in the vas deferens neuro-effector junction. The allosteric principles that apply to purinoceptors are also reviewed highlighting concepts derived from receptor theory at the light of the current available structural elements. Finally, we discuss clinical applications of these concepts.
[Show abstract][Hide abstract] ABSTRACT: Ionotropic glutamate receptors are widely distributed in the central nervous system and play a major role in excitatory synaptic transmission. All three ionotropic glutamate subfamilies (i.e. AMPA-type, kainate-type and NMDA-type) assemble as tetramers of four homologous subunits. There is good evidence that both heteromeric AMPA and kainate receptors have a 2:2 subunit stoichiometry and an alternating subunit arrangement. Recent studies based on presumed structural homology have indicated that NMDA receptors adopt the same arrangement. Here we use atomic force microscopy (AFM) imaging of receptor-antibody complexes to show that while the GluA1/GluA2 AMPA receptor assembles with an alternating (i.e. 1/2/1/2) subunit arrangement, the GluN1/GluN2A NMDA receptor adopts an adjacent (i.e. 1/1/2/2) arrangement. We conclude that the two types of ionotropic glutamate receptor are built in different ways from their constituent subunits. This surprising finding necessitates a reassessment of the assembly of these important receptors.
Journal of Biological Chemistry 06/2013; · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cellular membranes comprise hundreds of lipids in which protein complexes, such as ion channels, receptors, and scaffolding complexes, are embedded. These protein assemblies act as signalling and trafficking platforms for processes fundamental to life. Much effort in recent years has focused on identifying the protein components of these complexes after their extraction from the lipid membrane in detergent micelles. Spectacular advances have been made using X-ray crystallography, providing in some cases detailed information about the mechanism of pumping and channel gating. These structural studies are leading to a growing realisation that, to understand their function, it is not only the structures of the protein components that are important but also knowledge of the protein-lipid interactions. This review highlights recent insights gained from this knowledge, surveys methods being developed for probing these interactions, and focuses specifically on the potential of mass spectrometry in this growing area of research.
Trends in cell biology 09/2012; · 12.12 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: That membrane protein complexes could survive in the gas phase had always seemed impossible. The lack of chargeable residues, high hydrophobicity, and poor solubility and the vast excess of detergent contributed to the view that it would not be possible to obtain mass spectra of intact membrane complexes. With the recent success in recording mass spectra of these complexes, first from recombinant sources and later from the cellular environment, many surprising properties of these gas phase membrane complexes have been revealed. The first of these was that the interactions between membrane and soluble subunits could survive in vacuum, without detergent molecules adhering to the complex. The second unexpected feature was that their hydrophobicity and, consequently, lower charge state did not preclude ionization. The final surprising finding was that these gas phase membrane complexes carry with them lipids, bound specifically in subunit interfaces. This provides us with an opportunity to distinguish annular lipids that surround the membrane complexes, from structural lipids that have a role in maintaining structure and subunit interactions. In this perspective, we track these developments and suggest explanations for the various discoveries made during this research.
Journal of Molecular Biology 06/2012; 423(1):1-13. · 3.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The ability of electrospray to propel large viruses into a mass spectrometer is established and is rationalized by analogy to the atmospheric transmission of the common cold. Much less clear is the fate of membrane-embedded molecular machines in the gas phase. Here we show that rotary adenosine triphosphatases (ATPases)/synthases from Thermus thermophilus and Enterococcus hirae can be maintained intact with membrane and soluble subunit interactions preserved in vacuum. Mass spectra reveal subunit stoichiometries and the identity of tightly bound lipids within the membrane rotors. Moreover, subcomplexes formed in solution and gas phases reveal the regulatory effects of nucleotide binding on both ATP hydrolysis and proton translocation. Consequently, we can link specific lipid and nucleotide binding with distinct regulatory roles.
[Show abstract][Hide abstract] ABSTRACT: Rapid advances in structural genomics and in large-scale proteomic projects have yielded vast amounts of data on soluble proteins and their complexes. Despite these advances, progress in studying membrane proteins using mass spectrometry (MS) has been slow. This is due in part to the inherent solubility and dynamic properties of these proteins, but also to their low abundance and the absence of polar side chains in amino acid residues. Considerable progress in overcoming these challenges is, however, now being made for all levels of structural characterization. This progress includes MS studies of the primary structure of membrane proteins, wherein sophisticated enrichment and trapping procedures are allowing multiple posttranslational modifications to be defined through to the secondary structure level in which proteins and peptides have been probed using hydrogen exchange, covalent, or radiolytic labeling methods. Exciting possibilities now exist to go beyond primary and secondary structure to reveal the tertiary and quaternary interactions of soluble and membrane subunits within intact assemblies of more than 700 kDa.
