Functional promiscuity of small multidrug resistance transporters from Staphylococcus aureus, Pseudomonas aeruginosa, and Francisella tularensis.

Small multidrug resistance transporters efflux toxic compounds from bacteria and are a minimal system to understand multidrug transport. Most previous studies have focused on EmrE, the model SMR from Escherichia coli, finding that EmrE has a broader substrate profile than previously thought and that EmrE may perform multiple types of transport, resulting in substrate-dependent resistance or susceptibility. Here, we performed a broad screen to identify potential substrates of three other SMRs: PAsmr from Pseudomonas aeruginosa; FTsmr from Francisella tularensis; and SAsmr from Staphylococcus aureus. This screen tested metabolic differences in E. coli expressing each transporter versus an inactive mutant, for a clean comparison of sequence and substrate-specific differences in transporter function, and identified many substrates for each transporter. In general, resistance compounds were charged, and susceptibility substrates were uncharged, but hydrophobicity was not correlated with phenotype. Two resistance hits and two susceptibility hits were validated via growth assays and IC50 calculations. Susceptibility is proposed to occur via substrate-gated proton leak, and the addition of bicarbonate antagonizes the susceptibility phenotype, consistent with this hypothesis.

A Small Multidrug Resistance Transporter in Pseudomonas aeruginosa Confers Substrate-Specific Resistance or Susceptibility.

Small Multidrug Resistance (SMR) transporters are key players in the defense of multidrug-resistant pathogens to toxins and other homeostasis-perturbing compounds. However, recent evidence demonstrates that EmrE, an SMR from Escherichia coli and a model for understanding transport, can also induce susceptibility to some compounds by drug-gated proton leak. This runs down the ∆pH component of the Proton Motive Force (PMF), reducing viability of the affected bacteria. Proton leak may provide an unexplored drug target distinct from the targets of most known antibiotics. Activating proton leak requires an SMR to be merely present, rather than be the primary resistance mechanism, and dissipates the energy source for many other efflux pumps. PAsmr, an EmrE homolog from P. aeruginosa, transports many EmrE substrates in cells and purified systems. We hypothesized that PAsmr, like EmrE, may confer susceptibility to some compounds via drug-gated proton leak. Growth assays of E. coli expressing PAsmr displayed substrate-dependent resistance and susceptibility phenotypes, and in vitro solid-supported membrane electrophysiology experiments revealed that PAsmr performs both antiport and substrate-gated proton uniport, demonstrating the same functional promiscuity observed in EmrE. Growth assays of P. aeruginosa strain PA14 demonstrated that PAsmr contributes resistance to some antimicrobial compounds, but no growth defect is observed with susceptibility substrates, suggesting P. aeruginosa can compensate for the proton leak occurring through PAsmr. These phenotypic differences between P. aeruginosa and E. coli advance our understanding of underlying resistance mechanisms in P. aeruginosa and prompt further investigation into the role that SMRs play in antibiotic resistance in pathogens.

Solution Structure of Poly(UG) RNA.

Poly(UG) or "pUG" RNAs are UG or GU dinucleotide repeat sequences which are highly abundant in eukaryotes. Post-transcriptional addition of pUGs to RNA 3' ends marks mRNAs as vectors for gene silencing in C. elegans. We previously determined the crystal structure of pUG RNA bound to the ligand N-methyl mesoporphyrin IX (NMM), but the structure of free pUG RNA is unknown. Here we report the solution structure of the free pUG RNA (GU)12, as determined by nuclear magnetic resonance spectroscopy and small and wide-angle x-ray scattering (NMR-SAXS-WAXS). The low complexity sequence and 4-fold symmetry of the structure result in overlapped NMR signals that complicate chemical shift assignment. We therefore utilized single site-specific deoxyribose modifications which did not perturb the structure and introduced well-resolved methylene signals that are easily identified in NMR spectra. The solution structure ensemble has a root mean squared deviation (RMSD) of 0.62 Å and is a compact, left-handed quadruplex with a Z-form backbone, or "pUG fold." Overall, the structure agrees with the crystal structure of (GU)12 bound to NMM, indicating the pUG fold is unaltered by docking of the NMM ligand. The solution structure reveals conformational details that could not be resolved by x-ray crystallography, which explain how the pUG fold can form within longer RNAs.

