Scholarly Journals--Published

  • Watts, K. J. and M. S. Johnson. "Analyzing Protein Domain Interactions by in vivo PEGylation". Methods Mol Biol 1729:137-145. DOI: 10.1007/978-1-4939-7577-8_13 The instability of some proteins can hamper in vitro studies. This is true for the membrane-bound aerotaxis receptor, Aer, which exhibits significant proteolysis during the preparation of membrane vesicles. Permeabilized cells can closely mimic in vivo conditions, maintaining the intracellular milieu and geometry of interacting domains. Here, we describe an optimized method for determining solvent accessibility in permeabilized Escherichia coli cells. In this method, E. coli expressing Aer with a series of cysteine replacements are treated with toluene and ethanol, after which a large sulfhydryl reactive probe, PEG-mal, is added. PEGylated protein is separated from un-PEGylated protein by its apparent size difference on SDS-PAGE. The extent to which each cysteine residue becomes PEGylated is then used as a measure of solvent accessibility. When a library of single-Cys replacements is mapped, regions of low accessibility can suggest interacting protein surfaces. We successfully used this method to reveal inaccessible surfaces on both the Aer PAS and HAMP domains that were then shown by disulfide cross-linking to interact. (01/2019)
  • Greer-Phillips S, Sukomon N, Johnson MS, Crane BR and Watts KJ. 2018. The Aer2 from Vibrio cholerae is a dual PAS-heme oxygen receptor. Mol Micro. 109(2):209–224. doi: 10.1111/mmi.13978 The diarrheal pathogen Vibrio cholerae navigates complex environments using three chemosensory systems and 44–45 chemoreceptors. Chemosensory cluster II modulates chemotaxis, whereas clusters I and III have unknown functions. Ligands have been identified for only five V. cholerae chemoreceptors. Here we report that the cluster III receptor, VcAer2, binds and responds to O2. VcAer2 is an ortholog of Pseudomonas aeruginosa Aer2 (PaAer2), but differs in that VcAer2 has two, rather than one, N-terminal PAS domain. We have determined that both PAS1 and PAS2 form homodimers and bind penta-coordinate b-type heme via an Eη-His residue. Heme binding to PAS1 required the entire PAS core, but receptor function also required the N-terminal cap. PAS2 functioned as an O2-sensor [Kd(O2), 19 µM], utilizing the same Iβ Trp (W276) as PaAer2 to stabilize O2. The crystal structure of PAS2-W276L was similar to that of PaAer2-PAS, but resided in an active conformation mimicking the ligand-bound state, consistent with its signal-on phenotype. PAS1 also bound O2 [Kd(O2), 12 µM], although O2 binding was stabilized by either a Trp or Tyr residue. Moreover, PAS1 appeared to function as a signal modulator, regulating O2-mediated signaling from PAS2, and resulting in activation of the cluster III chemosensory pathway. (05/2018) (link)
  • Garcia, D., Orillard, E. Johnson, M. S. and K. J. Watts. "Gas Sensing and Signaling in the PAS-Heme Domain of the Pseudomonas aeruginosa Aer2 Receptor". J. Bacteriol, 199:3-17. doi: 10.1128/JB.00003-17 The Aer2 chemoreceptor from Pseudomonas aeruginosa contains a PAS sensing domain that coordinates b-type heme and signals in response to the binding of O2, CO, or NO. PAS-heme structures suggest that Aer2 uniquely coordinates heme via a His residue on a 310 helix (H234 on Eη), stabilizes O2 binding via a Trp residue (W283), and signals via both W283 and an adjacent Leu residue (L264). Ligand binding may displace L264 and reorient W283 for hydrogen bonding to the ligand. Here, we clarified the mechanisms by which Aer2-PAS binds heme, regulates ligand binding, and initiates conformational signaling. H234 coordinated heme, but additional hydrophobic residues in the heme cleft were also critical for stable heme binding. O2 appeared to be the native Aer2 ligand (dissociation constant [Kd] of 16 μM). With one exception, mutants that bound O2 could signal, whereas many mutants that bound CO could not. W283 stabilized O2 binding but not CO binding, and it was required for signal initiation; W283 mutants that could not stabilize O2 were rapidly oxidized to Fe(III). W283F was the only Trp mutant that bound O2 with wild-type affinity. The size and nature of residue 264 was important for gas binding and signaling: L264W blocked O2 binding, L264A and L264G caused O2-mediated oxidation, and L264K formed a hexacoordinate heme. Our data suggest that when O2 binds to Aer2, L264 moves concomitantly with W283 to initiate the conformational signal. The signal then propagates from the PAS domain to regulate the C-terminal HAMP and kinase control domains, ultimately modulating a cellular response. (09/2017) (link)