Annual review of biochemistry 06/2011; 80:247-71. · 29.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The multiple transferable resistance (mTR) pump from Neisseria gonorrhoeae MtrCDE multidrug pump is assembled from the inner and outer membrane proteins MtrD and MtrE and the periplasmic membrane fusion protein MtrC. Previously we established that while there is a weak interaction of MtrD and MtrE, MtrC binds with relatively high affinity to both MtrD and MtrE. MtrD conferred antibiotic resistance only when it was expressed with MtrE and MtrC, suggesting that these proteins form a functional tripartite complex in which MtrC bridges MtrD and MtrE. Furthermore, we demonstrated that MtrC interacts with an intraprotomer groove on the surface of MtrE, inducing channel opening. However, a second groove is apparent at the interface of the MtrE subunits, which might also be capable of engaging MtrC. We have now established that MtrC can be cross-linked to cysteines placed in this interprotomer groove and that mutation of residues in the groove impair the ability of the pump to confer antibiotic resistance by locking MtrE in the closed channel conformation. Moreover, MtrE K390C forms an intermolecular disulfide bond with MtrC E149C locking MtrE in the open channel conformation, suggesting that a functional salt bridge forms between these residues during the transition from closed to open channel conformations. MtrC forms dimers that assemble into hexamers, and electron microscopy studies of single particles revealed that these hexamers are arranged into ring-like structures with an internal aperture sufficiently large to accommodate the MtrE trimer. Cross-linking of single cysteine mutants of MtrC to stabilize the dimer interface in the presence of MtrE, trapped an MtrC-MtrE complex with a molecular mass consistent with a stoichiometry of 3:6 (MtrE(3)MtrC(6)), suggesting that dimers of MtrC interact with MtrE, presumably by binding to the two grooves. As both MtrE and MtrD are trimeric, our studies suggest that the functional pump is assembled with a stoichiometry of 3:6:3.
Journal of Biological Chemistry 05/2011; 286(30):26900-12. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The multiple transferable resistance (MTR) pump, from Neisseria gonorrhoeae, is typical of the specialized machinery used to translocate drugs across the inner and outer membranes of Gram-negative bacteria. It consists of a tripartite complex composed of an inner-membrane transporter, MtrD, a periplasmic membrane fusion protein, MtrC, and an outer-membrane channel, MtrE. We have expressed the components of the pump in Escherichia coli and used the antibiotic vancomycin, which is too large to cross the outer-membrane by passive diffusion, to test for opening of the MtrE channel. Cells expressing MtrCDE are not susceptible to vancomycin, indicating that the channel is closed; but become susceptible to vancomycin in the presence of transported substrates, consistent with drug-induced opening of the MtrE channel. A mutational analysis identified residues Asn-198, Glu-434, and Gln-441, lining an intraprotomer groove on the surface of MtrE, to be important for pump function; mutation of these residues yielded cells that were sensitive to vancomycin. Pull-down assays and micro-calorimetry measurements indicated that this functional impairment is not due to the inability of MtrC to interact with the MtrE mutants; nor was it due to the MtrE mutants adopting an open conformation, because cells expressing these MtrE mutants alone are relatively insensitive to vancomycin. However, cells expressing the MtrE mutants with MtrC are sensitive to vancomycin, indicating that residues lining the intra-protomer groove control opening of the MtrE channel in response to binding of MtrC.
Journal of Biological Chemistry 02/2011; 286(7):5484-93. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Our experimental approach is based on the atomic force microscope (AFM) imaging of epitope-tagged subunits within membrane protein complexes purified in small amounts and decorated by anti-tag antibodies. Furthermore, we can produce simultaneous decoration of protein complexes using Fab fragments and IgG antibodies, which, combined with chemical modification of the substrate, allows us to determine the protein orientation across the cell membrane. Here, we describe a detailed protocol for membrane protein purification, AFM data collection, analysis, and interpretation of results. The protocol also covers basic AFM instrument settings and best practices for both observation of membrane protein complexes by AFM and automatic detection of the structures by an in-house algorithm. Once a sufficient number of membrane protein complexes have been visualized by AFM, data acquisition and processing can be completed in approximately 10 min using a scanning surface of 1 μm(2).
