Portrait of Dr Christopher Mulligan

Dr Christopher Mulligan

Lecturer in Biochemistry
Plagiarism Officer


Dr. Christopher Mulligan joined the School of Biosciences in May 2017. Chris obtained a degree in Biochemistry from the University of York (2004). Chris performed his thesis research with Dr. Gavin Thomas at the University of York (2004-2008) and subsequently took up a postdoctoral position in the same lab (2008-2011). Here, Chris studied the structure and function of transporters involved in the uptake of virulence factors into human pathogens. Following his time in York, Chris obtained a visiting fellow position in Dr. Joseph Mindell’s lab in the Membrane Transport Biophysics Section of the National Institute of Neurological Disorders and Stroke (NINDS) at the National Institutes of Health (NIH) in Maryland, USA. During his time at NIH (2011-2017), Chris studied the structure and mechanism of transport protein families involved in a number of key cellular functions, including neurotransmission and metabolic regulation.

Orchid: orcid.org/0000-0001-5157-4651

Research interests

Transport proteins play a major role in crucial cellular processes in all forms of life; from the uptake of nutrients and virulence factors to the extrusion of toxins (including antibiotics) in bacteria, as well as the regulation of neurotransmission and metabolism in mammals.
In humans, improper regulation and mutations in transporters are often associated with disease states like ALS, Alzheimer’s disease and epilepsy. Whereas, in bacteria, transporters contribute substantially to the virulence of human pathogens and their resistance to antibiotics, making them prime targets for new therapeutics. In addition, transporters are also key players in industrial biotechnology. Bacterial cell factories that are used to produce high value chemicals for industrial and medical purposes rely heavily on transport proteins to take up precursors and extrude the final products.
Transport proteins are highly dynamic molecular machines, most of which actively pump their substrates across the membrane fueled by an energy source; usually either ATP hydrolysis or electrochemical gradients (H+ and Na+ gradients). My lab uses a combination of biochemical, biophysical and microbiological techniques to understand the molecular mechanisms of transport proteins; how they recognise compounds, how they harness an energy source to pump compounds across the membrane, and how they move during transport. The more we understand about the mechanism of transporters, the better placed we will be to manipulate their function; either inhibiting them if they have undesirable effects, for example, antimicrobial resistance; or harnessing and enhancing their capabilities if they are useful, for example, for applications in industrial biotechnology.


  • BI300 – Introduction to Biochemistry
  • BI604 – Biological Membranes (Module convenor) 


MSc-R projects available for 2020/21

Probing the mechanism of INDY (I’m not dead yet) transporters: a target for the treatment o cancer, diabetes and obesity
In eukaryotic cells, disrupting the activity of INDY transporters can extend lifespan, reduce cancer cell proliferation, and protect against metabolic disease such as diabetes and obesity. To develop inhibitors for INDY proteins, we first need to understand their transport mechanism. In this project, we will investigate the mechanism of the bacterial representative of this family, VcINDY. We will probe substrate and inhibitor interactions, and proteins dynamics using biochemical and biophysical approaches. The student will receive training in molecular biology techniques, such as site-directed mutagenesis, integral membrane protein expression and purification, transporter characterisation, protein biochemistry and biophysical techniques. 

Additional research costs: £1500 

The path to least resistance: probing the mechanism of integral membrane transport proteins essential for antimicrobial resistance in bacteria. Antimicrobial resistance is a major global health concern. One of the most effective mechanisms bacteria have developed to resist the effects of antimicrobial agents is to use drug efflux transporters to pump them out of the cell before they can do any damage. Understanding the structure and function of these proteins will lay the foundation for the development of future inhibitors, which could be used to enhance the efficacy of current antimicrobials and breathe new life into antimicrobials rendered ineffective due to the development of resistance.
In this project, we will elucidate the functional mechanism of a family of integral membrane transport proteins that strongly influence the antibiotic resistance of several bacterial pathogens. This project will take an integrated approach to probing the structure and function of these membrane proteins, which will include protein biochemistry, biophysical approaches and microbial phenotypic assays.

Additional Research costs: £1500  
Please get in touch with Dr Mulligan if you would like more details about the projects. 


  • Member of the Microbiology Society. 
  • Member of the Biochemical Society. 
  • Member of the British Biophysical Society. 
  • Member of Crossing Biological Membranes Network (CBMNet), a BBSRC Network in Industrial Biotechnology and Bioenergy.