  • Garcia, D., K. J. Watts, M. S. Johnson, and B. L. Taylor. "Delineating PAS-HAMP interaction surfaces and signalling-associated changes in the aerotaxis receptor Aer". Mol Micro 100:156-172 (2016). doi: 10.1111/mmi.13308 The Escherichia coli aerotaxis receptor, Aer, monitors cellular oxygen and redox potential via FAD bound to a cytosolic PAS domain. Here, we show that Aer-PAS controls aerotaxis through direct, lateral interactions with a HAMP domain. This contrasts with most chemoreceptors where signals propagate along the protein backbone from an N-terminal sensor to HAMP. We mapped the interaction surfaces of the Aer PAS, HAMP and proximal signalling domains in the kinase-off state by probing the solvent accessibility of 129 cysteine substitutions. Inaccessible PAS-HAMP surfaces overlapped with a cluster of PAS kinase-on lesions and with cysteine substitutions that crosslinked the PAS β-scaffold to the HAMP AS-2 helix. A refined Aer PAS-HAMP interaction model is presented. Compared to the kinase-off state, the kinase-on state increased the accessibility of HAMP residues (apparently relaxing PAS-HAMP interactions), but decreased the accessibility of proximal signalling domain residues. These data are consistent with an alternating static-dynamic model in which oxidized Aer-PAS interacts directly with HAMP AS-2, enforcing a static HAMP domain that in turn promotes a dynamic proximal signalling domain, resulting in a kinase-off output. When PAS-FAD is reduced, PAS interaction with HAMP is relaxed and a dynamic HAMP and static proximal signalling domain convey a kinase-on output. (04/2016) (link)
  • Garcia D, Watts K J, Johnson M S, & Taylor B L. (2015). Delineating PAS-HAMP interaction surfaces and signaling-associated changes in the aerotaxis receptor aer. Mol Microbiol, , . The Escherichia coli aerotaxis receptor, Aer, monitors cellular oxygen and redox potential via FAD bound to a cytosolic PAS domain. Here we show that Aer-PAS controls aerotaxis through direct, lateral interactions with a HAMP domain. This contrasts with most chemoreceptors where signals propagate along the protein backbone from an N-terminal sensor to HAMP. We mapped the interaction surfaces of the Aer PAS, HAMP and proximal signaling domains in the kinase-off state by probing the solvent accessibility of 129 cysteine substitutions. Inaccessible PAS-HAMP surfaces overlapped with a cluster of PAS kinase-on lesions and with cysteine substitutions that crosslinked the PAS beta-scaffold to the HAMP AS-2 helix. A refined Aer PAS-HAMP interaction model is presented. Compared to the kinase-off state, the kinase-on state increased the accessibility of HAMP residues (apparently relaxing PAS-HAMP interactions), but decreased the accessibility of proximal signaling domain residues. These data are consistent with an alternating static-dynamic model in which oxidized Aer-PAS interacts directly with HAMP AS-2, enforcing a static HAMP domain that in turn promotes a dynamic proximal signaling domain, resulting in a kinase-off output. When PAS-FAD is reduced, PAS interaction with HAMP is relaxed and a dynamic HAMP and static proximal signaling domain convey a kinase-on output. This article is protected by copyright. All rights reserved. (12/2015) (link)
