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Associate Professor Anthony George


Assoc Prof George has been at the University of Technology since its inception as a university in 1989. He has published over 60 papers. He graduated with a PhD degree from the University of Sydney in 1981 and completed postdoctoral fellowships at Tufts University Medical School (Boston) and the John Curtin School of Medical Research (Canberra).

His tenured academic position at UTS began in 1986. His early research was focused on multidrug resistance in pathogenic bacteria and this work continued at UTS, which more recently has moved into the study of pathogenic infections in serious medical syndromes such as cystic fibrosis.

A/P George’s research has taken two new directions, one beginning in 1995 and the second in 2007. In 1996, A/P George published the first and still only successful cloning, expression, and drug efflux for human P-glycoprotein in Eschericia coli. This MDR protein belongs to one of the largest gene/protein families in all species, namely ABC transporters. Most of his work since then has involved structure-function studies of ABC transporters, using traditional laboratory methods and the emerging and powerful computational molecular dynamic simulations that take X-ray crystal structures of proteins and “animate” them in computers to explore their function. We have been very successful in this field, having discovered the dimeric structural configuration of ABC transporters and elaborated a new model for the function of these proteins. A/P George’s work has been rewarded by prestigious invitations to give keynote talks at international conferences, to Chair a Gordon Research Meeting on multidrug resistance systems and invited on to the Scientific Advisory Board of the FEBS ABC Efflux Meetings, held every two years in Austria.

A/P George has published trailblazing studies and many scholarly reviews in high impact journals such as Proc Natl Acad Sci USA and Trend Biochem Sci. He has supervised many Honours, Masters, and PhDs, and has a Research Associate of long standing for his mainstream ABC transporter research. Total research funding and fellowships for all projects exceeds $5m.

In 2007, A/P George began a new project intended to alleviate the onset and progression of incurable asbestos-related diseases. Research began by studying the effects of asbestos fibres and counteracting agents in human lung cells; and has moved these past years to an animal model. The work has been rewarded with a Trailblazer Award (2008) and with substantial funding ($712,000) from philanthropic sources. The mouse model work has illustrated remarkable success in slowing or preventing the onset of mesothelioma, an incurable disease. A provisional patent on our new treatment has recently been converted to a full patent. Presently, we are seeking funding to take the project to a first human trial in 2017.


I have held memberships of a number of professional societies including, the Federation of European Microbiologists Society, American Chemical Society, Biophysical Society, and American Society for Biochemistry and Molecular Biology.

Image of Anthony George
Associate Professor, School of Life Sciences
Associate Member, CHT - Centre for Health Technologies
Core Member, ithree - Institute of Infection, Immunity and Innovation
B.Sc(Hons), MSc (Syd), PhD (Syd)
+61 2 9514 4158

Research Interests

1. ABC transporters and cancer
ABC transporters are involved in diverse cellular processes including resistance to cytotoxic drugs. Inhibition of these proteins will improve the efficacy of primary drug treatment and render these proteins as targets for new drugs. We are using recent atomic structures as the basis for molecular dynamics calculations and cross-linking experiments designed to identify crucial mobile regions, enabling us to identify and test small molecules that interfere with the normal movement of critical regions in these proteins. [Collaboration with Dr Richard Callaghan, Oxford University; Dr Ian Kerr, Nottingham University; and Dr Megan O’Mara, QLD University].

2. Cystic Fibrosis infections
Cystic fibrosis is the most common lethal inherited disorder in Caucasians, affecting 1 in 2,500 births. Whilst median survival has increased from 1-2 years (1960) to a current 36-38 years, chronic lung disease still causes the majority of deaths associated with CF. Most patients are infected chronically with P. aeruginosa. Resistance to chemotherapeutic antibiotics is commonplace. We are testing the efficacy of the combination of tobramycin and amiloride against P. aeruginosa in clinical and laboratory studies.
[in collaboration with Assoc Prof Cythia Whitworth and Dr Lynne Turnbull, IBID/UTS; and Assoc Profs Peter Middleton and Jon Iredell, Sydney University].

3. Ameliorating Asbestosis
When asbestos is mined or pulverized, it becomes suspended in the air as "parachutes", leading to inhalation into the lungs where it can induce fibrosis (asbestosis) or malignancy (mesothelioma). Asbestos fibres generate reactive oxygen radicals that cause cellular and DNA damage. We have made a serendipitous discovery for ameliorating the adverse effects of asbestos fibres that we now want to test in lung cell cultures, and then in animal trials.

I have been involved in a number of teaching subjects at UTS over past years. Currently, I am the subject coordinator for Molecular Biology 1 and DNA Profiling. Most of my teaching is in Molecular Biology 1, a subject that was originally created by me, and whose content has been written, upgraded and revised at regular intervals. It has become one of the largest second year subjects with over 300 students. DNA Profiling in the Forensics Biology Degree is a subject that I inherited from an ex-staff member. The lectures and tutorials are given by external forensic scientists, which allows for an up to date interface with the developments in the subject area. The practical course is my major focus and it has been revised substantially in recent years. I make small contributions in terms of specialist lectures in the subjects Molecular Biology 2 and Biotechnology.


Ling, V., George, A.M., Jones, P.M., Slotbloom, D.J., Poolman, B., Van Veen, H.W., Otto, M., Hohl, M., Kerr, I.D., Gottesman, M.M., Chiba, P., Callaghan, R., Bates, S.E., Ambudkar, S.V., Schuetz, J.D., Ishikawa, T., Ford, R., Sarkadi, B. & Homolya, L. 2016, ABC Transporters - 40 Years On, Springer International Publishing.


George, A.M. & Jones, P.M. 2014, 'Bacterial ABC Transporters: Structure and Function' in Han Renaut (ed), Bacterial Membranes: Structural and Molecular Biology, Horizon Scientific Press, Norwich, UK.
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ATP-Binding-Cassette (ABC) membrane transporters belong to one of the largest and most ancient gene families, occurring in bacteria, archaea, and eukaryota. In addition to nutrient uptake, ABC transporters are involved in other diverse processes such as the export of toxins, peptides, proteins, antibiotics, polysaccharides and lipids, and in cell division, bacterial immunity and nodulation in plants. While prokaryotic ABC transporters encompass both importers and exporters, eukaryotes harbour only exporters. Bacterial ABC transporters are intricately involved either directly or indirectly in all aspects of cellular physiology, metabolism, homeostasis, drug resistance, secretion, and cellular division. Whilst several complete ABC transporter structures have been solved over the past decade, their functional mechanism of transport is still somewhat controversial and this aspect is discussed in detail.
Callaghan, R., George, A.M. & Kerr, I. 2011, 'Molecular aspects of the translocation process by ABC proteins' in Ferguson, S. (ed), Comprehensive Biophysics, American Chemical Society, Oxford, pp. 145-173.
George, A.M. 2005, 'Multiple Antimicrobial Resistance' in White, D.G., Alekshun, M.N. & McDermott, P.F. (eds), Frontiers in Antimicrobial Resistance: a Tribute to Stuart B. Levy, ASM Press, Washington DC, USA, pp. 151-164.
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Journal articles

