Maltose Transport Research Articles

Fundamental Difference of Transport Mechanisms Between ABC Importers and Exporters

ABC transporters use the energy of ATP to drive the active transport of various substrates across the membrane. All members of this superfamily share a common machinery of two nucleotide binding domains (NBDs) at their cytoplasmic side, which close and open in response to ATP binding and hydrolysis. Despite highly homologous NBDs, substrates can be either imported or exported in different subfamilies. Although crystal structures of ABC transporters have been resolved in different conformational/functional states, it is still puzzling how a common set of motor domains can drive transport in opposite directions. We use comparative molecular dynamics simulations of an ABC-importer (maltose transporter) and an ABC-exporter (P-glycoprotein), to investigate functionally relevant dynamical differences. The two transporters exhibit very different structural flexibility and fluctuations in their inward-facing states, especially with regard to the separation of the NBDs. Comparing the dynamics of NBDs, it becomes evident that ABC importers fluctuate significantly lower than ABC exporters, suggesting that the conformational ensembel obtained for the former represents a deeper energy well. In contrast, the NBDs of P-glycoprotein are able to reach near dimerized conformations even in the absence of the nucleotide due to the overall higher structural flexibility of the protein architecture. The dynamical difference between the two ABC transporters appears to present a fundamental mechanistic difference between the subfamilies they belong to, which can be described as different energy landscapes along their pathways for NBD dimerization. The difference in energy landscapes is evidenced by their transport activities in that the NBDs of the maltose transporter dimerize only in the presence of its substrate, whereas P-glycoprotein possesses a remarkable substrate-independent ATPase activity.

Competition between pentoses and glucose during uptake and catabolism in recombinant Saccharomyces cerevisiae

BackgroundIn mixed sugar fermentations with recombinant Saccharomyces cerevisiae strains able to ferment D-xylose and L-arabinose the pentose sugars are normally only utilized after depletion of D-glucose. This has been attributed to competitive inhibition of pentose uptake by D-glucose as pentose sugars are taken up into yeast cells by individual members of the yeast hexose transporter family. We wanted to investigate whether D-glucose inhibits pentose utilization only by blocking its uptake or also by interfering with its further metabolism.ResultsTo distinguish between inhibitory effects of D-glucose on pentose uptake and pentose catabolism, maltose was used as an alternative carbon source in maltose-pentose co-consumption experiments. Maltose is taken up by a specific maltose transport system and hydrolyzed only intracellularly into two D-glucose molecules. Pentose consumption decreased by about 20 - 30% during the simultaneous utilization of maltose indicating that hexose catabolism can impede pentose utilization. To test whether intracellular D-glucose might impair pentose utilization, hexo-/glucokinase deletion mutants were constructed. Those mutants are known to accumulate intracellular D-glucose when incubated with maltose. However, pentose utilization was not effected in the presence of maltose. Addition of increasing concentrations of D-glucose to the hexo-/glucokinase mutants finally completely blocked D-xylose as well as L-arabinose consumption, indicating a pronounced inhibitory effect of D-glucose on pentose uptake. Nevertheless, constitutive overexpression of pentose-transporting hexose transporters like Hxt7 and Gal2 could improve pentose consumption in the presence of D-glucose.ConclusionOur results confirm that D-glucose impairs the simultaneous utilization of pentoses mainly due to inhibition of pentose uptake. Whereas intracellular D-glucose does not seem to have an inhibitory effect on pentose utilization, further catabolism of D-glucose can also impede pentose utilization. Nevertheless, the results suggest that co-fermentation of pentoses in the presence of D-glucose can significantly be improved by the overexpression of pentose transporters, especially if they are not inhibited by D-glucose.