Medicago Sativa Defensin1 as a tumor sensitizer for improving chemotherapy: translation from anti-fungal agent to a potential anti-cancer agent.

Plant defensins including Medicago Sativa defensin 1 (MsDef1) are cysteine-rich antifungal peptides which are known for potent broad-spectrum antifungal activity against bacterial or fungal pathogens of plants. The antimicrobial activities of these cationic defensins are attributed to their capacity to bind to cell membranes to create potentially structural defects tin the cell membranes to interact with intracellular target (s) and mediates cytotoxic effects. Our earlier work identified Glucosylceramide (GlcCer) of fungus F. graminearum as a potential target for biological activity. Multi-drug resistant (MDR) cancer cells overexpress GlcCer on the surface of plasma membrane. Hence, MsDef1 may have a potential to bind to GlcCer of MDR cancer cells to induce cell death. We have characterized the three-dimensional structure of MsDef1 and the solution dynamics using of 15N-labeled MsDef1 nuclear magnetic resonance (NMR) spectroscopy which showed that GlcCer binds MsDef1 at two specific sites on the peptide molecule. The ability of MsDef1 to permeate MDR cancer cells was demonstrated by measuring the release of apoptotic ceramide in drug resistant MCF-7R cells. It was also shown that MsDef1 activated dual cell death pathways ceramide and Apoptosis Stimulating Kinase ASK1 by disintegrating GlcCer and oxidizing tumor specific biomarker thioredoxin (Trx) respectively. As a result, MsDef1 sensitizes MDR cancer cells to evoke a better response from Doxorubicin, a front-line chemotherapy for triple negative breast cancer (TNBC) treatment. The combination of MsDef1 and Doxorubicin induced 5 to10-fold greater apoptosis in vitro MDR cells MDA-MB-231R compared to either MsDef1 or Doxorubicin alone. Confocal microscopy revealed that MsDef1 facilitates a) influx of Doxorubicin in MDR cancer cells, b) preferential uptake by MDR cells but not by normal fibroblasts and breast epithelial cells (MCF-10A). These results suggest that MsDef1 targets MDR cancer cells and may find utility as a neoadjuvant chemotherapy. Hence, the extension of antifungal properties of MsDef1 to cancer my result in addressing the MDR problems in cancer.

Microsecond Motion of the Bacterial Transporter EmrE in Lipid Bilayers.

The bacterial transporter EmrE is a homo-dimeric membrane protein that effluxes cationic polyaromatic substrates against the concentration gradient by coupling to proton transport. As the archetype of the small multidrug resistance family of transporters, EmrE structure and dynamics provide atomic insights into the mechanism of transport by this family of proteins. We recently determined high-resolution structures of EmrE in complex with a cationic substrate, tetra(4-fluorophenyl)phosphonium (F4-TPP+), using solid-state NMR spectroscopy and an S64V-EmrE mutant. The substrate-bound protein exhibits distinct structures at acidic and basic pH, reflecting changes upon binding or release of a proton from residue E14, respectively. To obtain insight into the protein dynamics that mediate substrate transport, here we measure 15N rotating-frame spin-lattice relaxation (R1ρ) rates of F4-TPP+-bound S64V-EmrE in lipid bilayers under magic-angle spinning (MAS). Using perdeuterated and back-exchanged protein and 1H-detected 15N spin-lock experiments under 55 kHz MAS, we measured 15N R1ρ rates site-specifically. Many residues show spin-lock field-dependent 15N R1ρ relaxation rates. This relaxation dispersion indicates the presence of backbone motions at a rate of about 6000 s-1 at 280 K for the protein at both acidic and basic pH. This motional rate is 3 orders of magnitude faster than the alternating access rate but is within the range estimated for substrate binding. We propose that these microsecond motions may allow EmrE to sample different conformations to facilitate substrate binding and release from the transport pore.

Charge neutralization of the active site glutamates does not limit substrate binding and transport by small multidrug resistance transporter EmrE.