Mittra, R., Pavy, M., Subramanian, N., George, A.M., O'Mara, M.L., Kerr, I.D. & Callaghan, R. 2017, 'Location of contact residues in pharmacologically distinct drug binding sites on P-glycoprotein.', Biochem Pharmacol, vol. 123, pp. 19-28.
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The multidrug resistance P-glycoprotein (P-gp) is characterised by the ability to bind and/or transport an astonishing array of drugs. This poly-specificity is imparted by at least four pharmacologically distinct binding sites within the transmembrane domain. Whether or not these sites are spatially distinct has remained unclear. Biochemical and structural investigations have implicated a central cavity as the likely location for the binding sites. In the present investigation, a number of contact residues that are involved in drug binding were identified through biochemical assays using purified, reconstituted P-gp. Drugs were selected to represent each of the four pharmacologically distinct sites. Contact residues important in rhodamine123 binding were identified in the central cavity of P-gp. However, contact residues for the binding of vinblastine, paclitaxel and nicardipine were located at the lipid-protein interface rather than the central cavity. A key residue (F978) within the central cavity is believed to be involved in coupling drug binding to nucleotide hydrolysis. Data observed in this investigation suggest the presence of spatially distinct drug binding sites connecting through to a single translocation pore in the central cavity.
Jones, P.M. & George, A.M. 2017, 'How Intrinsic Dynamics Mediates the Allosteric Mechanism in the ABC Transporter Nucleotide Binding Domain Dimer.', J Chem Theory Comput, vol. 13, no. 4, pp. 1712-1722.
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A protein's architecture facilitates specific motions-intrinsic dynamic modes-that are employed to effect function. Here we used molecular dynamics (MD) simulations to investigate the dynamics of the MJ0796 ABC transporter nucleotide-binding domain (NBD). ABC transporter NBDs form a rotationally symmetric dimer whereby two equivalent active sites are formed at their interface; in complex with a dimer of transmembrane domains they hydrolyze ATP to energize translocation of substrates across cellular membranes. Our data suggest the ABC NBD's ensemble of functional states can be understood predominately in terms of conformational changes between its major subdomains, occurring along two orthogonal dynamic modes. The data show that ligands and oligomeric interactions modulate the equilibrium conformation of the NBD with respect to these motions, suggesting that allostery is achieved by affecting the energetic profile along these two modes. The observed dynamics and allostery integrate consonantly and logically within a mechanistic framework for the ABC NBD dimer, which is supported by a large body of experimental and theoretical data, providing a higher resolution view of the enzyme's dynamic cycle. Our study shows how valuable mechanistic inferences can be derived from accessible short-time scale MD simulations of an enzyme's substructures.
Jones, P.M. & George, A.M. 2016, 'Computational analysis of the MCoTI-II plant defence knottin reveals a novel intermediate conformation that facilitates trypsin binding.', Scientific reports, vol. 6, p. 23174.
MCoTI-I and II are plant defence proteins, potent trypsin inhibitors from the bitter gourd Momordica cochinchinensis. They are members of the Knottin Family, which display exceptional stability due to unique topology comprising three interlocked disulfide bridges. Knottins show promise as scaffolds for new drug development. A crystal structure of trypsin-bound MCoTI-II suggested that loop 1, which engages the trypsin active site, would show decreased dynamics in the bound state, an inference at odds with an NMR analysis of MCoTI-I, which revealed increased dynamics of loop 1 in the presence of trypsin. To investigate this question, we performed unrestrained MD simulations of trypsin-bound and free MCoTI-II. This analysis found that loop 1 of MCoTI-II is not more dynamic in the trypsin-bound state than in the free state. However, it revealed an intermediate conformation, transitional between the free and bound MCoTI-II states. The data suggest that MCoTI-II binding involves a process in which initial interaction with trypsin induces transitions between the free and intermediate conformations, and fluctuations between these states account for the increase in dynamics of loop 1 observed for trypsin-bound MCoTI-I. The MD analysis thus revealed new aspects of the inhibitors' dynamics that may be of utility in drug design.
Sharpe, L.J., Rao, G., Jones, P.M., Glancey, E., Aleidi, S.M., George, A.M., Brown, A.J. & Gelissen, I.C. 2015, 'Cholesterol sensing by the ABCG1 lipid transporter: Requirement of a CRAC motif in the final transmembrane domain.', Biochimica et biophysica acta, vol. 1851, no. 7, pp. 956-964.
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The ATP-binding cassette (ABC) transporter, ABCG1, is a lipid exporter involved in removal of cholesterol from cells that has been investigated for its role in foam cells formation and atherosclerosis. The mechanism by which ABC lipid transporters bind and recognise their substrates is currently unknown. In this study, we identify a critical region in the final transmembrane domain of ABCG1, which is essential for its export function and stabilisation by cholesterol, a post-translational regulatory mechanism that we have recently identified as dependent on protein ubiquitination. This transmembrane region contains several Cholesterol Recognition/interaction Amino acid Consensus (CRAC) motifs, and its inverse CARC motifs. Mutational analyses identify one CRAC motif in particular with Y667 at its core, that is especially important for transport activity to HDL as well as stability of the protein in the presence of cholesterol. In addition, we present a model of how cholesterol docks to this CRAC motif in an energetically favourable manner. This study identifies for the first time how ABCG1 can interact with cholesterol via a functional CRAC domain, which provides the first insight into the substrate-transporter interaction of an ABC lipid exporter.
Jones, P.M. & George, A.M. 2015, 'The Nucleotide-Free State of the Multidrug Resistance ABC Transporter LmrA: Sulfhydryl Cross-Linking Supports a Constant Contact, Head-to-Tail Configuration of the Nucleotide-Binding Domains.', PloS one, vol. 10, no. 6, p. e0131505.
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ABC transporters are integral membrane pumps that are responsible for the import or export of a diverse range of molecules across cell membranes. ABC transporters have been implicated in many phenomena of medical importance, including cystic fibrosis and multidrug resistance in humans. The molecular architecture of ABC transporters comprises two transmembrane domains and two ATP-binding cassettes, or nucleotide-binding domains (NBDs), which are highly conserved and contain motifs that are crucial to ATP binding and hydrolysis. Despite the improved clarity of recent structural, biophysical, and biochemical data, the seemingly simple process of ATP binding and hydrolysis remains controversial, with a major unresolved issue being whether the NBD protomers separate during the catalytic cycle. Here chemical cross-linking data is presented for the bacterial ABC multidrug resistance (MDR) transporter LmrA. These indicate that in the absence of nucleotide or substrate, the NBDs come into contact to a significant extent, even at 4°C, where ATPase activity is abrogated. The data are clearly not in accord with an inward-closed conformation akin to that observed in a crystal structure of V. cholerae MsbA. Rather, they suggest a head-to-tail configuration 'sandwich' dimer similar to that observed in crystal structures of nucleotide-bound ABC NBDs. We argue the data are more readily reconciled with the notion that the NBDs are in proximity while undergoing intra-domain motions, than with an NBD 'Switch' mechanism in which the NBD monomers separate in between ATP hydrolysis cycles.
George, A.M. 2014, 'A reciprocating twin-channel model for ABC transporters', Quarterly Reviews of Biophysics.
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Jones, P.M. & George, A.M. 2013, 'Mechanism of the ABC transporter ATPase domains: catalytic models and the biochemical and biophysical record', Critical Reviews in Biochemistry and Molecular Biology, vol. 48, no. 1, pp. 39-50.