  • Watts, K. J., B. L. Taylor, and M. S. Johnson. "PAS/poly-HAMP signalling in Aer-2, a soluble haem-based sensor". Mol Microbiol, 79:686-99 (2011).  doi: 10.1111/j.1365-2958.2010.07477.x Poly-HAMP domains are widespread in bacterial chemoreceptors, but previous studies have focused on receptors with single HAMP domains. The Pseudomonas aeruginosa chemoreceptor, Aer-2, has an unusual domain architecture consisting of a PAS sensing domain sandwiched between three N-terminal and two C-terminal HAMP domains, followed by a conserved kinase control module. The structure of the N-terminal HAMP domains was recently solved, making Aer-2 the first protein with resolved poly-HAMP structure. The role of Aer-2 in P. aeruginosa is unclear, but here we show that Aer-2 can interact with the chemotaxis system of Escherichia coli to mediate repellent responses to oxygen, carbon monoxide and nitric oxide. Using this model system to investigate signaling and poly-HAMP function, we determined that the Aer-2 PAS domain binds penta-coordinated b-type heme and that reversible signaling requires four of the five HAMP domains. Deleting HAMP 2 and/or 3 resulted in a kinase-off phenotype, whereas deleting HAMP 4 and/or 5 resulted in a kinase-on phenotype. Overall, these data support a model in which ligand-bound Aer-2 PAS and HAMP 2 and 3 act together to relieve inhibition of the kinase control module by HAMP 4 and 5, resulting in the kinase-on state of the Aer-2 receptor. (02/2011) (link)
  • Campbell, A. J., K. J. Watts, M. S. Johnson, and B. L. Taylor. "Role of the F1 region in the Escherichia coli aerotaxis receptor Aer". J Bacteriol, 193:358-66 (2011). doi: 10.1128/JB.01028-10 In Escherichia coli, the aerotaxis receptor Aer is an atypical receptor because it senses intracellular redox potential. The Aer sensor is a cytoplasmic, N-terminal PAS domain that is tethered to the membrane by a 47-residue F1 linker. Here we investigated the function, topology, and orientation of F1 by employing random mutagenesis, cysteine scanning, and disulfide cross-linking. No native residue was obligatory for function, most deleterious substitutions had radically different side chain properties, and all F1 mutants but one were functionally rescued by the chemoreceptor Tar. Cross-linking studies were consistent with the predicted α-helical structure in the N-terminal F1 region and demonstrated trigonal interactions among the F1 linkers from three Aer monomers, presumably within trimer-of-dimer units, as well as binary interactions between subunits. Using heterodimer analyses, we also demonstrated the importance of arginine residues near the membrane interface, which may properly anchor the Aer protein in the membrane. By incorporating these data into a homology model of Aer, we developed a model for the orientation of the Aer F1 and PAS regions in an Aer lattice that is compatible with the known dimensions of the chemoreceptor lattice. We propose that the F1 region facilitates the orientation of PAS and HAMP domains during folding and thereby promotes the stability of the PAS and HAMP domains in Aer. (01/2011) (link)
  • Campbell, A. J., K. J. Watts, M. S. Johnson, and B. L. Taylor. "Gain-of-function mutations cluster in distinct regions associated with the signalling pathway in the PAS domain of the aerotaxis receptor, Aer". Mol Microbiol, 77:575-586 (2010). doi: 10.1111/j.1365-2958.2010.07231.x The Aer receptor monitors internal energy (redox) levels in Escherichia coli with an FAD-containing PAS domain. Here, we randomly mutagenized the region encoding residues 14–119 of the PAS domain and found 72 aerotaxis-defective mutants, 24 of which were gain-of-function, signal-on mutants. The muta- tions were mapped onto an Aer homology model based on the structure of the PAS–FAD domain in NifL from Azotobacter vinlandii. Signal-on lesions clustered in the FAD binding pocket, the b-scaffolding and in the N-cap loop. We suggest that the signal-on lesions mimic the ‘signal-on’ state of the PAS domain, and therefore may be markers for the signal-in and signal-out regions of this domain. We propose that the reduction of FAD rearranges the FAD binding pocket in a way that repositions the b-scaffolding and the N-cap loop. The resulting conformational changes are likely to be conveyed directly to the HAMP domain, and on to the kinase control module. In support of this hypothesis, we demonstrated disulphide band formation between cysteines substituted at residues N98C or I114C in the PAS b-scaffold and residue Q248C in the HAMP AS-2 helix. (08/2010) (link)