  • Fitzgerald, G., Mulligan, C. and Mindell, J. (2017). A general method for determining secondary active transporter substrate stoichiometry. eLife [Online] 6:e21016. Available at: http://dx.doi.org/10.7554/eLife.21016.
    The number of ions required to drive substrate transport through a secondary active transporter determines the protein's ability to create a substrate gradient, a feature essential to its physiological function, and places fundamental constraints on the transporter's mechanism. Stoichiometry is known for a wide array of mammalian transporters, but, due to a lack of readily available tools, not for most of the prokaryotic transporters for which high-resolution structures are available. Here, we describe a general method for using radiolabeled substrate flux assays to determine coupling stoichiometries of electrogenic secondary active transporters reconstituted in proteoliposomes by measuring transporter equilibrium potentials. We demonstrate the utility of this method by determining the coupling stoichiometry of VcINDY, a bacterial Na(+)-coupled succinate transporter, and further validate it by confirming the coupling stoichiometry of vSGLT, a bacterial sugar transporter. This robust thermodynamic method should be especially useful in probing the mechanisms of transporters with available structures.
  • Mulligan, C., Fenollar-Ferrer, C., Fitzgerald, G., Vergara-Jaque, A., Kaufmann, D., Li, Y., Forrest, L. and Mindell, J. (2016). The bacterial dicarboxylate transporter VcINDY uses a two-domain elevator-type mechanism. Nature structural & molecular biology [Online] 23:256-263. Available at: http://dx.doi.org/10.1038/nsmb.3166.
    Secondary transporters use alternating-access mechanisms to couple uphill substrate movement to downhill ion flux. Most known transporters use a 'rocking bundle' motion, wherein the protein moves around an immobile substrate-binding site. However, the glutamate-transporter homolog GltPh translocates its substrate-binding site vertically across the membrane, through an 'elevator' mechanism. Here, we used the 'repeat swap' approach to computationally predict the outward-facing state of the Na(+)/succinate transporter VcINDY, from Vibrio cholerae. Our model predicts a substantial elevator-like movement of VcINDY's substrate-binding site, with a vertical translation of ~15 Å and a rotation of ~43°. Our observation that multiple disulfide cross-links completely inhibit transport provides experimental confirmation of the model and demonstrates that such movement is essential. In contrast, cross-links across the VcINDY dimer interface preserve transport, thus revealing an absence of large-scale coupling between protomers.
  • Vergara-Jaque, A., Fenollar-Ferrer, C., Mulligan, C., Mindell, J. and Forrest, L. (2015). Family resemblances: A common fold for some dimeric ion-coupled secondary transporters. The Journal of General Physiology [Online] 146:423-434. Available at: http://dx.doi.org/10.1085/jgp.201511481.
    Membrane transporter proteins catalyze the passage of a broad range of solutes across cell membranes, allowing the uptake and efflux of crucial compounds. Because of the difficulty of expressing, purifying, and crystallizing integral membrane proteins, relatively few transporter structures have been elucidated to date. Although every membrane transporter has unique characteristics, structural and mechanistic similarities between evolutionarily diverse transporters have been identified. Here, we compare two recently reported structures of membrane proteins that act as antimicrobial efflux pumps, namely MtrF from Neisseria gonorrhoeae and YdaH from Alcanivorax borkumensis, both with each other and with the previously published structure of a sodium-dependent dicarboxylate transporter from Vibrio cholerae, VcINDY. MtrF and YdaH belong to the p-aminobenzoyl-glutamate transporter (AbgT) family and have been reported as having architectures distinct from those of all other families of transporters. However, our comparative analysis reveals a similar structural arrangement in all three proteins, with highly conserved secondary structure elements. Despite their differences in biological function, the overall "design principle" of MtrF and YdaH appears to be almost identical to that of VcINDY, with a dimeric quaternary structure, helical hairpins, and clear boundaries between the transport and scaffold domains. This observation demonstrates once more that the same secondary transporter architecture can be exploited for multiple distinct transport modes, including cotransport and antiport. Based on our comparisons, we detected conserved motifs in the substrate-binding region and predict specific residues likely to be involved in cation or substrate binding. These findings should prove useful for the future characterization of the transport mechanisms of these families of secondary active transporters.
  • Mulligan, C., Fitzgerald, G., Wang, D. and Mindell, J. (2014). Functional characterization of a Na+-dependent dicarboxylate transporter from Vibrio cholerae. The Journal of General Physiology [Online] 143:745-759. Available at: http://dx.doi.org/10.1085/jgp.201311141.