Nanodiscs and SILAC-Based Mass Spectrometry to Identify a Membrane Protein Interactome

Integral membrane proteins are challenging to work with biochemically given their insoluble nature; the nanodisc circumvents the difficulty by stabilizing them in small patches of lipid bilayer. Here, we show that nanodiscs combined with SILAC-based quantitative proteomics can be used to identify the soluble interacting partners of virtually any membrane protein. As a proof of principle, we applied the method to the bacterial SecYEG protein-conducting channel, the maltose transporter MalFGK(2) and the membrane integrase YidC. In contrast to the detergent micelles, which tend to destabilize interactions, the nanodisc was able to capture out of a complex whole cell extract the proteins SecA, Syd, and MalE with a high degree of confidence and specificity. The method was sensitive enough to isolate these interactors as a function of the lipid composition in the disc and the culture conditions. In agreement with a previous photo-cross linking analysis, YidC did not show any high-affinity interactions with cytosolic or periplasmic proteins. These three examples illustrate the utility of nanoscale lipid bilayers to identify the soluble peripheral partners of proteins intergrated in the lipid bilayer.

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.

A genetically encoded, high-signal-to-noise maltose sensor.

We describe the generation of a family of high-signal-to-noise single-wavelength genetically encoded indicators for maltose. This was achieved by insertion of circularly permuted fluorescent proteins into a bacterial periplasmic binding protein (PBP), Escherichia coli maltodextrin-binding protein, resulting in a four-color family of maltose indicators. The sensors were iteratively optimized to have sufficient brightness and maltose-dependent fluorescence increases for imaging, under both one- and two-photon illumination. We demonstrate that maltose affinity of the sensors can be tuned in a fashion largely independent of the fluorescent readout mechanism. Using literature mutations, the binding specificity could be altered to moderate sucrose preference, but with a significant loss of affinity. We use the soluble sensors in individual E. coli bacteria to observe rapid maltose transport across the plasma membrane, and membrane fusion versions of the sensors on mammalian cells to visualize the addition of maltose to extracellular media. The PBP superfamily includes scaffolds specific for a number of analytes whose visualization would be critical to the reverse engineering of complex systems such as neural networks, biosynthetic pathways, and signal transduction cascades. We expect the methodology outlined here to be useful in the development of indicators for many such analytes.

Snapshots of the maltose transporter during ATP hydrolysis

ATP-binding cassette transporters are powered by ATP, but the mechanism by which these transporters hydrolyze ATP is unclear. In this study, four crystal structures of the full-length wild-type maltose transporter, stabilized by adenosine 5'-(β,γ-imido)triphosphate or ADP in conjunction with phosphate analogs BeF(3)(-), VO(4)(3-), or AIF(4)(-), were determined to 2.2- to 2.4-Å resolution. These structures led to the assignment of two enzymatic states during ATP hydrolysis and demonstrate specific functional roles of highly conserved residues in the nucleotide-binding domain, suggesting that ATP-binding cassette transporters catalyze ATP hydrolysis via a general base mechanism.

Conformational Coupling of the Nucleotide-Binding and the Transmembrane Domains in ABC Transporters

Basic architecture of ABC transporters includes two transmembrane domains (TMDs) and two nucleotide-binding domains (NBDs). Although the transport process takes place in the TMDs, which provide the substrate translocation pathway across the cell membrane and control its accessibility between the two sides of the membrane, the energy required for the process is provided by conformational changes induced in the NBDs by binding and hydrolysis of ATP. Nucleotide-dependent conformational changes in the NBDs, therefore, need to be coupled to structural changes in the TMDs. Using molecular dynamics simulations, we have investigated the structural elements involved in the conformational coupling between the NBDs and the TMDs in the Escherichia coli maltose transporter, an ABC importer for which an intact structure is available both in inward-facing and outward-facing conformations. The prevailing model of coupling is primarily based on a single structural motif, known as the coupling helices, as the main structural element for the NBD-TMD coupling. Surprisingly, we find that in the absence of the NBDs the coupling helices can be conformationally decoupled from the rest of the TMDs, despite their covalent connection. That is, the structural integrity of the coupling helices and their tight coupling to the core of the TMDs rely on the contacts provided by the NBDs. Based on the conformational and dynamical analysis of the simulation trajectories, we propose that the core coupling elements in the maltose transporter involve contributions from several structural motifs located at the NBD-TMD interface, namely, the EAA loops from the TMDs, and the Q-loop and the ENI motifs from the NBDs. These three structural motifs in small ABC importers show a high degree of correlation in motion and mediate the necessary conformational coupling between the core of TMDs and the helical subdomains of NBDs. A comprehensive analysis of the structurally known ABC transporters shows a high degree of conservation of the identified 3-motif coupling elements only in the subfamily of small ABC importers, suggesting a distinct mode of NBD-TMD coupling from the other two major ABC transporter folds, namely large ABC importers and ABC exporters.