[Show abstract][Hide abstract] ABSTRACT: Here we examined the gas-phase structures of two tetrameric membrane protein complexes by ion mobility mass spectrometry. The collision cross sections measured for the ion channel are in accord with a compact configuration of subunits, suggesting that the native-like structure can be preserved under the harsh activation conditions required to release it from the detergent micelle into the gas phase. We also found that the quaternary structure of the transporter, which has fewer transmembrane subunits than the ion channel, is less stable once stripped of detergents and bulk water. These results highlight the potential of ion mobility mass spectrometry for characterizing the overall topologies of membrane protein complexes and the structural changes associated with nucleotide, lipid, and drug binding.
Journal of the American Chemical Society 10/2010; 132(44):15468-70. · 10.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Many multi-protein assemblies exhibit characteristics which hamper their structural and dynamical characterization. These impediments include low copy number, heterogeneity, polydispersity, hydrophobicity, and intrinsic disorder. It is becoming increasingly apparent that both novel and hybrid structural biology approaches need to be developed to tackle the most challenging targets. Nanoelectrospray mass spectrometry has matured over the last decade to enable the elucidation of connectivity and composition of large protein assemblies. Moreover, comparing mass spectrometry data with transmission electron microscopy images has enabled the mapping of subunits within topological models. Here we describe a preparative form of mass spectrometry designed to isolate specific protein complexes from within a heterogeneous ensemble, and to 'soft-land' these target complexes for ex situ imaging. By building a retractable probe incorporating a versatile target holder, and modifying the ion optics of a commercial mass spectrometer, we show that we can steer the macromolecular ion beam onto a target for imaging by means of transmission electron microscopy and atomic force microscopy. Our data for the tetradecameric chaperonin GroEL show that not only are the molecular volumes of the landed particles consistent with the overall dimensions of the complex, but also that their gross topological features can be maintained.
Journal of Structural Biology 03/2010; 172(2):161-8. · 3.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 3,4-Methylenedioxymethamphetamine (MDMA), an important recreational psychostimulant drug, was examined for its ability to alter visuo-spatial learning and synaptic plasticity. Young rats received MDMA (0.2 and 2mg/kg s.c.) twice per day for 6 days while their visuo-spatial learning was tested using the Morris Water Maze. After this, animals were sacrificed and LTP induced in hippocampal slices. Visuo-spatial learning was impaired and LTP reduced, both dose-dependently, without changes in serotonin levels or paired-pulse facilitation. We conclude that low, nontoxic doses of MDMA, applied during several days, slow learning by impairing postsynaptic plasticity.
[Show abstract][Hide abstract] ABSTRACT: We describe a general mass spectrometry approach to determine subunit stoichiometry and lipid binding in intact membrane protein complexes. By exploring conditions for preserving interactions during transmission into the gas phase and for optimally stripping away detergent, by subjecting the complex to multiple collisions, we released the intact complex largely devoid of detergent. This enabled us to characterize both subunit stoichiometry and lipid binding in 4 membrane protein complexes.
[Show abstract][Hide abstract] ABSTRACT: LmrA is a multidrug ATP-binding cassette (ABC) transporter from Lactococcus lactis with no known physiological substrate, which can transport a wide range of chemotherapeutic agents and toxins from the cell. The protein can functionally replace the human homologue ABCB1 (also termed multidrug resistance P-glycoprotein MDR1) in lung fibroblast cells. Even though LmrA mediates ATP-dependent transport, it can use the proton-motive force to transport substrates, such as ethidium bromide, across the membrane by a reversible, H(+)-dependent, secondary-active transport reaction. The mechanism and physiological context of this reaction are not known.
We examined ion transport by LmrA in electrophysiological experiments and in transport studies using radioactive ions and fluorescent ion-selective probes. Here we show that LmrA itself can transport NaCl by a similar secondary-active mechanism as observed for ethidium bromide, by mediating apparent H(+)-Na(+)-Cl(-) symport. Remarkably, LmrA activity significantly enhances survival of high-salt adapted lactococcal cells during ionic downshift.