    The SLC13 transporter family, whose members play key physiological roles in the regulation of fatty acid synthesis, adiposity, insulin resistance, and other processes, catalyzes the transport of Krebs cycle intermediates and sulfate across the plasma membrane of mammalian cells. SLC13 transporters are part of the divalent anion:Na(+) symporter (DASS) family that includes several well-characterized bacterial members. Despite sharing significant sequence similarity, the functional characteristics of DASS family members differ with regard to their substrate and coupling ion dependence. The publication of a high resolution structure of dimer VcINDY, a bacterial DASS family member, provides crucial structural insight into this transporter family. However, marrying this structural insight to the current functional understanding of this family also demands a comprehensive analysis of the transporter's functional properties. To this end, we purified VcINDY, reconstituted it into liposomes, and determined its basic functional characteristics. Our data demonstrate that VcINDY is a high affinity, Na(+)-dependent transporter with a preference for C4- and C5-dicarboxylates. Transport of the model substrate, succinate, is highly pH dependent, consistent with VcINDY strongly preferring the substrate's dianionic form. VcINDY transport is electrogenic with succinate coupled to the transport of three or more Na(+) ions. In contrast to succinate, citrate, bound in the VcINDY crystal structure (in an inward-facing conformation), seems to interact only weakly with the transporter in vitro. These transport properties together provide a functional framework for future experimental and computational examinations of the VcINDY transport mechanism.
  • Mulligan, C. and Mindell, J. (2013). Mechanism of transport modulation by an extracellular loop in an archaeal excitatory amino acid transporter (EAAT) homolog. Journal of Biological Chemistry [Online] 288:35266-35276. Available at: http://dx.doi.org/10.1074/jbc.M113.508408.
    Secondary transporters in the excitatory amino acid transporter family terminate glutamatergic synaptic transmission by catalyzing Na(+)-dependent removal of glutamate from the synaptic cleft. Recent structural studies of the aspartate-specific archaeal homolog, Glt(Ph), suggest that transport is achieved by a rigid body, piston-like movement of the transport domain, which houses the substrate-binding site, between the extracellular and cytoplasmic sides of the membrane. This transport domain is connected to an immobile scaffold by three loops, one of which, the 3-4 loop (3L4), undergoes substrate-sensitive conformational change. Proteolytic cleavage of the 3L4 was found to abolish transport activity indicating an essential function for this loop in the transport mechanism. Here, we demonstrate that despite the presence of fully cleaved 3L4, Glt(Ph) is still able to sample conformations relevant for transport. Optimized reconstitution conditions reveal that fully cleaved Glt(Ph) retains some transport activity. Analysis of the kinetics and temperature dependence of transport accompanied by direct measurements of substrate binding reveal that this decreased transport activity is not due to alteration of the substrate binding characteristics but is caused by the significantly reduced turnover rate. By measuring solute counterflow activity and cross-link formation rates, we demonstrate that cleaving 3L4 severely and specifically compromises one or more steps contributing to the movement of the substrate-loaded transport domain between the outward- and inward-facing conformational states, sparing the equivalent step(s) during the movement of the empty transport domain. These results reveal a hitherto unknown role for the 3L4 in modulating an essential step in the transport process.
  • Mulligan, C., Leech, A., Kelly, D. and Thomas, G. (2012). The membrane proteins SiaQ and SiaM form an essential stoichiometric complex in the sialic acid tripartite ATP-independent periplasmic (TRAP) transporter SiaPQM (VC1777-1779) from Vibrio cholerae. Journal of Biological Chemistry [Online] 287:3598-3608. Available at: http://dx.doi.org/10.1074/jbc.M111.281030.
    Tripartite ATP-independent periplasmic (TRAP) transporters are widespread in bacteria but poorly characterized. They contain three subunits, a small membrane protein, a large membrane protein, and a substrate-binding protein (SBP). Although the function of the SBP is well established, the membrane components have only been studied in detail for the sialic acid TRAP transporter SiaPQM from Haemophilus influenzae, where the membrane proteins are genetically fused. Herein, we report the first in vitro characterization of a truly tripartite TRAP transporter, the SiaPQM system (VC1777-1779) from the human pathogen Vibrio cholerae. The active reconstituted transporter catalyzes unidirectional Na(+)-dependent sialic acid uptake having similar biochemical features to the orthologous system in H. influenzae. However, using this tripartite transporter, we demonstrate the tight association of the small, SiaQ, and large, SiaM, membrane proteins that form a 1:1 complex. Using reconstituted proteoliposomes containing particular combinations of the three subunits, we demonstrate biochemically that all three subunits are likely to be essential to form a functional TRAP transporter.
  • Mulligan, C., Fischer, M. and Thomas, G. (2010). Tripartite ATP-independent periplasmic (TRAP) transporters in bacteria and archaea. FEMS microbiology reviews [Online] 35:68-86. Available at: http://dx.doi.org/10.1111/j.1574-6976.2010.00236.x.