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ABC transporters comprise a large, diverse, and ubiquitous superfamily of membrane active transporters. Their core architecture is a dimer of dimers, comprising two transmembrane domains that bind substrate and form the channel, and two ATP-binding cassettes, which bind and hydrolyze ATP to energize the translocase function. The prevailing paradigm for the ABC transport mechanism is the Switch Model, in which the nucleotide binding domains are proposed to dimerise upon binding of two ATP molecules, and thence dissociate upon sequential hydrolysis of the ATP. This idea appears consistent with crystal structures of both isolated subunits and whole transporters, as well as with a significant body of biochemical data. Nonetheless, an alternative Constant Contact Model has been proposed, in which the nucleotide binding domains do not fully dissociate, and ATP hydrolysis occurs alternately at each of the two active sites. Here, we review the biochemical and biophysical data relating to the ABC catalytic mechanism, to show how they may be construed as consistent with a Constant Contact Model, and to assess to what extent they support the Switch Model.
George, A.M. & Jones, P.M. 2013, 'An asymmetric post-hydrolysis state of the ABC transporter ATPase dimer', PLoS One, vol. 8, no. 4.
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ABC transporters are a superfamily of enzyme pumps that hydrolyse ATP in exchange for translocation of substrates across cellular membranes. Architecturally, ABC transporters are a dimer of transmembrane domains coupled to a dimer of nucleotide binding domains (NBDs): the NBD dimer contains two ATP-binding sites at the intersubunit interface. A current controversy is whether the protomers of the NBD dimer separate during ATP hydrolysis cycling, or remain in constant contact. In order to investigate the ABC ATPase catalytic mechanism, MD simulations using the recent structure of the ADP+Pi-bound MJ0796 isolated NBD dimer were performed. In three independent simulations of the ADP+Pi/apo state, comprising a total of .0.5 ms, significant opening of the apo (empty) active site was observed; occurring by way of intrasubunit rotations between the core and helical subdomains within both NBD monomers. In contrast, in three equivalent simulations of the ATP/apo state, the NBD dimer remained close to the crystal structure, and no opening of either active site occurred. The results thus showed allosteric coupling between the active sites, mediated by intrasubunit conformational changes. Opening of the apo site is exquisitely tuned to the nature of the ligand, and thus to the stage of the reaction cycle, in the opposite site. In addition to this, in also showing how one active site can open, sufficient to bind nucleotide, while the opposite site remains occluded and bound to the hydrolysis products ADP+Pi, the results are consistent with a Constant Contact Model. Conversely, they show how there may be no requirement for the NBD protomers to separate to complete the catalytic cycle.
Gong, J., Luk, F., Jaiswal, R., George, A.M., Grau, G. & Bebawy, M. 2013, 'Microparticle drug sequestration provides a parallel pathway in the acquisition of cancer drug resistance', European Journal of Pharmacology, vol. 721, no. 1-3, pp. 116-125.
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Gong, J., Luk, F., Jaiswal, R., George, A.M., Grau, G. & Bebawy, M. 2013, 'Microparticle drug sequestration provides a parallel pathway in the acquisition of cancer drug resistance', European Journal of Pharmacology, vol. 721, no. 1-3, pp. 116-125.
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Jones, P.M., Curmi, P.M., Valenzuela, S.M. & George, A.M. 2013, 'Computational analysis of the soluble form of the intracellular chloride ion channel protein CLIC1.', BioMed research international, vol. 2013, p. 170586.
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The chloride intracellular channel (CLIC) family of proteins has the remarkable property of maintaining both a soluble form and an integral membrane form acting as an ion channel. The soluble form is structurally related to the glutathione-S-transferase family, and CLIC can covalently bind glutathione via an active site cysteine. We report approximately 0.6s of molecular dynamics simulations, encompassing the three possible ligand-bound states of CLIC1, using the structure of GSH-bound human CLIC1. Noncovalently bound GSH was rapidly released from the protein, whereas the covalently ligand-bound protein remained close to the starting structure over 0.25s of simulation. In the unliganded state, conformational changes in the vicinity of the glutathione-binding site resulted in reduced reactivity of the active site thiol. Elastic network analysis indicated that the changes in the unliganded state are intrinsic to the protein architecture and likely represent functional transitions. Overall, our results are consistent with a model of CLIC function in which covalent binding of glutathione does not occur spontaneously but requires interaction with another protein to stabilise the GSH binding site and/or transfer of the ligand. The results do not indicate how CLIC1 undergoes a radical conformational change to form a transmembrane chloride channel but further elucidate the mechanism by which CLICs are redox controlled.
Jones, P.M. & George, A.M. 2012, 'Role of the D-loops in allosteric control of ATP hydrolysis in an ABC transporter', Journal of Physical Chemistry A, vol. 116, no. 11, pp. 3004-3013.
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ABC transporters couple ATP hydrolysis to movement of substrates across cell membranes. They comprise two transmembrane domains and two cytosolic nucleotide-binding domains forming two active sites that hydrolyze ATP cooperatively. The mechanism of ATP hydrolysis is controversial and the structural dynamic basis of its allosteric control unknown. Here we report molecular dynamics simulations of the ATP/apo and ATP/ADP states of the bacterial ABC exporter Sav1866, in which the cytoplasmic region of the protein was simulated in explicit water for 150 ns. In the simulation of the ATP/apo state, we observed, for the first time, conformers of the active site with the canonical geometry for an in-line nucleophilic attack on the ATP gamma-phosphate. The conserved glutamate immediately downstream of the Walker B motif is the catalytic base, forming a dyad with the H-loop histidine, whereas the Q-loop glutamine has an organizing role. Each D-loop provides a coordinating residue of the attacking water, and comparison with the simulation of the ATP/ADP state suggests that via their flexibility, the D-loops modulate formation of the hydrolysis-competent state. A global switch involving a coupling helix delineates the signal transmission route by which allosteric control of ATP hydrolysis in ABC transporters is mediated.
George, A.M. & Jones, P.M. 2012, 'Perspectives on the structureefunction of ABC transporters: The Switch and Constant Contact Models', Progress in Biophysics and Molecular Biology, vol. 109, pp. 95-107.
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ABC transporters constitute one of the largest protein families across the kingdoms of archaea, eubacteria and eukarya. They couple ATP hydrolysis to vectorial translocation of diverse substrates across membranes. The ABC transporter architecture comprises two transmembrane domains and two cytosolic ATP-binding cassettes. During 2002-2012, nine prokaryotic ABC transporter structures and two eukaryotic structures have been solved to medium resolution. Despite a wealth of biochemical, biophysical, and structural data, fundamental questions remain regarding the coupling of ATP hydrolysis to unidirectional substrate translocation, and the mechanistic suite of steps involved. The mechanics of the ATP cassette dimer is defined most popularly by the 'Switch Model', which proposes that hydrolysis in each protomer is sequential, and that as the sites are freed of nucleotide, the protomers lose contact across a large solvent-filled gap of 20-30 angstrom: as captured in several X-ray solved structures. Our 'Constant Contact' model for the operational mechanics of ATP binding and hydrolysis in the ATP-binding cassettes is derived from the 'alternating sites' model, proposed in 1995, and which requires an intrinsic asymmetry in the ATP sites, but does not require the partner protomers to lose contact. Thus one of the most debated issues regarding the function of ABC transporters is whether the cooperative mechanics of ATP hydrolysis requires the ATP cassettes to separate or remain in constant contact and this dilemma is discussed at length in this review.
Nolan, L.M., Croft, L., Jones, P.M., George, A.M., Turnbull, L. & Whitchurch, C.B. 2012, 'Extragenic suppressor mutations that restore twitching motility to fimL mutants of Pseudomonas aeruginosa are associated with elevated intracellular cyclic AMP levels', Microbiology Open, vol. 1, no. 4, pp. 490-501.