  • Watts, K. J., M. S. Johnson & B. L. Taylor, "Structure-function relationships in the HAMP and proximal signaling domains of the aerotaxis receptor Aer", J Bacteriol 190: 2118-2127, 2008.  doi: 10.1128/JB.01858-07   Aer, the Escherichia coli aerotaxis receptor, faces the cytoplasm, where the PAS (Per-ARNT-Sim)-flavin adenine dinucleotide (FAD) domain senses redox changes in the electron transport system or cytoplasm. PAS-FAD interacts with a HAMP (histidine kinase, adenylyl cyclase, methyl-accepting protein, and phosphatase) domain to form an input-output module for Aer signaling. In this study, the structure of the Aer HAMP and proximal signaling domains was probed to elucidate structure-function relationships important for signaling. Aer residues 210 to 290 were individually replaced with cysteine and then cross-linked in vivo. The results confirmed that the Aer HAMP domain is composed of two alpha-helices separated by a structured loop. The proximal signaling domain consisted of two alpha-helices separated by a short undetermined structure. The Af1503 HAMP domain from Archaeoglobus fulgidus was recently shown to be a four-helix bundle. To test whether the Af1503 HAMP domain is a prototype for the Aer HAMP domain, the latter was modeled using coordinates from Af1503. Several findings supported the hypothesis that Aer has a four-helix HAMP structure: (i) cross-linking independently identified the same residues at the dimer interface that were predicted by the model, (ii) the rate of cross-linking for residue pairs was inversely proportional to the beta-carbon distances measured on the model, and (iii) clockwise lesions that were not contiguous in the linear Aer sequence were clustered in one region in the folded HAMP model, defining a potential site of PAS-HAMP interaction during signaling. In silico modeling of mutant Aer proteins indicated that the four-helix HAMP structure was important for Aer stability or maturation. The significance of the HAMP and proximal signaling domain structure for signal transduction is discussed. (01/2008 - 12/2008) (link)
  • Amin, D. N., B. L. Taylor, and M. S. Johnson.  "Organization of the aerotaxis receptor aer in the membrane of Escherichia coli". J Bacteriol 189:7206-12  (2007). doi: 10.1128/JB.00871-07 The Aer receptor guides Escherichia coli to specific oxygen and energy-generating niches. The input sensor in Aer is an FAD-binding, PAS domain, which is separated from a HAMP/signaling output domain by two membrane spanning segments that flank a short (four amino acid) periplasmic loop. In this study, we determined the overall membrane organization of Aer by introducing combinations of residues that allowed us to differentiate intra- from inter-dimeric collisions. Collisions between proximal residues in the membrane anchor were exclusively intra- or inter-dimeric, but, with one exception, not both. Crosslinking profiles were consistent with a rigid, rather than flexible periplasmic loop, and a tilted TM2 helix that crossed TM2'''' at residue V197C, near the center of the lipid bilayer. The periplasmic loop formed a stable neighborhood that i) included a maximum of three Aer dimers, ii) did not swap neighbors over time and iii) appeared to be constrained by interactions in the cytosolic signaling domain. (08/2007) (link)
  • Taylor, B. L., K. J. Watts, M. S. Johnson. "Oxygen and Redox Sensing by Two-Component Systems That Regulate Behavioral Responses: Behavioral Assays and Structural Studies of Aer Using In Vivo Disulfide Cross-Linking." Methods in Enzymology 422. (2007): 190-232. A remarkable increase in the number of annotated aerotaxis (oxygen-seeking) and redox taxis sensors can be attributed to recent advances in bacterial genomics. However, in silico predictions should be supported by behavioral assays and genetic analyses that confirm an aerotaxis or redox taxis function. This chapter presents a collection of procedures that have been highly successful in characterizing aerotaxis and redox taxis in Escherichia coli. The methods are described in enough detail to enable investigators of other species to adapt the procedures for their use. A gas flow cell is used to quantitate the temporal responses of bacteria to a step increase or decrease in oxygen partial pressure or redox potential. Bacterial behavior in spatial gradients is analyzed using optically flat capillaries and soft agar plates (succinate agar or tryptone agar). We describe two approaches to estimate the preferred partial pressure of oxygen that attracts a bacterial species; this concentration is important for understanding microbial ecology. At