Crystal Structure of the Maltose Transporter in a Pretranslocation Intermediate State

Adenosine triphosphate (ATP)-binding cassette (ABC) transporters convert chemical energy from ATP hydrolysis to mechanical work for substrate translocation. They function by alternating between two states, exposing the substrate-binding site to either side of the membrane. A key question that remains to be addressed is how substrates initiate the transport cycle. Using x-ray crystallography, we have captured the maltose transporter in an intermediate step between the inward- and outward-facing states. We show that interactions with substrate-loaded maltose-binding protein in the periplasm induce a partial closure of the MalK dimer in the cytoplasm. ATP binding to this conformation then promotes progression to the outward-facing state. These results, interpreted in light of biochemical and functional studies, provide a structural basis to understand allosteric communication in ABC transporters.

Both Lobes of Maltose Binding Protein are Engaged in Stabilization of the Semi-Open State of the Malk Dimer in the Maltose ABC Transporter

ATP-binding cassette (ABC) transporters involved in uptake in bacteria are capable of accumulating substrates against a concentration gradient at the expense of ATP. In most systems, high-affinity binding protein located in the periplasmic space or outside the cell scavenges solutes for delivery to and through the transmembrane components. One such system is the maltose transporter in Escherichia coli, which is composed of a periplasmic maltose binding protein (MBP), two transmembrane domains, MalF and MalG, and a nucleotide-binding homodimer, MalK2. Interaction of the ligand-bound MBP with the MalFGK2 transporter in the presence of MgATP triggers conformational changes that result in reorientation of transmembrane helices to receive maltose from MBP and closure of the nucleotide-binding interface in MalK2 to hydrolyze ATP. Following ATP hydrolysis, the MalK dimer opens and transmembrane helices reorient, bringing maltose into the cell. Recently, evidence of a semi-open state of MalK2 was found when the transporter was allowed to hydrolyze ATP in the presence of MBP. We have used site-directed spin labeling and electron paramagnetic resonance spectroscopy to further characterize this “post-hydrolysis” state. By placing EPR probes in different locations along the interface where MBP is known to interact with MalFGK2, we found that whereas only the N-lobe is bound to the transporter in the resting state conformation, both the N- and C-terminal lobes of MBP are engaged with the transporter under conditions where the semi-open state of MalK2 is stabilized. Interpreted in light of how MBP may stimulate the ATPase activity of the transporter, it appears that both lobes of MBP are involved in dictating the conformation of the MalK dimer on the opposite site of the membrane.

Exposure to Glycolytic Carbon Sources Reveals a Novel Layer of Regulation for the MalT Regulon

Bacteria adapt to changing environments by means of tightly coordinated regulatory circuits. The use of synthetic lethality, a genetic phenomenon in which the combination of two nonlethal mutations causes cell death, facilitates identification and study of such circuitry. In this study, we show that the E. coli ompR malT con double mutant exhibits a synthetic lethal phenotype that is environmentally conditional. MalTcon, the constitutively active form of the maltose system regulator MalT, causes elevated expression of the outer membrane porin LamB, which leads to death in the absence of the osmoregulator OmpR. However, the presence and metabolism of glycolytic carbon sources, such as sorbitol, promotes viability and unveils a novel layer of regulation within the complex circuitry that controls maltose transport and metabolism.

Binding Protein-Dependent Uptake of Maltose into Cells via an ATP-Binding Cassette Transporter.