EmrE, a small multidrug resistance transporter from Escherichia coli, confers broad-spectrum resistance to polyaromatic cations and quaternary ammonium compounds. Previous transport assays demonstrate that EmrE transports a +1 and a +2 substrate with the same stoichiometry of two protons:one cationic substrate. This suggests that EmrE substrate binding capacity is limited to neutralization of the two essential glutamates, E14A and E14B (one from each subunit in the antiparallel homodimer), in the primary binding site. Here, we explicitly test this hypothesis, since EmrE has repeatedly broken expectations for membrane protein structure and transport mechanism. We previously showed that EmrE can bind a +1 cationic substrate and proton simultaneously, with cationic substrate strongly associated with one E14 residue, whereas the other remains accessible to bind and transport a proton. Here, we demonstrate that EmrE can bind a +2 cation substrate and a proton simultaneously using NMR pH titrations of EmrE saturated with divalent substrates, for a net +1 charge in the transport pore. Furthermore, we find that EmrE can alternate access and transport a +2 substrate and proton at the same time. Together, these results lead us to conclude that E14 charge neutralization does not limit the binding and transport capacity of EmrE.

Activating alternative transport modes in a multidrug resistance efflux pump to confer chemical susceptibility.

Small multidrug resistance (SMR) transporters contribute to antibiotic resistance through proton-coupled efflux of toxic compounds. Previous biophysical studies of the E. coli SMR transporter EmrE suggest that it should also be able to perform proton/toxin symport or uniport, leading to toxin susceptibility rather than resistance in vivo. Here we show EmrE does confer susceptibility to several previously uncharacterized small-molecule substrates in E. coli, including harmane. In vitro electrophysiology assays demonstrate that harmane binding triggers uncoupled proton flux through EmrE. Assays in E. coli are consistent with EmrE-mediated dissipation of the transmembrane pH gradient as the mechanism underlying the in vivo phenotype of harmane susceptibility. Furthermore, checkerboard assays show this alternative EmrE transport mode can synergize with some existing antibiotics, such as kanamycin. These results demonstrate that it is possible to not just inhibit multidrug efflux, but to activate alternative transport modes detrimental to bacteria, suggesting a strategy to address antibiotic resistance.

Full opening of helix bundle crossing does not lead to NaK channel activation.

A critical part of ion channel function is the ability to open and close in response to stimuli and thus conduct ions in a regulated fashion. While x-ray diffraction studies of ion channels suggested a general steric gating mechanism located at the helix bundle crossing (HBC), recent functional studies on several channels indicate that the helix bundle crossing is wide-open even in functionally nonconductive channels. Two NaK channel variants were crystallized in very different open and closed conformations, which served as important models of the HBC gating hypothesis. However, neither of these NaK variants is conductive in liposomes unless phenylalanine 92 is mutated to alanine (F92A). Here, we use NMR to probe distances at near-atomic resolution of the two NaK variants in lipid bicelles. We demonstrate that in contrast to the crystal structures, both NaK variants are in a fully open conformation, akin to Ca2+-bound MthK channel structure where the HBC is widely open. While we were not able to determine what a conductive NaK structure is like, our further inquiry into the gating mechanism suggests that the selectivity filter and pore helix are coupled to the M2 helix below and undergo changes in the structure when F92 is mutated. Overall, our data show that NaK exhibits coupling between the selectivity filter and HBC, similar to K+ channels, and has a more complex gating mechanism than previously thought, where the full opening of HBC does not lead to channel activation.

2-Propylphenol Allosterically Modulates COQ8A to Enhance ATPase Activity.

COQ8A is an atypical kinase-like protein that aids the biosynthesis of coenzyme Q, an essential cellular cofactor and antioxidant. COQ8A's mode of action remains unclear, in part due to the lack of small molecule tools to probe its function. Here, we blend NMR and hydrogen-deuterium exchange mass spectrometry to help determine how a small CoQ precursor mimetic, 2-propylphenol, modulates COQ8A activity. We identify a likely 2-propylphenol binding site and reveal that this compound modulates a conserved COQ8A domain to increase nucleotide affinity and ATPase activity. Our findings promise to aid further investigations into COQ8A's precise enzymatic function and the design of compounds capable of boosting endogenous CoQ production for therapeutic gain.

High-pH structure of EmrE reveals the mechanism of proton-coupled substrate transport.