the molecular level, we describe procedures used to determine the structure and topology of Aer, a membrane receptor for aerotaxis. Cysteine-scanning mutagenesis and in vivo disulfide cross-linking procedures utilize the oxidant Cu(II)-(1,10-phenanthroline)3 and bifunctional sulfhydryl-reactive probes. Finally, we describe methods used to determine the boundaries of transmembrane segments of receptors such as Aer. These include 5-iodoacetamidofluorescein, 4-acetamido-4-disulfonic acid, disodium salt (AMS), and methoxy polyethylene glycol maleimide, a 5-kDa molecular mass probe that alters the mobility of Aer on SDS-PAGE. (07/2007) (link)
  • Watts, K. J., K. Sommer, S. L Fry, M. S Johnson, B. L Taylor. "Function of the N-Terminal Cap of the PAS Domain in Signaling by the Aerotaxis Receptor Aer." J. Bacteriol. 188. (2006): 2154-2162. Aer, the Escherichia coli receptor for behavioral responses to oxygen (aerotaxis), energy, and redox potential, contains a PAS sensory-input domain. Within the PAS superfamily, the N-terminal segment (N-cap) is poorly conserved and its role is not well understood. We investigated the role of the N-cap (residues 1 to 19) in the Aer PAS domain by missense and truncation mutagenesis. Aer-PAS N-cap truncations and an Aer-M21P substitution resulted in low cellular levels of the mutant proteins, suggesting that the N-terminal region was important for stabilizing the structure of the PAS domain. The junction of the N-cap and PAS core was critical for signaling in Aer. Mutations and truncations in the sequence encoding residues 15 to 21 introduced a range of phenotypes, including defects in FAD binding, constant tumbling motility, and an inverse response in which E. coli cells migrated away from oxygen concentrations to which they are normally attracted. The proximity of two N-cap regions in an Aer dimer was assessed in vivo by oxidatively cross-linking serial cysteine substitutions. Cross-linking of several cysteine replacements at 23 degrees C was attenuated at 10 degrees C, indicating contact was not at a stable dimer interface but required lateral mobility. We observed large multimers of Aer when we combined cross-linking of N-cap residues with a cysteine replacement that cross-links exclusively at the Aer dimer interface. This suggests that the PAS N-cap faces outwards in a dimer and that PAS-PAS contacts can occur between adjacent dimers. (03/2006) (link)
  • Watts, K. J., M. S. Johnson, and B. L. Taylor . "Minimal requirements for oxygen sensing by the aerotaxis receptor Aer." Molecular Microbiology 59. (2006): 1317-1326. The PAS and HAMP domain superfamilies are signal transduction modules found in all kingdoms of life. The Aer receptor, which contains both domains, initiates rapid behavioural responses to oxygen (aerotaxis) and other electron acceptors, guiding Escherichia coli to niches where it can generate optimal cellular energy. We used intragenic complementation to investigate the signal transduction pathway from the Aer PAS domain to the signalling domain. These studies showed that the HAMP domain of one monomer in the Aer dimer stabilized FAD binding to the PAS domain of the cognate monomer. In contrast, the signal transduction pathway was intra-subunit, involving the PAS and signalling domains from the same monomer. The minimal requirements for signalling were investigated in heterodimers containing a full-length and truncated monomer. Either the PAS or signalling domains could be deleted from the non-signalling subunit of the heterodimer, but removing 16 residues from the C-terminus of the signalling subunit abolished aerotaxis. Although both HAMP domains were required for aerotaxis, signalling was not disrupted by missense mutations in the HAMP domain from the signalling subunit. Possible models for Aer signal transduction are compared. (02/2006) (link)