Maltose and maltodextrins are actively transported across the cytoplasmic membrane of Escherichia coli and Salmonella by a periplasmic binding protein (BP)- dependent transport system. Since 1996, there have been many advances in the understanding of the structure and mechanism of the maltose transporter, in the assembly of the membrane-associated transporter complex, and in the mechanism of regulation of transport both at the DNA and the protein level. The transporter has been studied in detergent and reconstituted in liposome vesicles, and while many features, including the ability of maltose-binding protein (MBP) to stimulate ATPase activity, are retained in detergent, it has been noted that the basal ATPase activity of the transporter is elevated in detergent compared with liposomes. This review focuses on these recent developments, which have culminated in a high resolution structure of MBP in a complex with the MalFGK2 transporter. While this review focuses on the maltose system, complementary work has been carried out on many different ATP binding cassette (ABC) transporters, all of which has contributed in important ways to the understanding of the maltose transport system. The regulation of the maltose transport system, at the DNA level, is implemented by the synergistic action of MalT and cAMP/CAP complex and, at the protein level, by interactions of MalK with unphosphorylated EIIAglc, a signal-transducing component of the phosphoenolpyruvate-glucose phosphotransferase system.

Crystallization of Escherichia coli maltoporin in the trigonal space group R3.

Maltoporin is an outer-membrane protein that forms a β-barrel composed of three monomers and ensures the transport of maltose and maltodextrin in Gram-negative bacteria. Previously, the crystallization of Escherichia coli or Salmonella typhimurium maltoporin has been achieved in the presence of a mixture of the detergents β-decylmaltoside and dodecyl nonaoxyethylene. These crystals all belonged to the orthorhombic space group C222(1) and gave rise to several structures of maltoporin in complex with different carbohydrates determined at resolutions between 3.2 and 2.4 Å. Here, the crystallization of E.coli maltoporin in a new crystal form is reported; the crystals belonged to the trigonal R3 space group and diffracted to 1.9 Å resolution. These crystals were obtained using n-dodecyl-β-D-maltoside as a detergent. Crystals with a lens orpyramidal morphology could be obtained using sitting or hanging drops, respectively, and despite their very different shapes they presented the same space group and very similar unit-cell parameters.

Role of the plastidic glucose translocator in the export of starch degradation products from the chloroplasts in Arabidopsis thaliana

In higher plants, the plastidic glucose translocator (pGlcT) is assumed to play a role in the export of starch degradation products, but this has not yet been studied in detail. To elucidate the role of pGlcT in the leaves of Arabidopsis thaliana, we generated single and double mutants lacking three plastidic sugar transporters, pGlcT, the triose-phosphate/phosphate translocator (TPT), and the maltose transporter (MEX1), and analyzed their growth phenotypes, photosynthetic properties and metabolite contents. In contrast to the pglct-1 and pglct-2 single mutants lacking a visible growth phenotype, the double mutants pglct-1/mex1 and tpt-2/mex1 displayed markedly inhibited plant growth. Notably, pglct-1/mex1 exhibited more severe growth retardation than that seen for the other mutants. In parallel, the most severe reductions in sucrose content and starch turnover were observed in the pglct-1/mex1 mutant. The concurrent loss of pGlcT and MEX1 also resulted in severely reduced photosynthetic activities and extreme chloroplast abnormalities. These findings suggest that pGlcT, together with MEX1, contributes significantly to the export of starch degradation products from chloroplasts in A. thaliana leaves, and that this starch-mediated pathway for photoassimilate export via pGlcT and MEX1 is essential for the growth and development of A. thaliana.

Synergistic antitumor effects of Escherichia coli maltose binding protein and Bacillus Calmette–Guerin in a mouse lung carcinoma model

Maltose binding protein (MBP) is a component of the maltose transport system in the periplasm of Escherichia coli. It is commonly believed that MBP has minimal effects on the bioactivity, thus, it is widely used in the purification of recombinant proteins. Here, we found that the combined immunization with MBP and Bacillus Calmette–Guerin (BCG) significantly inhibited tumor growth compared with MBP or BCG immunization alone in a mouse lung carcinoma model. Further studies showed that MBP nonspecifically activated T helper 1 (Th1) cells and enhanced the BCG-induced Th1 cell activation. Moreover, MBP or BCG immunization alone increased the activities of natural killer (NK) cells and macrophages, and the combined immunization with MBP and BCG induced a synergistic effect on the activities of NK cells and macrophages. These results suggest that MBP possesses potent immune enhancement activities, and that the combination of MBP and BCG-induced synergistic antitumor effects might be mediated mainly through the activation of Th1 cells, NK cells and macrophages.