    The tripartite ATP-independent periplasmic (TRAP) transporters are the best-studied family of substrate-binding protein (SBP)-dependent secondary transporters and are ubiquitous in prokaryotes, but absent from eukaryotes. They are comprised of an SBP of the DctP or TAXI families and two integral membrane proteins of unequal sizes that form the DctQ and DctM protein families, respectively. The SBP component has a structure comprised of two domains connected by a hinge that closes upon substrate binding. In DctP-TRAP transporters, substrate binding is mediated through a conserved and specific arginine/carboxylate interaction in the SBP. While the SBP component has now been relatively well characterized, the membrane components of TRAP transporters are still poorly understood both in terms of their structure and function. We review the expanding repertoire of substrates and physiological roles for experimentally characterized TRAP transporters in bacteria and discuss mechanistic aspects of these transporters using data primarily from the sialic acid-specific TRAP transporter SiaPQM from Haemophilus influenzae, which suggest that TRAP transporters are high-affinity, Na(+)-dependent unidirectional secondary transporters.
  • Mulligan, C., Geertsma, E., Severi, E., Kelly, D., Poolman, B. and Thomas, G. (2009). The substrate-binding protein imposes directionality on an electrochemical sodium gradient-driven TRAP transporter. Proceedings of the National Academy of Sciences of the United States of America [Online] 106:1778-1783. Available at: http://dx.doi.org/10.1073/pnas.0809979106.
    Substrate-binding protein-dependent secondary transporters are widespread in prokaryotes and are represented most frequently by members of the tripartite ATP-independent periplasmic (TRAP) transporter family. Here, we report the membrane reconstitution of a TRAP transporter, the sialic acid-specific SiaPQM system from Haemophilus influenzae, and elucidate its mechanism of energy coupling. Uptake of sialic acid via membrane-reconstituted SiaQM depends on the presence of the sialic acid-binding protein, SiaP, and is driven by the electrochemical sodium gradient. The interaction between SiaP and SiaQM is specific as transport is not reconstituted using the orthologous sialic acid-binding protein VC1779. Importantly, the binding protein also confers directionality on the transporter, and reversal of sialic acid transport from import to export is only possible in the presence of an excess of unliganded SiaP.
  • Mulligan, C., Kelly, D. and Thomas, G. (2007). Tripartite ATP-independent periplasmic transporters: application of a relational database for genome-wide analysis of transporter gene frequency and organization. Journal of molecular microbiology and biotechnology [Online] 12:218-226. Available at: http://dx.doi.org/10.1159/000099643.
    Tripartite ATP-independent periplasmic (TRAP) transporters are a family of extracytoplasmic solute receptor-dependent secondary transporters that are widespread in the prokaryotic world but which have not been extensively studied. Here, we present results of a genome-wide analysis of TRAP sequences and genome organization from application of TRAPDb, a relational database created for the collection, curation and analysis of TRAP sequences. This has revealed a specific enrichment in the number of TRAP transporters in several bacteria which is consistent with increased use of TRAP transporters in saline environments. Additionally, we report a number of new organizations of TRAP transporter genes and proteins which suggest the recruitment of TRAP transporter components for use in other biological contexts.
  • Müller, A., Severi, E., Mulligan, C., Watts, A., Kelly, D., Wilson, K., Wilkinson, A. and Thomas, G. (2006). Conservation of structure and mechanism in primary and secondary transporters exemplified by SiaP, a sialic acid binding virulence factor from Haemophilus influenzae. The Journal of biological chemistry [Online] 281:22212-22222. Available at: http://dx.doi.org/10.1074/jbc.M603463200.
    Extracytoplasmic solute receptors (ESRs) are important components of solute uptake systems in bacteria, having been studied extensively as parts of ATP binding cassette transporters. Herein we report the first crystal structure of an ESR protein from a functionally characterized electrochemical ion gradient dependent secondary transporter. This protein, SiaP, forms part of a tripartite ATP-independent periplasmic transporter specific for sialic acid in Haemophilus influenzae. Surprisingly, the structure reveals an overall topology similar to ATP binding cassette ESR proteins, which is not apparent from the sequence, demonstrating that primary and secondary transporters can share a common structural component. The structure of SiaP in the presence of the sialic acid analogue 2,3-didehydro-2-deoxy-N-acetylneuraminic acid reveals the ligand bound in a deep cavity with its carboxylate group forming a salt bridge with a highly conserved Arg residue. Sialic acid binding, which obeys simple bimolecular association kinetics as determined by stopped-flow fluorescence spectroscopy, is accompanied by domain closure about a hinge region and the kinking of an alpha-helix hinge component. The structure provides insight into the evolution, mechanism, and substrate specificity of ESR-dependent secondary transporters that are widespread in prokaryotes.