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Cyclic AMP (cAMP) is a signaling molecule that is involved in the regulation of multiple virulence systems of the opportunistic pathogen Pseudomonas aeruginosa. The intracellular concentration of cAMP in P. aeruginosa cells is tightly controlled at the levels of cAMP synthesis and degradation through regulation of the activity and/or expression of the adenylate cyclases CyaA and CyaB or the cAMP phosphodiesterase CpdA. Interestingly, mutants of fimL, which usually demonstrate defective twitching motility, frequently revert to a wild-type twitching-motility phenotype presumably via the acquisition of an extragenic suppressor mutation(s). In this study, we have characterized five independent fimL twitching-motility revertants and have determined that all have increased intracellular cAMP levels compared with the parent fimL mutant. Whole-genome sequencing revealed that only one of these fimL revertants has acquired a loss-of-function mutation in cpdA that accounts for the elevated levels of intracellular cAMP. As mutation of cpdA did not account for the restoration of twitching motility observed in the other four fimL revertants, these observations suggest that there is at least another, as yet unidentified, site of extragenic suppressor mutation that can cause phenotypic reversion in fimL mutants and modulation of intracellular cAMP levels of P. aeruginosa.
Jones, P.M. & George, A.M. 2011, 'Molecular-dynamics simulations of the ATP/apo state of a multidrug ATP-binding cassette transporter provide a structural and mechanistic basis for the asymmetric occluded state', Biophysical Journal, vol. 100, no. 12, pp. 3025-3034.
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ATP-binding cassette transporters use the energy of ATP hydrolysis to transport substrates across cellular membranes. They have two transmembrane domains and two cytosolic nucleotide-binding domains.Biochemical studies have characterized an occluded state of the transporter in which nucleotide is tenaciously bound in one active site, whereas the opposite active site is empty or binds nucleotide loosely. Here, we report molecular-dynamics simulations of the bacterial multidrug ATP-binding cassette transporter Sav1866. In two simulations of the ATP/apo state, the empty site opened substantially by way of rotation of the nucleotide-binding domain (NBD) core subdomain, whereas the ATP-bound site remained occluded and intact. We correlate our findings with elastic network and molecular-dynamics simulation analyses of the 5av1866 NBD monomer, and with existing experimental data, to argue that the observed transition is physiological, and that the final structure observed in the ATP/apo simulations corresponds to the tight/loose state of the NBD dimer characterized experimentally..
Robinson, M.W., Corvo, I., Jones, P.M., George, A.M., Padula, M., To, J., Cancela, M., Rinaldi, G., Tort, J.F., Roche, L. & Dalton, J.P. 2011, 'Collagenolytic Activities of the Major Secreted Cathepsin L Peptidases Involved in the Virulence of the Helminth Pathogen, Fasciola hepatica', Plos Neglected Tropical Diseases, vol. 5, no. 4, pp. 1-13.
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Background: The temporal expression and secretion of distinct members of a family of virulence-associated cathepsin L cysteine peptidases (FhCL) correlates with the entry and migration of the helminth pathogen Fasciola hepatica in the host. Thus, infective larvae traversing the gut wall secrete cathepsin L3 (FhCL3), liver migrating juvenile parasites secrete both FhCL1 and FhCL2 while the mature bile duct parasites, which are obligate blood feeders, secrete predominantly FhCL1 but also FhCL2. Methodology/Principal Findings: Here we show that FhCL1, FhCL2 and FhCL3 exhibit differences in their kinetic parameters towards a range of peptide substrates. Uniquely, FhCL2 and FhCL3 readily cleave substrates with Pro in the P2 position and peptide substrates mimicking the repeating Gly-Pro-Xaa motifs that occur within the primary sequence of collagen. FhCL1, FhCL2 and FhCL3 hydrolysed native type I and II collagen at neutral pH but while FhCL1 cleaved only non-collagenous (NC, non-Gly-X-Y) domains FhCL2 and FhCL3 exhibited collagenase activity by cleaving at multiple sites within the alpha 1 and alpha 2 triple helix regions (Col domains). Molecular simulations created for FhCL1, FhCL2 and FhCL3 complexed to various seven-residue peptides supports the idea that Trp67 and Tyr67 in the S2 subsite of the active sites of FhCL3 and FhCL2, respectively, are critical to conferring the unique collagenase-like activity to these enzymes by accommodating either Gly or Pro residues at P2 in the substrate. The data also suggests that FhCL3 accommodates hydroxyproline (Hyp)-Gly at P3-P2 better than FhCL2 explaining the observed greater ability of FhCL3 to digest type I and II collagens compared to FhCL2 and why these enzymes cleave at different positions within the Col domains. Conclusions/Significance: These studies further our understanding of how this helminth parasite regulates peptidase expression to ensure infection, migration and establishment in host tissues.
Jones, P.M., Robinson, M.W., Dalton, J.P. & George, A.M. 2011, 'The Plasmodium falciparum malaria M1 alanyl aminopeptidase (PfA-M1): Insights of catalytic mechanism and function from MD simulations', PLoS ONE, vol. 6, no. 12, p. e28589.
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Malaria caused by several species of Plasmodium is major parasitic disease of humans, causing 1-3 million deaths worldwide annually. The widespread resistance of the human parasite to current drug therapies is of major concern making the identification of new drug targets urgent. While the parasite grows and multiplies inside the host erythrocyte it degrades the host cell hemoglobin and utilizes the released amino acids to synthesize its own proteins. The P. falciparum malarial M1 alanyl-aminopeptidase (PfA-M1) is an enzyme involved in the terminal stages of hemoglobin digestion and the generation of an amino acid pool within the parasite. The enzyme has been validated as a potential drug target since inhibitors of the enzyme block parasite growth in vitro and in vivo. In order to gain further understanding of this enzyme, molecular dynamics simulations using data from a recent crystal structure of PfA-M1 were performed. The results elucidate the pentahedral coordination of the catalytic Zn in these metallo-proteases and provide new insights into the roles of this cation and important active site residues in ligand binding and in the hydrolysis of the peptide bond. Based on the data, we propose a two-step catalytic mechanism, in which the conformation of the active site is altered between the Michaelis complex and the transition state. In addition, the simulations identify global changes in the protein in which conformational transitions in the catalytic domain are transmitted at the opening of the N-terminal 8 angstrom-long channel and at the opening of the 30 angstrom-long C-terminal internal chamber that facilitates entry of peptides to the active site and exit of released amino acids. The possible implications of these global changes with regard to enzyme function are discussed.
George, A.M. & Jones, P.M. 2011, 'Type II ABC permeases: Are they really so different?', Structure, vol. 19, pp. 1540-1542.
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ATP binding-cassette (ABC) transporters couple ATP hydrolysis to vectorial movement of substrates across cell membranes. Of ancient origin and ubiquitous occurrence, they have been adapted to traffic a vast array of compounds, performing roles such as nutrient import and removal of toxins, antigen presentation, hormone release, signal reception, channel gating, and many others (Higgins, 1992; Jones and George. 2004). Their central roles in many physiological process has brought ABC transporters to the forefront in biomedical research in diverse areas including multidrug resistance in cancers and human genetic disorders, such as cystic fibrosis.
Kerr, I., Jones, P.M. & George, A.M. 2010, 'Multidrug efflux pumps: The structures of prokaryotic ATP-binding cassette transporter efflux pumps and implications for our understanding of eukaryotic P-glycoproteins and homologues.', FEBS Journal, vol. 277, no. 3, pp. 550-563.