Uncoupling Substrate Transport from ATP Hydrolysis in the Escherichia coli Maltose Transporter

Members of the ATP-binding cassette superfamily couple the energy from ATP hydrolysis to the active transport of substrates across the membrane. The maltose transporter, a well characterized model system, consists of a periplasmic maltose-binding protein (MBP) and a multisubunit membrane transporter, MalFGK(2). On the basis of the structure of the MBP-MalFGK(2) complex in an outward-facing conformation (Oldham, M. L., Khare, D., Quiocho, F. A., Davidson, A. L., and Chen, J. (2007) Nature 450, 515-521), we identified two mutants in transmembrane domains MalF and MalG that generated futile cycling; although interaction with MBP stimulated the ATPase activity of the transporter, maltose was not transported. Both mutants appeared to disrupt the normal transfer of maltose from MBP to MalFGK(2). In the first case, substitution of aspartate for glycine in the maltose-binding site of MalF likely generated a futile cycle by preventing maltose from binding to MalFGK(2) during the catalytic cycle. In the second case, a four-residue deletion of a periplasmic loop of MalG limited its reach into the maltose-binding pocket of MBP, allowing maltose to remain associated with MBP during the catalytic cycle. Retention of maltose in the MBP binding site in the deletion mutant, as well as insertion of this loop into the binding site in the wild type, was detected by EPR as a change in mobility of a nitroxide spin label positioned near the maltose-binding pocket of MBP.

Identification and Functions of Amino Acid Residues in PotB and PotC Involved in Spermidine Uptake Activity

Amino acid residues on PotB and PotC involved in spermidine uptake were identified by random and site-directed mutagenesis. It was found that Trp(8), Tyr(43), Trp(100), Leu(110), and Tyr(261) in PotB and Trp(46), Asp(108), Glu(169), Ser(196), Asp(198), and Asp(199) in PotC were strongly involved in spermidine uptake and that Tyr(160), Glu(172), and Leu(274) in PotB and Tyr(19), Tyr(88), Tyr(148), Glu(160), Leu(195), and Tyr(211) in PotC were moderately involved in spermidine uptake. Among 11 amino acid residues that were strongly involved in spermidine uptake, Trp(8) in PotB was important for insertion of PotB and PotC into membranes. Tyr(43), Trp(100), and Leu(110) in PotB and Trp(46), Asp(108), Ser(196), and Asp(198) in PotC were found to be involved in the interaction with PotD. Leu(110) and Tyr(261) in PotB and Asp(108), Asp(198), and Asp(199) in PotC were involved in the recognition of spermidine, and Trp(100) and Tyr(261) in PotB and Asp(108), Glu(169), and Asp(198) in PotC were involved in ATPase activity of PotA. Accordingly, Trp(100) in PotB was involved in both PotD recognition and ATPase activity, Leu(110) in PotB was involved in both PotD and spermidine recognition, and Tyr(261) in PotB was involved in both spermidine recognition and ATPase activity. Asp(108) and Asp(198) in PotC were involved in PotD and spermidine recognition as well as ATPase activity. These results suggest that spermidine passage from PotD to the cytoplasm is coupled to the ATPase activity of PotA through a structural change of PotA by its ATPase activity.

Exploring the Potential of C. Pasteurianum to Produce Biofuels from Waste Products

Biofuels which can be produced by microorganisms from agricultural waste products will provide a renewable and non controversial source of energy for the future. The saccharolytic Clostridium sp. can ferment a wide range of sugars to produce organic acids and solvents. They are also anaerobic and in some cases thermophilic which is advantageous for large-scale industrial processes. C.pasteurianum shows potentially high solvent and hydrogen gas production capacity. The fermentation end products of C.pasteurianum, when grown in media of high sugar content, included significant quantities of acetone, butanol and ethanol, acetate, butyrate, CO2 and H2. The aim of this research was to investigate maltose uptake and metabolism in Clostridium pasteurianum NCIB 9486, to increase understanding of its starch utilisation. The organism was grown in different substrates and assayed for induction of glucosidase activities. Results confirmed that enzyme synthesis is induced by substrates.MaltaseandpNPGasewere found largely in soluble form (89.5% and 86.6% respectively) proving that maltase is produced intracellularly and hydrolysis of maltose takes place in the cytpoplasm. Fractionationof the soluble extract ofmaltose growncells yielded two maltase enzymes, maltase I (120-150 KDa) and maltase II (60 KDa). Further characterisation of maltase II showed high substrate specificity for maltose and its analogues, and sucrose. Maltase II exhibited similar rates of hydrolysis of maltose andmaltotriose: values for Km of 0.8 and 0.7 mmol l-1;Vmax of 3.3 and 4.2 nmol min-1, respectively. There is indication of specific activity of maltase II in cleaving the -1,4 (maltose) and -1,6-(isomaltose) linkages, but not -linkages (cellobiose). PEP-dependent phosphorylation of maltose was demonstrated suggesting the presence of a PEP-dependent PTS for maltose transport. This species has potential for use in biofuel production and a better understanding of its carbohydrate metabolism will facilitate its exploitation.