The homo-dimeric bacterial membrane protein EmrE effluxes polyaromatic cationic substrates in a proton-coupled manner to cause multidrug resistance. We recently determined the structure of substrate-bound EmrE in phospholipid bilayers by measuring hundreds of protein-ligand HN-F distances for a fluorinated substrate, 4-fluoro-tetraphenylphosphonium (F4-TPP+), using solid-state NMR. This structure was solved at low pH where one of the two proton-binding Glu14 residues is protonated. Here, to understand how substrate transport depends on pH, we determine the structure of the EmrE-TPP complex at high pH, where both Glu14 residues are deprotonated. The high-pH complex exhibits an elongated and hydrated binding pocket in which the substrate is similarly exposed to the two sides of the membrane. In contrast, the low-pH complex asymmetrically exposes the substrate to one side of the membrane. These pH-dependent EmrE conformations provide detailed insights into the alternating-access model, and suggest that the high-pH conformation may facilitate proton binding in the presence of the substrate, thus accelerating the conformational change of EmrE to export the substrate.

Ion-dependent structure, dynamics, and allosteric coupling in a non-selective cation channel.

The selectivity filter (SF) determines which ions are efficiently conducted through ion channel pores. NaK is a non-selective cation channel that conducts Na+ and K+ with equal efficiency. Crystal structures of NaK suggested a rigid SF structure, but later solid-state NMR and MD simulations questioned this interpretation. Here, we use solution NMR to characterize how bound Na+ vs. K+ affects NaK SF structure and dynamics. We find that the extracellular end of the SF is flexible on the ps-ns timescale regardless of bound ion. On a slower timescale, we observe a structural change between the Na+ and K+-bound states, accompanied by increased structural heterogeneity in Na+. We also show direct evidence that the SF structure is communicated to the pore via I88 on the M2 helix. These results support a dynamic SF with multiple conformations involved in non-selective conduction. Our data also demonstrate allosteric coupling between the SF and pore-lining helices in a non-selective cation channel that is analogous to the allosteric coupling previously demonstrated for K+-selective channels, supporting the generality of this model.

A solid-supported membrane electrophysiology assay for efficient characterization of ion-coupled transport.

Transport stoichiometry determination can provide great insight into the mechanism and function of ion-coupled transporters. Traditional reversal potential assays are a reliable, general method for determining the transport stoichiometry of ion-coupled transporters, but the time and material costs of this technique hinder investigations of transporter behavior under multiple experimental conditions. Solid-supported membrane electrophysiology (SSME) allows multiple recordings of liposomal or membrane samples adsorbed onto a sensor and is sensitive enough to detect transport currents from moderate-flux transporters that are inaccessible to traditional electrophysiology techniques. Here, we use SSME to develop a new method for measuring transport stoichiometry with greatly improved throughput. Using this technique, we were able to verify the recent report of a fixed 2:1 stoichiometry for the proton:guanidinium antiporter Gdx, reproduce the 1H+:2Cl- antiport stoichiometry of CLC-ec1, and confirm loose proton:nitrate coupling for CLC-ec1. Furthermore, we were able to demonstrate quantitative exchange of internal contents of liposomes adsorbed onto SSME sensors to allow multiple experimental conditions to be tested on a single sample. Our SSME method provides a fast, easy, general method for measuring transport stoichiometry, which will facilitate future mechanistic and functional studies of ion-coupled transporters.

Mapping temperature-dependent conformational change in the voltage-sensing domain of an engineered heat-activated K+ channel.

Temperature-dependent regulation of ion channel activity is critical for a variety of physiological processes ranging from immune response to perception of noxious stimuli. Our understanding of the structural mechanisms that underlie temperature sensing remains limited, in part due to the difficulty of combining high-resolution structural analysis with temperature stimulus. Here, we use NMR to compare the temperature-dependent behavior of Shaker potassium channel voltage sensor domain (WT-VSD) to its engineered temperature sensitive (TS-VSD) variant. Further insight into the molecular basis for temperature-dependent behavior is obtained by analyzing the experimental results together with molecular dynamics simulations. Our studies reveal that the overall secondary structure of the engineered TS-VSD is identical to the wild-type channels except for local changes in backbone torsion angles near the site of substitution (V369S and F370S). Remarkably however, these structural differences result in increased hydration of the voltage-sensing arginines and the S4-S5 linker helix in the TS-VSD at higher temperatures, in contrast to the WT-VSD. These findings highlight how subtle differences in the primary structure can result in large-scale changes in solvation and thereby confer increased temperature-dependent activity beyond that predicted by linear summation of solvation energies of individual substituents.