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One of the Holy Grails of ATP-binding cassette transporter research is a structural understanding of drug binding and transport in a eukaryotic multidrug resistance pump. These transporters are front-line mediators of drug resistance in cancers and represent an important therapeutic target in future chemotherapy. Although there has been intensive biochemical research into the human multidrug pumps, their 3D structure at atomic resolution remains unknown. The recent determination of the structure of a mouse P-glycoprotein at subatomic resolution is complemented by structures for a number of prokaryotic homologues. These structures have provided advances into our knowledge of the ATP-binding cassette exporter structure and mechanism, and have provided the template data for a number of homology modelling studies designed to reconcile biochemical data on these clinically important proteins
George, A.M., Jones, P.M. & Middleton, P. 2009, 'Cystic fibrosis infections: treatment strategies and prospects', FEMS Microbiology Letters, vol. 300, no. 2, pp. 153-164.
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Pseudomonas aeruginosa and Burkholderia cepacia are the two major Gram-negative rods that colonize/infect the lungs of patients with cystic fibrosis (CF). These organisms may cause progressive respiratory failure, although occasionally more rapid infections result in the 'Cepacia' syndrome. Many antibiotics have been used against Pseudomonas and Burkholderia, but once chronic colonization has been established, eradication of these organisms is rare. Drug therapy for CF patients is compromised by a number of bacterial factors that render the infectious agents resistant to antibiotics, including efflux pumps that remove antibiotics, lack of penetration of antibiotics into bacterial biofilms, and changes in the cell envelope that reduce the permeability of antibiotics. Any combination of these mechanisms increases the likelihood of bacterial survival. Therefore, combinations of antibiotics or of antibiotic and nonantibiotic compounds are currently being tested against Pseudomonas and Burkholderia. However, progress has been slow, with only occasional combinations showing promise for the eradication of persistent Gram-negative rods in the airways of CF patients. This review will summarize the current knowledge of CF infections and speculate on potential future pathways to treat these chronic infections.
Jones, P.M. & George, A.M. 2009, 'Opening of the ADP-bound active site in the ABC transporter ATPase dimer: Evidence for a constant contact, alternating sites model for the catalytic cycle', Proteins-Structure Function And Bioinformatics, vol. 75, no. 2, pp. 387-396.
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ABC transporters are ubiquitous, ATP-dependent transmembrane pumps. The mechanism by which ATP hydrolysis in the nucleotide-binding domain (NBD) effects conformational changes in the transmembrane domain that lead to allocrite translocation remains largely unknown. A possible aspect of this mechanism was suggested by previous molecular dynamics simulations of the MJ0796 NBD dimer, which revealed a novel, nucleotide-dependent intrasubunit conformational change involving the relative rotation of the helical and catalytic subdomains. Here, we find that in four of five simulations of the ADP/ATP-bound dimer, the relative rotation of the helical and catalytic subdomains in the ADP-bound monomer results in opening of the ADP-bound active site, probably sufficient or close to sufficient to allow nucleotide exchange. We also observe that in all five simulations of the ADP/ATP-bound dimer, the intimate contact of the LSGGQ signature sequence with the ATP gamma-phosphate is weakened by the intrasubunit conformational change within the ADP-bound monomer. We discuss how these results support a constant contact model for the function of the NBD dimer in contrast to switch models, in which the NBDs are proposed to fully disassociate during the catalytic cycle.
Kerr, I., Jones, P.M. & George, A.M. 2009, 'Multidrug efflux pumps: The structures of prokaryotic ATP-binding cassette transporter efflux pumps and implications for our understanding of eukaryotic P-glycoproteins and homologues', FEBS Journal, vol. 277, pp. 550-563.
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One of the Holy Grails of ATP-binding cassette transporter research is a structural understanding of drug binding and transport in a eukaryotic multidrug resistance pump. These transporters are front-line mediators of drug resistance in cancers and represent an important therapeutic target in future chemotherapy. Although there has been intensive biochemical research into the human multidrug pumps, their 3D structure at atomic resolution remains unknown. The recent determination of the structure of a mouse P-glycoprotein at subatomic resolution is complemented by structures for a number of prokaryotic homologues. These structures have provided advances into our knowledge of the ATP-binding cassette exporter structure and mechanism, and have provided the template data for a number of homology modelling studies designed to reconcile biochemical data on these clinically important proteins.
Jones, P.M., O'Mara, M. & George, A.M. 2009, 'ABC transporters: A riddle wrapped in a mystery inside an enigma', Trends in Biochemical Sciences, vol. 34, no. 10, pp. 520-531.
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ATP-binding cassette (ABC) transporters form one of the largest and most ancient of protein families. ABC transporters couple hydrolysis of ATP to vectorial translocation of diverse substrates across cellular membranes. Many human ABC transporters are medically important in causing, for example, multidrug resistance to cytotoxic drugs. Seven complete prokaryotic structures and one eukaryotic structure have been solved for transporters from 2002 to date, and a wealth of research is being conducted on and around these structures to resolve the mechanistic conundrum of how these transporters couple ATP hydrolysis in cytosolic domains to substrate translocation through the transmembrane pore. Many questions remained unanswered about this mechanism, despite a plethora of data and a number of interesting and controversial models.
Treerat, P., Widmer, F., Middleton, P., Iredell, J. & George, A.M. 2008, 'In vitro interactions of tobramycin with various nonantibiotics against Pseudomonas aeruginosa and Burkholderia cenocepacia', FEMS Microbiology Letters, vol. 285, no. 1, pp. 40-50.
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Pseudomonas aeruginosa and Burkholderia cepacia are the major pathogens that colonize the airway surface and cause progressive respiratory failure and high mortality, especially in cystic fibrosis (CF) patients. Tobramycin is the treatment of choice, but persistent usage enables the infectious organisms to activate defence mechanisms, making eradication rarely successful. Combinations of antibiotic and nonantibiotic compounds have been tested in vitro against P. aeruginosa and B. cepacia, but with mixed results. Sodium ions interfere with the bacterial tobramycin uptake system, but amiloride partially reverses this antagonism. In this pilot study, we extend previous findings of the effectiveness of tobramycin in combination with amiloride and other nonantibiotics against a P. aeruginosa type strain, and against four P. aeruginosa strains and one Burkholderia cenocepacia strain isolated from CF patients. Significantly, the four clinical P. aeruginosa strains were tobramycin resistant. We also find that Na+ and K+, but not Cl-, are the chief antagonists of tobramycin efficacy. These results suggest that chemotherapy for CF patients might not only be compromised by antibiotic-resistant pathogens alone, but by a lack of penetration of antibiotics caused either by bacterial biofilms or the high sodium flux in the CF lung, or by antagonistic effects of some drug combinations, any of which could allow the persistence of drug-susceptible bacteria.
Jones, P.M. & George, A.M. 2007, 'Nucleotide-dependent allostery within the ABC transporter ATP-binding cassette-A computational study of the MJ0796 dimer', Journal of Biological Chemistry, vol. 282, no. 31, pp. 22793-22803.
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ATP-binding cassette transporters perform energy-dependent transmembrane solute trafficking in all organisms. These proteins often mediate cellular resistance to therapeutic drugs and are involved in a range of human genetic diseases. Enzymological studies have implicated a helical subdomain within the ATP-binding cassette nucleotide-binding domain in coupling ATP hydrolysis to solute transport in the transmembrane domains. Consistent with this, structural and computational analyses have indicated that the helical subdomain undergoes nucleotide-dependent movement relative to the core of the nucleotide-binding domain fold. Here we use theoretical methods to examine the allosteric nucleotide dependence of helical subdomain transitions to further elucidate its role in interactions between the transmembrane and nucleotide-binding domains. Unrestrained 30-ns molecular dynamics simulations of the ATP-bound, ADP-bound, and apo states of the MJ0796 monomer support the idea that interaction of a conserved glutamine residue with the catalytic metal mediates the rotation of the helical subdomain in response to nucleotide binding and hydrolysis. Simulations of the nucleotide-binding domain dimer revealed that ATP hydrolysis induces a large transition of one helical subdomain, resulting in an asymmetric conformation of the dimer not observed previously. A coarse-grained elastic network analysis supports this finding, revealing the existence of corresponding dynamic modes intrinsic to the contact topology of the protein. The implications of these findings for the coupling of ATP hydrolysis to conformational changes in the transmembrane domains required for solute transport are discussed in light of recent whole transporter structures.