  • Amin, D.N., B. L. Taylor, and M. S. johnson. "Topology and boundaries of the aerotaxis receptor Aer in the membrane of Escherichia coli." J. Bacteriol. 188. (2006): 894-901. Escherichia coli chemoreceptors are type I membrane receptors that have a periplasmic sensing domain, a cytosolic signaling domain, and two transmembrane segments. The aerotaxis receptor, Aer, is different in that both its sensing and signaling regions are proposed to be cytosolic. This receptor has a 38-residue hydrophobic segment that is thought to form a membrane anchor. Most transmembrane prediction programs predict a single transmembrane-spanning segment, but such a topology is inconsistent with recent studies indicating that there is direct communication between the membrane flanking PAS and HAMP domains. We studied the overall topology and membrane boundaries of the Aer membrane anchor by a cysteine-scanning approach. The proximity of 48 cognate cysteine replacements in Aer dimers was determined in vivo by measuring the rate and extent of disulfide cross-linking after adding the oxidant copper phenanthroline, both at room temperature and to decrease lateral diffusion in the membrane, at 4°C. Membrane boundaries were identified in membrane vesicles using 5-iodoacetamidofluorescein and methoxy polyethylene glycol 5000 (mPEG). To map periplasmic residues, accessible cysteines were blocked in whole cells by pretreatment with 4-acetamido-4'-maleimidylstilbene-2, 2' disulfonic acid before the cells were lysed in the presence of mPEG. The data were consistent with two membrane-spanning segments, separated by a short periplasmic loop. Although the membrane anchor contains a central proline residue that reaches the periplasm, its position was permissive to several amino acid and peptide replacements (02/2006) (link)
  • Ma Q, Johnson MS, Taylor BL. "Genetic analysis of the HAMP domain of the Aer aerotaxis sensor localizes flavin adenine dinucleotide-binding determinants to the AS-2 helix." J Bacteriol. 187.1 (2005): 193-201. HAMP domains are signal transduction domains typically located between the membrane anchor and cytoplasmic signaling domain of the proteins in which they occur. The prototypical structure consists of two helical amphipathic sequences (AS-1 and AS-2) connected by a region of undetermined structure. The Escherichia coli aerotaxis receptor, Aer, has a HAMP domain and a PAS domain with a flavin adenine dinucleotide (FAD) cofactor that senses the intracellular energy level. Previous studies reported mutations in the HAMP domain that abolished FAD binding to the PAS domain. In this study, using random and site-directed mutagenesis, we identified the distal helix, AS-2, as the component of the HAMP domain that stabilizes FAD binding. AS-2 in Aer is not amphipathic and is predicted to be buried. Mutations in the sequence coding for the contiguous proximal signaling domain altered signaling by Aer but did not affect FAD binding. The V264M residue replacement in this region resulted in an inverted response in which E. coli cells expressing the mutant Aer protein were repelled by oxygen. Bioinformatics analysis of aligned HAMP domains indicated that the proximal signaling domain is conserved in other HAMP domains that are not involved in chemotaxis or aerotaxis. Only one null mutation was found in the coding sequence for the HAMP AS-1 and connector regions, suggesting that these are not active signal transduction sites. We consider a model in which the signal from FAD is transmitted across a PAS-HAMP interface to AS-2 or the proximal signaling domain (01/2005) (link)
  • Ma Q, Roy F, Herrmann S, Taylor BL, Johnson MS. "The Aer protein of Escherichia coli forms a homodimer independent of the signaling domain and flavin adenine dinucleotide binding." J Bacteriol. 186.21 (2004): 7456-7459. In vivo cross-linking between native cysteines in the Aer receptor of Escherichia coli showed dimer formation at the membrane anchor and in the putative HAMP domain. Dimers also formed in mutants that did not bind flavin adenine dinucleotide and in truncated peptides without a signaling domain and part of the HAMP domain (11/2004) (link)