Structure and dynamics of the drug-bound bacterial transporter EmrE in lipid bilayers.

The dimeric transporter, EmrE, effluxes polyaromatic cationic drugs in a proton-coupled manner to confer multidrug resistance in bacteria. Although the protein is known to adopt an antiparallel asymmetric topology, its high-resolution drug-bound structure is so far unknown, limiting our understanding of the molecular basis of promiscuous transport. Here we report an experimental structure of drug-bound EmrE in phospholipid bilayers, determined using 19F and 1H solid-state NMR and a fluorinated substrate, tetra(4-fluorophenyl) phosphonium (F4-TPP+). The drug-binding site, constrained by 214 protein-substrate distances, is dominated by aromatic residues such as W63 and Y60, but is sufficiently spacious for the tetrahedral drug to reorient at physiological temperature. F4-TPP+ lies closer to the proton-binding residue E14 in subunit A than in subunit B, explaining the asymmetric protonation of the protein. The structure gives insight into the molecular mechanism of multidrug recognition by EmrE and establishes the basis for future design of substrate inhibitors to combat antibiotic resistance.

1H, 13C, and 15N backbone and side chain chemical shift assignments of the SARS-CoV-2 non-structural protein 7.

The SARS-CoV-2 genome encodes for approximately 30 proteins. Within the international project covid19-nmr, we distribute the spectroscopic analysis of the viral proteins and RNA. Here, we report NMR chemical shift assignments for the protein nsp7. The 83 amino acid nsp7 protein is an essential cofactor in the RNA-dependent RNA polymerase. The polymerase activity and processivity of nsp12 are greatly enhanced by binding 1 copy of nsp7 and 2 copies of nsp8 to form a 160 kD complex. A separate hexadecameric complex of nsp7 and nsp8 (8 copies of each) forms a large ring-like structure. Thus, nsp7 is an important component of several large protein complexes that are required for replication of the large and complex coronavirus genome. We here report the near-complete NMR backbone and sidechain resonance assignment (1H,13C,15N) of isolated nsp7 from SARS-CoV-2 in solution. Further, we derive the secondary structure and compare it to the previously reported assignments and structure of the SARS-CoV nsp7.

Highly coupled transport can be achieved in free-exchange transport models.

Secondary active transporters couple the transport of an ion species down its concentration gradient to the uphill transport of another substrate. Despite the importance of secondary active transport to multidrug resistance, metabolite transport, and nutrient acquisition, among other biological processes, the microscopic steps of the coupling mechanism are not well understood. Often, transport models illustrate coupling mechanisms through a limited number of "major" conformations or states, yet recent studies have indicated that at least some transporters violate these models. The small multidrug resistance transporter EmrE has been shown to couple proton influx to multidrug efflux via a mechanism that incorporates both "major" and "minor" conformational states and transitions. The resulting free exchange transport model includes multiple leak pathways and theoretically allows for both exchange and cotransport of ion and substrate. To better understand how coupled transport can be achieved in such a model, we numerically simulate a free-exchange model of transport to determine the step-by-step requirements for coupled transport. We find that only moderate biasing of rate constants for key transitions produce highly efficient net transport approaching a perfectly coupled, stoichiometric model. We show how a free-exchange model can enable complex phenotypes, including switching transport direction with changing environmental conditions or substrates. This research has broad implications for synthetic biology, as it demonstrates the utility of free-exchange transport models and the fine tuning required for perfectly coupled transport.

NMR Structural Analysis of Isolated Shaker Voltage-Sensing Domain in LPPG Micelles.