Jones, P.M., Turner, K.M., Djordjevic, J.T., Sorrell, T.C., Wright, L.C. & George, A.M. 2007, 'Role of conserved active site residues in catalysis by phospholiphase B1 from Cryptococcus neoformans', Biochemistry, vol. 46, no. 35, pp. 10024-10032.
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Phospholiphase B1 (PLB1), secreted by the pathogenic yeast Crysptococcus neoformans, has an established role in virulence. Although the mechanism of it phospholiphase B, lysophospholipase, and lysophospholipase transacylase activities is unknown, it possesses lipase, subtilisin protease aspartate, and phospholipase motifs containing putative catalytic residues S146, D392 and R108 respectively, conserved in fungal PLBs and essential for human cytosolic phopholiphase A2 (cPLA2) catalysis. To determine the role of these residues in PLB1 catalysis, each was substituted with alanine, and the mutant cDNAs were expressed in Sacchromyces cerevisiae. The mutant PLB1s were deficient in all threee enzymatic activities. As the active site structure of PLB1 is unknown, a homilogy model was developed, based o the X-ray structure of the cPLA2 catalytic domain. This shows that the two proteins share a closely related fold, with the three catalytic residues located in identical positions as part of a single active site with S146 and D392 forming a catalytic dyad. The model suggests that PLB1 lacks the "lid" region which ocucludes the cPLA2 active site and provides a mechanism of interfacial activation. In silico substrate docking studies with cPLA2 reveal the binding mode of the lipid headgroup, confirming the catalytic dyad mechanism ofor the cleavage of the sn-2 ester bond within one of two separate binding tracts for the lipid acyl chains. Residues specific for binding arachidonic and palmitic acids, preferred substrated for cPLA2 and PLB1, respectively. are identified. These results provide an explanation for difference in substrate specificity between lipases sharing the cPLA2 catalytic domain and for the differential effect of inhibitors on PLB1 enzymatic activities.
George, A.M. & Jones, P.M. 2006, 'Molecular dynamics simulations and analysis of ABC transporters', Current Computer-Aided Drug Design, vol. 2, no. 3, pp. 203-214.
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The increasingavailability of atomic-level protein derived from X-ray crystallography and NMR spectroscopy, together with advances in computational power, have ushered in a new era of powerful theoretical apporaches to study protein mechanism,s and, by extension, use a computer-aided structural approach to drug design. Calssical molecular dynamics calculations, in which Newton's equations of motion are solved for all atoms in the system, has emerged as an important tool for analysing protein dynamics at physiologically relevant timescales, in ways that are either very difficult or impossible to do experimentally. Indeed, the computer is becoming a kind of virtual microscope that can observe things not observable by any other means. The availability of more sophisticated parallel computer clusters and program suites will lead to simulations thatw ill be capable of examining entire processes such as polypeptide folding pathways and reaction mechanisms. In this review, the incipient applicationof molecular dynamics analysis of ABC (ATP-Binding Cassette) trasporters is surveyed and discussed, with particular relevance to unresolved and controversial issues.
Jones, P.M. & George, A.M. 2005, 'Multidrug resistance in parasites: ABC transporters, P-glycoproteins and molecular modelling', International Journal For Parasitology, vol. 35, no. 5, pp. 555-566.
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Parasitic diseases, caused by protozoa, helminths and arthropods, rank among the most important problems in human and veterinary medicine, and in agriculture, leading to debilitating sicknesses and loss of life. In the absence of vaccines and with the ge
Jones, P.M. & George, A.M. 2004, 'The ABC transporter structure and mechanism: perspectives on recent research', Cellular and Molecular Life Sciences, vol. 61, pp. 682-699.
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ATP-binding cassetee (ABC) transporters are multidomain integral membrane proteins that utilise the energy of ATP hydrolysis to translocate solutes across cellular membranes in all phyla. ABC transporters form one of the largest of all protein families and are central to many important biomedical phenomena, including resistance of cancers and pathogenic mocribes to drugs. Elucidation of the sturcture and mechanism of ABC transporters is essential to the rational design of agents to control their fuctiom. While a wealth of high-resolution structures of ABC proteins have been produced in recent years, many fundamental questions regarding the protein's mechanism remain unasnwered. I this review, we examine the recent structural data concerning ABC transporters and related proteins in te ligh of other experimental and theoretical data, and discuss these data in relation to current ideas concerning the transporters' molecular mechanism.
George, A.M. & Hall, R.M. 2002, 'Efflux of chloramphenicol by the Cm1A1 protein', FEMS Microbiology Letter, vol. 209, no. N/A, pp. 209-213.
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Jones, P.M. & George, A.M. 2002, 'Mechanism of ABC transporters: a molecular dynamics simulation of a well characterised nucleotide-binding subunit', Proceedings of the National Academy of Sciences, vol. 99, no. 20, pp. 12639-12644.
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ATP-binding cassette (ABC) transporters are membrane-bound molecular pumps that form one of the largest of all protein families. Several of them are central to phenomena of biomedical interest, including cystic fibrosis and resistance to chemotherapeutic drugs. ABC transporters share a common architecture comprising two hydrophilic nucleotide-binding domains (NBDs) and two hydrophobic transmembrane domains (TMDs) that form the substrate pathway across the membrane. The conformational changes in the NBDs induced by ATP hydrolysis and the means by which they are transmitted to the TMDs to effect substrate translocation remain largely unknown. We have performed a molecular dynamics simulation of HisP, the well studied NBD of the bacterial histidine permease, to identify hinges and switches of the NBD conformational transitions and subunitsubunit interfaces.
George, A.M. & Hall, R.M. 2002, 'Efflux of chloramphenicol by the CmlA1 protein.', FEMS microbiology letters, vol. 209, no. 2, pp. 209-213.
The cmlA1 gene cassette contains the cmlA1 gene, that confers resistance to chloramphenicol, as well as a promoter and translational attenuation signals, and expression of cmlA1 is inducible by low concentrations of chloramphenicol. The CmlA1 protein encoded by cmlA1 was localised in the inner membrane. Active efflux of chloramphenicol, additional to the endogenous efflux from Escherichia coli cells, was observed when the cmlA1 gene was present and the production of CmlA1 had been preinduced with subinhibitory concentrations of chloramphenicol. Both endogenous and CmlA1-mediated export of chloramphenicol was driven by the proton-motive force.
Jones, P.M. & George, A.M. 2000, 'Symmetry and Sturcture in P-Glycoprotein and ABC Transporters. What goes Around Comes Around', European Journal fo Biochemistry, vol. 267, no. 0, pp. 5298-5305.
Jones, P.M. & George, A.M. 2000, 'Symmetry and structure in P-glycoprotein and ABC transporters what goes around comes around.', European journal of biochemistry / FEBS, vol. 267, no. 17, pp. 5298-5305.