The maltose ATP‐binding cassette transporter in the 21st century – towards a structural dynamic perspective on its mode of action

The maltose/maltodextrin transport system of Escherichia coli/Salmonella, composed of periplasmic maltose-binding protein, MalE, the pore-forming subunits MalF and MalG, and a homodimer of the nucleotide-binding subunit, MalK, serves as a model for canonical ATP-binding cassette importers in general. The wealth of knowledge accumulated on the maltose transporter in more than three decades by genetic, molecular genetic and biochemical means was complemented more recently by crystal structures of the isolated MalK dimer and of two conformational states of the full transporter. Here, we summarize insights into the transport mechanism provided by these structures and draw the reader's attention to experimental tools by which the dynamics of the transporter can be studied during substrate translocation. A transport model is presented that integrates currently available biochemical, biophysical and structural data. We also present the state of knowledge on regulatory functions of the maltose transporter associated with the C-terminal domain of MalK. Finally, we will address the application of coarse-grained modelling to visualize the progression of the conformational changes of an ABC transporter with special emphasis on the maltose system, which can provide a model platform for testing and validating the bioinformatic tools.

Comparison of the MTT1- and MAL31-like maltose transporter genes in lager yeast strains

Maltose transporter genes were isolated from four lager yeast strains and sequenced. All four strains contain at least two different types of maltose transporter genes, MTT1 and MAL31. In addition, 'long' 2.7 kb, and 'short' 2.4 kb, versions of each type exist. The size difference is caused by the insertion of two repeats of 147 bp into the promoter regions of the long versions of the genes. As a consequence of the insertion, two Mal63-binding sites move 294 bp away from the transcription initiation site. The 2.4- and 2.7-kb versions are further highly similar. Only the 2.4-kb versions and not the 2.7-kb versions of MTT1 could restore the rapid growth of lager yeast strain A15 on maltotriose in the presence of antimycin A. These results suggest that insertion of the two repeats into the promoter region of the 'long versions' of MTT1 genes led to a diminished expression of these genes. None of the tested long and short versions of the MAL31 genes were able to restore this growth. As the promoter regions of the MTT1 and MAL31 genes are identical, small differences in the protein sequence may be responsible for the different properties of these genes.

Functional Reconstitution of an ABC Transporter in Nanodiscs for Use in Electron Paramagnetic Resonance Spectroscopy

Electron paramagnetic resonance (EPR) spectroscopy is a powerful biophysical technique for study of the structural dynamics of membrane proteins. Many of these proteins interact with ligands or proteins on one or both sides of the membrane. Membrane proteins are typically reconstituted in proteoliposomes to observe their function in a physiologically relevant environment. However, membrane proteins can insert into liposomes in two different orientations, and surfaces facing the lumen of the vesicle can be inaccessible to ligands. This heterogeneity can lead to subpopulations that do not respond to ligand binding, complicating EPR spectral analysis, particularly for distance measurements. Using the well-characterized maltose transporter, an ATP binding cassette (ABC) transporter that interacts with ligands on both sides of the membrane, we provide evidence that reconstitution into nanodiscs, which are soluble disk-shaped phospholipid bilayers, is an ideal solution to these problems. We describe the functional reconstitution of the maltose transporter into nanodiscs and demonstrate that this system is ideally suited to study conformational changes and intramolecular distances by EPR.