  • Watts KJ, Ma Q, Johnson MS, Taylor BL. "Interactions between the PAS and HAMP domains of the Escherichia coli aerotaxis receptor Aer." J Bacteriol. 186.21 (2004): 7440-7449. The Escherichia coli energy-sensing Aer protein initiates aerotaxis towards environments supporting optimal cellular energy. The Aer sensor is an N-terminal, FAD-binding, PAS domain. The PAS domain is linked by an F1 region to a membrane anchor, and in the C-terminal half of Aer, a HAMP domain links the membrane anchor to the signaling domain. The F1 region, membrane anchor, and HAMP domain are required for FAD binding. Presumably, alterations in the redox potential of FAD induce conformational changes in the PAS domain that are transmitted to the HAMP and C-terminal signaling domains. In this study we used random mutagenesis and intragenic pseudoreversion analysis to examine functional interactions between the HAMP domain and the N-terminal half of Aer. Missense mutations in the HAMP domain clustered in the AS-2 alpha-helix and abolished FAD binding to Aer, as previously reported. Three amino acid replacements in the Aer-PAS domain, S28G, A65V, and A99V, restored FAD binding and aerotaxis to the HAMP mutants. These suppressors are predicted to surround a cleft in the PAS domain that may bind FAD. On the other hand, suppression of an Aer-C253R HAMP mutant was specific to an N34D substitution with a predicted location on the PAS surface, suggesting that residues C253 and N34 interact or are in close proximity. No suppressor mutations were identified in the F1 region or membrane anchor. We propose that functional interactions between the PAS domain and the HAMP AS-2 helix are required for FAD binding and aerotactic signaling by Aer. (11/2004) (link)
  • Herrmann S, Ma Q, Johnson MS, Repik AV, Taylor BL. "PAS domain of the Aer redox sensor requires C-terminal residues for native-fold formation and flavin adenine dinucleotide binding." J Bacteriol. 186.20 (2004): 6782-6791. The Aer protein in Escherichia coli is a membrane-bound, FAD-containing aerotaxis and energy sensor that putatively monitors the redox state of the electron transport system. Binding of FAD to Aer requires the N-terminal PAS domain and residues in the F1 region and C-terminal HAMP domain. The PAS domains of other PAS proteins are soluble in water. To investigate properties of the PAS domain, we subcloned segments of the aer gene from E. coli that encode the PAS domain with and without His6 tags and expressed the PAS peptides in E. coli. The 20-kDa His6-Aer2-166 PAS-F1 fragment was purified as an 800-kDa complex by gel filtration chromatography, and the associating protein was identified by N-terminal sequencing as the chaperone protein GroEL. None of the N-terminal fragments of Aer found in the soluble fraction was released from GroEL, suggesting that these peptides do not fold correctly in an aqueous environment and require a motif external to the PAS domain for proper folding. Consistent with this model, peptide fragments that included the membrane binding region and part (Aer2-231) or all (Aer2-285) of the HAMP domain inserted into the membrane, indicating that they were released by GroEL. Aer2-285, but not Aer2-231, bound FAD, confirming the requirement for the HAMP domain in stabilizing FAD binding. The results raise an interesting possibility that residues outside the PAS domain that are required for FAD binding are essential for formation of the PAS native fol (10/2004) (link)


  • Johnson M, Ng D, Schaepper M, & Kirsch W. (2009). ALPHA DEFENSINS AS A POTENTIAL BIOMARKER IN SCHIZOPHRENIC SWEAT. Journal of Investigative Medicine, 57(1), 132-133. (01/2009)
  • (PEER REVIEWED) Pierre, K., B. L. Taylor, M. S. Johnson. "The nitrate-dependent vanishing act of the aspartate ring in Escherichia coli." BLAST IX Meeting . (2007): p. 91-. (01/2007) (link)
  • (PEER REVIEWED) Campbell, A. J., K. J. Watts, M. S. Johnson, B. L. Taylor. "The Aer F1 domain- proximity, location and determinants for membrane insertion." BLAST IX Meeting . (2007): p. 137-. (01/2007) (link)
  • (PEER REVIEWED) Amin, D., B. L. Taylor, and M. S. johnson. "Organization and boundaries of the transmembrane domain of the Aer receptor in Escherichia coli." In Abstracts of the 105th General Meeting . (2005): -. (06/2005)
  • (PEER REVIEWED) Edwards, J. C., M. S. Johnson, and B. L. Taylor. "Tsr- and Aer-mediated aerotaxis in Escherichia coli correlates with changes in membrane potential and redox changes, respectively." In Abstracts of the 105th General Meeting . (2005): -. (06/2005)
  • (PEER REVIEWED) Watts, K. J., K. Sommer, M. S. Johnson, and B. L. Taylor . "The essential Aer PAS N-cap in Escherichia coli faces outward and can be crosslinked with the N-cap from an adjacent dimer.." In Abstracts of the 105th General Meeting . (2005): -. (06/2005)