The ABC superfamily of membrane transporters is one of the largest classes of proteins across all species and one of the most intensely researched. ABC proteins are involved in the trafficking of a diverse variety of biological molecules across cell membranes, with some members implicated in medical syndromes such as cystic fibrosis and multidrug resistance to anti-cancer drugs. In the absence of X-ray crystallographic data, structural information has come from spectroscopy, electron microscopy, secondary structure prediction algorithms and residue substitution, epitope labelling and cysteine cross-linking studies. These have generally supported a model for the topology of the transmembrane domains of ABC transporters in which a single aqueous pore is formed by a toroidal ring of 12 alpha helices, deployed in two arcs of six helices each. Although this so-called 6 + 6 helix model can be arranged in either mirror or rotational symmetry configurations, experimental data supports the former. In this review, we put forward arguments against both configurations of this 6 + 6 helix model, based on what is known generally about symmetry relationships in proteins. We relate these arguments to P-glycoprotein, in particular, and discuss alternative models for the structure of ABC transporters in the light of the most recent research.
Oakey, H.J., Gibson, L.F. & George, A.M. 1999, 'DNA probes specific for Aeromonas hydrophila (HG1).', Journal of applied microbiology, vol. 86, no. 2, pp. 187-193.
Aeromonas hydrophila (HG1)-specific RAPD-PCR fragments were investigated for their potential as DNA probes. From 20 RAPD-PCR fragment bands, it was found that two were specific to all isolates of Aeromonas hydrophila (HG1) tested. Cloning and nucleotide sequence determination of one of these bands showed that co-migration of similar sized amplicons had occurred and that this band (designated '7e') contained at least four fragments of different sequences. Three of these individual amplicons had a sequence specific to Aer. hydrophila (HG1) isolates. The sequence of one of these amplicons ('7e5') was used to design primers for a specific polymerase chain reaction (PCR). The specificity of the PCR was achieved using a modified hot-start procedure. The identity of the PCR amplicons was confirmed by high stringency hybridization with a digoxygenin-labelled 7e5 probe.
Oakey, J., Gibson, L. & George, A.M. 1998, 'RAPD-PCR derived specific probes for Aeromonas hydrophila', Journal Of Applied Microbiology, vol. 84, no. 1, pp. 187-193.
George, A.M. & Gibson, L. 1998, 'Melanin and novel melanin precursors from Aeromonas media', FEMS Microbiology Letters, vol. 169, pp. 261-268.
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Gibson, L., Whitworth, J. & George, A.M. 1998, 'Probiotic activity of Aeromonas media on the Pacific oyster, Crassostrea gigas, challenged with V. tubiashii', Aquaculture, pp. 111-120.
Oakey, J., Gibson, L. & George, A.M. 1998, 'Co-migration of RAPD-PCR amplicons from Aeromonas hydrophilia', FEMS Microbiology Letters, vol. 164, pp. 35-38.
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Jones, P.M. & George, A.M. 1998, 'A new structural model for P-glycoprotein', Journal Of Membrane Biology, vol. 166, no. 2, pp. 133-147.
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Multidrug resistance to anti-cancer drugs is a major medical problem. Resistance is manifested largely by the product of the human MDR1 gene, P-glycoprotein, an ABC transporter that is an integral membrane protein of 1280 amino acids arranged into two homologous halves, each comprising 6 putative transmembrane ?-helices and an ATP binding domain. Despite the plethora of data from site-directed, scanning and domain replacement mutagenesis, epitope mapping and photoaffinity labeling, a clear structural model for P-glycoprotein remains largely elusive. In this report, we propose a new model for P-glycoprotein that is supported by the vast body of previous data. The model comprises 2 membrane-embedded 16-strand ?-barrels, attached by short loops to two 6-helix bundles beneath each barrel. Each ATP binding domain contributes 2 ?-strands and 1 ?-helix to the structure. This model, together with an analysis of the amino acid sequence alignment of P-glycoprotein isoforms, is used to delineate drug binding and translocation sites. We show that the locations of these sites are consistent with mutational, kinetic and labeling data.
Gibson, L.F. & George, A.M. 1998, 'Melanin and novel melanin precursors from Aeromonas media', FEMS Microbiology Letters, vol. 169, no. 2, pp. 261-268.
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Many bacteria produce reddish brown to black pigments and some of these have been characterised. This report describes the isolation and characterisation of a diffusible brown melanin-like pigment from the bacterium Aeromonas media. Physico-chemical testing suggested that the pigment is a true melanin. New butanol-soluble yellow, red and brown pigments were isolated from the A. media strain under reducing conditions during melanogenesis and these pigments were shown to be unstable precursors of the polymeric brown melanin product. Copyright (C) 1998 Federation of European Microbiological Societies.
Oakey, H.J., Gibson, L.F. & George, A.M. 1998, 'Co-migration of RAPD-PCR amplicons from Aeromonas hydrophila', FEMS Microbiology Letters, vol. 164, no. 1, pp. 35-38.
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Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) uses arbitrary primers and low stringency annealing conditions to amplify anonymous DNA fragments which are then depicted in agarose gels. RAPD-PCR fingerprints have been used for typing and differentiation of bacteria and, increasingly, for the study of genetic relationships between strains and species of microorganisms, plants and animals. The analysis of such fingerprints is based upon the assumption that comigration of amplicons does not occur and that any given band contains a single amplicon. This report shows that comigration of fragments of nearly identical size, but different nucleotide sequences, occurs between different isolates and within single RAPD-PCR bands from Aeromonas hydrophila. The possibility of the same phenomenon occurring for other prokaryotic or eukaryotic genomes argues for caution in the interpretation of RAPD-PCR fingerprints.
George, A.M., Davey, M. & Mir, A. 1996, 'Functional expression of the human MDR1 gene in Escherichia coli', Archives Of Biochemistry And Biophysics, vol. 333, pp. 66-74.
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George, A.M. 1996, 'Multidrug resistance in enteric and other Gram-negative bacteria', FEMS Microbiology Letters, vol. 139, pp. 1-10.
George, A.M., Hall, R.M. & Stokes, H. 1995, 'Multidrug resistance in Klebsiella pneumoniae: a novel gene, ramA, confers a multidrug resistance phenotype in Escherichia coli', Microbiology-uk, vol. 141, pp. 1909-1920.
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Spontaneous multidrug-resistant (Mdr) mutants of Klebsiella pneumoniae strain ECL8 arose at a frequency of 2-2 x 10-8 and showed increased resistance to a range of unrelated antibiotics, including chloramphenicol, tetracycline, nalidixic acid, ampicillin, norfloxacin, trimethoprim and puromycin. A chromosomal fragment from one such mutant was cloned, and found to confer an Mdr phenotype on Escherichia coli K12 cells that was essentially identical to that of the K. pneumoniae mutant. Almost complete loss of the OmpF porin in the E. coli transformant, and of the corresponding porin in the K. pneumoniae mutant, was observed. The presence of the Mdr mutation in K. pneumoniae or the cloned K. pneumoniae ramA (resistance antibiotic multiple) locus in E. coli also resulted in active efflux of tetracycline, and increased active efflux of chloramphenicol. After transformation of a ramA plasmid into E. coli, expression of chloramphenicol resistance occurred later than expression of resistance to tetracycline, puromycin, trimethoprim and nalidixic acid. The ramA gene was localized and sequenced. It encodes a putative positive transcriptional activator that is weakly related to the E. coli MarA and SoxS proteins. A ramA gene was also found to be present in an Enterobacter cloacae fragment that has previously been shown to confer an Mdr phenotype, and it appears that ramA, rather than the romA gene identified in that study, is responsible for multidrug resistance. The ramA gene from the wild-type K. pneumoniae was identical to that of the mutant strain and also conferred an Mdr phenotype on E. coli, indicating that the mutation responsible for Mdr in K. pneumoniae had not been cloned.
McMurry, L.M., George, A.M. & Levy, S.B. 1994, 'Active efflux of chloramphenicol in susceptible Escherichia coli strains and in multiple-antibiotic-resistant (Mar) mutants.', Antimicrobial agents and chemotherapy, vol. 38, no. 3, pp. 542-546.