The voltage-sensing domain (VSD) is a conserved structural module that regulates the gating of voltage-dependent ion channels in response to a change in membrane potential. Although the structures of many VSD-containing ion channels are now available, our understanding of the structural dynamics associated with gating transitions remains limited. To probe dynamics with site-specific resolution, we utilized NMR spectroscopy to characterize the VSD derived from Shaker potassium channel in 1-palmitoyl-2-hydroxy-sn-glycero-3-phospho-(1'-rac-glycerol) (LPPG) micelles. The backbone dihedral angles predicted based on secondary chemical shifts using torsion angle likeliness obtained from shift (TALOS+) showed that the Shaker-VSD shares many structural features with the homologous Kv1.2/2.1 chimera, including a transition from α-helix to 310 helix in the C-terminal portion of the fourth transmembrane helix. Nevertheless, there are clear differences between the Shaker-VSD and Kv1.2/2.1 chimera in the S2-S3 linker and S3 transmembrane region, where the organization of secondary structure elements in Shaker-VSD appears to more closely resemble the KvAP-VSD. Comparison of microsecond-long molecular dynamics simulations of Kv 1.2-VSD in LPPG micelles and a 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) bilayer showed that LPPG micelles do not induce significant structural distortion in the isolated voltage sensor. To assess the integrity of the tertiary fold, we directly probed the binding of BrMT analog 2-[2-({[3-(2-amino-ethyl)-6-bromo-1H-indol-2-yl]methoxy}k7methyl)-6-bromo-1H-indol-3-yl]ethan-1-amine (BrET), a gating modifier toxin, and identified the location of the putative binding site. Our results suggest that the Shaker-VSD in LPPG micelles is in a native-like fold and is likely to provide valuable insights into the dynamics of voltage-gating and its regulation.

Identification of an Alternating-Access Dynamics Mutant of EmrE with Impaired Transport.

Proteins that perform active transport must alternate the access of a binding site, first to one side of a membrane and then to the other, resulting in the transport of bound substrates across the membrane. To better understand this process, we sought to identify mutants of the small multidrug resistance transporter EmrE with reduced rates of alternating access. We performed extensive scanning mutagenesis by changing every amino acid residue to Val, Ala, or Gly, and then screening the drug resistance phenotypes of the resulting mutants. We identified EmrE mutants that had impaired transport activity but retained the ability to bind substrate and further tested their alternating access rates using NMR. Ultimately, we were able to identify a single mutation, S64V, which significantly reduced the rate of alternating access but did not impair substrate binding. Six other transport-impaired mutants did not have reduced alternating access rates, highlighting the importance of other aspects of the transport cycle to achieve drug resistance activity in vivo. To better understand the transport cycle of EmrE, efforts are now underway to determine a high-resolution structure using the S64V mutant identified here.

The C terminus of the bacterial multidrug transporter EmrE couples drug binding to proton release.

Ion-coupled transporters must regulate access of ions and substrates into and out of the binding site to actively transport substrates and minimize dissipative leak of ions. Within the single-site alternating access model, competitive substrate binding forms the foundation of ion-coupled antiport. Strict competition between substrates leads to stoichiometric antiport without slippage. However, recent NMR studies of the bacterial multidrug transporter EmrE have demonstrated that this multidrug transporter can simultaneously bind drug and proton, which will affect the transport stoichiometry and efficiency of coupled antiport. Here, we investigated the nature of substrate competition in EmrE using multiple methods to measure proton release upon the addition of saturating concentrations of drug as a function of pH. The resulting proton-release profile confirmed simultaneous binding of drug and proton, but suggested that a residue outside EmrE's Glu-14 binding site may release protons upon drug binding. Using NMR-monitored pH titrations, we trace this drug-induced deprotonation event to His-110, EmrE's C-terminal residue. Further NMR experiments disclosed that the C-terminal tail is strongly coupled to EmrE's drug-binding domain. Consideration of our results alongside those from previous studies of EmrE suggests that this conserved tail participates in secondary gating of EmrE-mediated proton/drug transport, occluding the binding pocket of fully protonated EmrE in the absence of drug to prevent dissipative proton transport.

A mass spectrometry based transport assay for studying EmrE transport of unlabeled substrates.

Membrane transporters are an important class of proteins which remain challenging to study. Transport assays are crucial to developing our understanding of such proteins as they allow direct measurement of their transport activity. However, currently available methods for monitoring liposomal loading of organic substrates primarily rely on detection of radioactively or fluorescently labeled substrates. The requirement of a labeled substrate significantly restricts the systems and substrates that can be studied. Here we present a mass spectrometry based detection method for liposomal uptake assays that eliminates the need for labeled substrates. We demonstrate the efficacy of the assay with EmrE, a small multidrug resistance transporter found in E. coli that has become a model transport system for the study of secondary active transport. Furthermore, we develop a method for differentiation between bound and transported substrate, enhancing the information gained from the liposomal uptake assay. The transport assay presented here is readily applicable to other transport systems and substrates.