The observations on H(+)-Na(+)-Cl(-) co-transport substantiate earlier suggestions of H(+)-coupled transport by LmrA, and indicate a novel link between the activity of LmrA and salt stress. Our findings demonstrate the relevance of investigations into the bioenergetics of substrate translocation by ABC transporters for our understanding of fundamental mechanisms in this superfamily. This study represents the first use of electrophysiological techniques to analyze substrate transport by a purified multidrug transporter.
PLoS ONE 02/2009; 4(7):e6137. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Gram-negative bacteria utilize specialized machinery to translocate drugs and protein toxins across the inner and outer membranes, consisting of a tripartite complex composed of an inner membrane secondary or primary active transporter (IMP), a periplasmic membrane fusion protein, and an outer membrane channel. We have investigated the assembly and function of the MacAB/TolC system that confers resistance to macrolides in Escherichia coli. The membrane fusion protein MacA not only stabilizes the tripartite assembly by interacting with both the inner membrane protein MacB and the outer membrane protein TolC, but also has a role in regulating the function of MacB, apparently increasing its affinity for both erythromycin and ATP. Analysis of the kinetic behavior of ATP hydrolysis indicated that MacA promotes and stabilizes the ATP-binding form of the MacB transporter. For the first time, we have established unambiguously the dimeric nature of a noncanonic ABC transporter, MacB that has an N-terminal nucleotide binding domain, by means of nondissociating mass spectrometry, analytical ultracentrifugation, and atomic force microscopy. Structural studies of ABC transporters indicate that ATP is bound between a pair of nucleotide binding domains to stabilize a conformation in which the substrate-binding site is outward-facing. Consequently, our data suggest that in the presence of ATP the same conformation of MacB is promoted and stabilized by MacA. Thus, MacA would facilitate the delivery of drugs by MacB to TolC by enhancing the binding of drugs to it and inducing a conformation of MacB that is primed and competent for binding TolC. Our structural studies are an important first step in understanding how the tripartite complex is assembled.
Journal of Biological Chemistry 11/2008; 284(2):1145-54. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ionotropic receptors mediate rapid communication between neurons. These receptors are oligomers and are usually assembled from multiple subunit types. Receptors built from different subunit combinations have distinct functional properties, such as single-channel conductances, rates of desensitization and sensitivities to activators and inactivators; they can also have different intracellular locations. Methods are now available for determining not only the subunit stoichiometry but also the subunit arrangement within ionotropic receptors. This information will inform experiments designed to understand the molecular basis of receptor assembly and function. It will also permit the modelling of potential ligand-binding sites at the interfaces between the subunits and should lead to a more rational approach to drug development.
Trends in Neurosciences 10/2008; 31(11):569-76. · 13.58 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: There has been confusion about the subunit stoichiometry of the degenerin family of ion channels. Recently, a crystal structure of acid-sensing ion channel (ASIC) 1a revealed that it assembles as a trimer. Here, we used atomic force microscopy (AFM) to image unprocessed ASIC1a bound to mica. We detected a mixture of subunit monomers, dimers and trimers. In some cases, triple-subunit clusters were clearly visible, confirming the trimeric structure of the channel, and indicating that the trimer sometimes disaggregated after adhesion to the mica surface. This AFM-based technique will now enable us to determine the subunit arrangement within heteromeric ASICs.
Biochemical and Biophysical Research Communications 09/2008; 372(4):752-5. · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The ability to maintain interactions between soluble protein subunits in the gas phase of a mass spectrometer gives critical insight into the stoichiometry and interaction networks of protein complexes. Conversely, for membrane protein complexes in micelles, the transition into the gas phase usually leads to the disruption of interactions, particularly between cytoplasmic and membrane subunits, and a mass spectrum dominated by large aggregates of detergent molecules. We show that by applying nanoelectrospray to a micellar solution of a membrane protein complex, the heteromeric adenosine 5'-triphosphate (ATP)-binding cassette transporter BtuC2D2, we can maintain the complex intact in the gas phase of a mass spectrometer. Dissociation of either transmembrane (BtuC) or cytoplasmic (BtuD) subunits uncovers modifications to the transmembrane subunits and cooperative binding of ATP. By protecting a membrane protein complex within a n-dodecyl-beta-d-maltoside micelle, we demonstrated a powerful strategy that will enable the subunit stoichiometry and ligand-binding properties of membrane complexes to be determined directly, by precise determination of the masses of intact complexes and dissociated subunits.