The multiple-antibiotic resistance (mar) locus (min 34) regulates a resistance to chloramphenicol in Escherichia coli that does not involve acetyltransferase. Transport studies showed that wild-type cells had an apparent endogenous active efflux of chloramphenicol which depended on the proton motive force. This efflux was not altered by a 39-kb chromosomal deletion which included the mar locus. Nevertheless, mutations at the mar locus led to a stronger net chloramphenicol efflux. Therefore, a gene encoding the putative efflux system cannot be at the mar locus but may be positively influenced by that locus.
George, A.M. & Levy, S.B. 1983, 'Amplifiable resistance to tetracycline, chloramphenicol, and other antibiotics in Escherichia coli: involvement of a non-plasmid-determined efflux of tetracycline.', Journal of bacteriology, vol. 155, no. 2, pp. 531-540.
Increasing levels of resistance to tetracycline and to a number of other unrelated antibiotics, including chloramphenicol, beta-lactams, puromycin, and nalidixic acid, occurred in Escherichia coli after 50 to 200 generations of growth in the presence of subinhibitory concentrations of tetracycline or chloramphenicol. In the absence of selective pressure, resistances fell to low levels within 100 generations of growth. This amplification of resistance was observed in laboratory and naturally occurring E. coli strains as well as in polA and recA strains. With the exception of previously identified cmlA and cmlB mutations, tetracycline or chloramphenicol resistances were not P1 transducible. Coincident with the emergence of resistance was the appearance of a previously cryptic energy-dependent efflux system for tetracycline. The expression of resistance phenotypes and the tetracycline efflux system were temperature sensitive at 42 degrees C.
George, A.M. & Levy, S.B. 1983, 'Gene in the major cotransduction gap of the Escherichia coli K-12 linkage map required for the expression of chromosomal resistance to tetracycline and other antibiotics.', Journal of bacteriology, vol. 155, no. 2, pp. 541-548.
In Escherichia coli K-12, amplifiable resistance to tetracycline, chloramphenicol, and other unrelated antibiotics was mediated by at least four spatially separated loci. Tetracycline-sensitive mutants were isolated by Tn5 insertional inactivation of an amplified multiply resistant strain. One of these, studied in detail, showed coordinate loss of expression of all other resistance phenotypes. The Tn5 element in this mutant mapped to 34 min on the E. coli K-12 linkage map. We have designated the locus marA (multiple antibiotic resistance). Tetracycline-sensitive mutants containing marA::Tn5 regained all resistance phenotypes at frequencies of 10(-8) to 10(-7) upon precise excision of Tn5. Moreover, a newly described tetracycline efflux system (A. M. George and S. B. Levy, J. Bacteriol. 155:531-540, 1983) was inactivated in tetracycline-sensitive mutants, but recovered in tetracycline-resistant revertants. In merodiploids, F-prime marA+ expressed partial or complete dominance over corresponding mutant chromosomal alleles. Dominance tests also established that a previously amplified host and a mutant marA allele were preconditions for the expression of phenotypic resistances.
Obbink, D.J., George, A.M. & Coombe, R.G. 1983, 'Aminoglycoside-modifying enzymes associated with hospital isolates of Gram-negative rods.', The Journal of antimicrobial chemotherapy, vol. 11, no. 6, pp. 525-533.
Multi-resistant Enterobacteriaceae and Pseudomonas isolated from clinical specimens at RNS were characterized for resistance phenotype and transferred resistant phenotype. These isolates were obtained over a two year period from different specimens and from patients with varying clinical syndromes. Twenty-six of these isolates were further characterized with respect to their aminoglycoside modifying enzymes. Only three enzymes were detected: AAC(3)-I, APH(3')-I and AAD(2")-II. The substrate range of these enzymes was investigated and not always found to coincide with the transferred phenotype. AAD(2")-II was found only in association with APH(3')-I and not alone. The substrate range of AAC(3)-I from Pseudomonas was different from AAC(3)-I from Enterobacteriaceae but not sufficiently dissimilar to classify them as separate isoenzymes. The relatively few aminoglycoside-inactivating enzymes suggest that within this hospital there may be a limited gene pool for these enzymes.
Coombe, R.G. & George, A.M. 1982, 'Purification and properties of an aminoglycoside acetyltransferase from Pseudomonas aeruginosa.', Biochemistry, vol. 21, no. 5, pp. 871-875.
An aminoglycoside 3-acetyltransferase [AAC(3)], possibly a new isoenzymic species of the 3-N-acetyltransferase group, was purified to apparent homogeneity from a crude extract of Pseudomonas aeruginosa, a gentamicin-resistant clinical isolate. The method of purification was consecutive column chromatography--(i) gel filtration, (ii) affinity chromatography, and (iii) ion-exchange chromatography--to give two protein peaks, one of which was coincident with activity and which indicated a purification of 600 (specific activity = 9.743 units mg-1 at pH 7.2, 34 degrees C). Polyacrylamide disc gel electrophoresis indicated a single protein band coincident with enzymic activity. The molecular weight of the enzyme was about 39 000. AAC(3)-V (provisonal designation) was further characterized by stability, substrate, pH, and kinetic studies. The Km was 0.724 microM (sisomicin), and the Vmax was 0.102 mumol min-1 mg-1 (sisomicin) at pH 7.2 and 34 degrees C. Substrate inhibition was exhibited by kanamycin A and tobramycin. Studies showed that enzyme activity was significantly stabilized when preparations contained substrate.
Coombe, R.G. & George, A.M. 1981, 'New plasmid-mediated aminoglycoside adenylyltransferase of broad substrate range that adenylylates amikacin.', Antimicrobial agents and chemotherapy, vol. 20, no. 1, pp. 75-80.
The same aminoglycoside 2"-adenylyltransferase was isolated from four gram-negative species which were among a random group of gentamicin-resistant isolates from the same hospital. The enzyme was partially purified from a crude extract which also contained a second modifying enzyme identified as APH(3')-I. The substrate range of the new aminoglycoside 2"-adenylyltransferase included the newer aminoglycosides sisomicin and amikacin, but showed much-reduced activity against gentamicins C2 and Cla. The pH optimum was 7.8 to 8.0 for every substrate, and the molecular weight of the enzyme molecule was estimated at approximately 29,000. Genetic experiments clearly established that both enzymes were expressed by a conjugative plasmid.
Coombe, R.G. & George, A.M. 1981, 'Enzymic determination of gentamicin in human plasma', Australian Journal of Pharmaceutical Sciences, vol. 10, no. 4, pp. 98-100.
Coombe, R.G. & George, A.M. 1981, 'Mass spectral and 13c n.m.r. analyses of aminoglycoside aminocyclitols modified by enzyme-catalysed reactions', Australian Journal of Chemistry, vol. 34, no. 3, pp. 547-554.
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Chemical and physical analytical methods, including mass spectrometry and 13C nnr., have been applied to the evaluation of the structures of the inactivated products of enzyme-mediated trans-ferase reactions. Evidence is presented for the assignment of structures for the products of three enzymic reaction mechanisms: O-adenylylation, O-phosphorylation and N-acetylation. © 1981, CSIRO. All rights reserved.
Coombe, R.G. & George, A.M. 1976, 'A rapid method for the purification of beta-lactamase from Bacillus cereus by affinity chromatography.', Analytical biochemistry, vol. 75, no. 2, pp. 652-655.
Coombe, R.G. & George, A.M. 1976, 'An alternative coupling procedure for preparing activated sepharose for affinity chromatography of penicillinase.', Australian journal of biological sciences, vol. 29, no. 4, pp. 305-316.
Most applications of affinity chromatography employ the cyanogen bromide activation scheme first devised by Axèn et al. (1967). Porath and Sunberg (1972) reported an alternative procedure in which phloroglucinol and divinylsulphone are used in activating reactions. The advantages of this scheme and parameters relevant to the activating reactions are reported here. Conditions for the attachment of various ligand molecules to sepharose using a divinylsulphone activation method are defined, and a comparison with cyanogen bromide activating and coupling techniques is drawn. alpha-Chymotrypsin is immobilized by covalent attachment to activated sepharose. The optimum coupling pH is 8-0-8-6 and the reaction is virtually complete after 20 h at room temperature. Conjugates containing as much as 2 g of enzyme per gram dry weight of polymer were obtained. The immobilized enzyme retained 41% of the free enzymic activity. An affinity column of divinylsulphone-activated methicillin-sepharose was used to demonstrate the reversible adsorption of penicillinase.