Book section

  • Mulligan, C. and Mindell, J. (2017). Pinning down the mechanism of transport: probing the structure and function of transporters using cysteine crosslinking and site-specific labelling. In: Ziegler, C. ed. Methods in Enzymology: A Structure-Function Toolbox for Membrane Transporter and Channels. Elsevier, pp. 165-202. Available at: https://doi.org/10.1016/bs.mie.2017.05.012.
    Transporters are crucial in a number of cellular functions, including nutrient uptake, cell signaling, and toxin removal. As such, transporters are important drug targets and their malfunction is related to several disease states. Treating transporter-related diseases and developing pharmaceuticals targeting transporters require an understanding of their mechanism. Achieving a detailed understanding of transporter mechanism depends on an integrative approach involving structural and computational approaches as well as biochemical and biophysical methodologies. Many of the elements of this toolkit exploit the unique and useful chemistry of the amino acid cysteine. Cysteine offers researchers a specific molecular handle with which to precisely modify the protein, which enables the introduction of biophysical probes to assess ligand binding and the conformational ensemble of the transporter, to topologically map transporters and validate structural models, and to assess essential conformational changes. Here, we summarize several uses for cysteine-based labeling and cross-linking in the pursuit of understanding transporter mechanism, the common cysteine-reactive reagents used to probe transporter mechanism, and strategies that can be used to confirm cysteine cross-link formation. In addition, we provide methodological considerations for each approach and a detailed procedure for the cross-linking of introduced cysteines, and a simple screening method to assess cross-link formation.


  • Battley, M. (2019). Exploring the Physiological Roles of the Antibacterial Resistance Associated DedA Genes in Escherichia Coli.
  • Scarsbrook, H. (2019). Structural and Functional Studies of the Antimicrobial Resistance Associated DedA Family.
    The advancement and adaptations of antimicrobial resistance mechanisms by bacteria causes a great health impact. To combat this,these mechanisms need to be understood. DedA are a family of proteins found within a variety of prokaryotes, shown to be possible drug sensitivity targets as although very little is known about these proteins, they are essential for cell viability and resistance to different antimicrobials. The potential of targeting these proteins and inhibiting the possible membrane transport mechanisms could possibly sensitise bacteria to a range of different antimicrobials, with particular reference to clinically relevant pathogens. This research studies the role of the DedA protein family with particular focus on the DedA protein; YqjA. This work utilised two key areas of analysis; topological analysis alongside biochemical and microbiological analysis to evaluate the role of the DedA protein YqjA in Escherichia coli (E. coli). Topological analysis of DedA protein family members was first analysed to form predictions for the structural features of YqjA. These predictions were then tested using substituted cysteine accessibility measurements to determine the position of introduced cysteines in relation to the membrane. Further work included sensitivity analysis of YqjA with single point mutations to different antimicrobials and stressors to determine the importance of specific amino acids for the functioning of the protein. This research identifies YqjA as a membrane protein with five transmembrane helices. Results from cysteine accessibility measurements highlighted the boundaries of each of the transmembrane helices as well as the boundaries between the helices. The work studying the function of different amino acids in YqjA highlighted two which are shown to be vital for the functioning of YqjA; YqjA R130 and YqjA R136. Other work using pH sensitivity tests determined which areas of the DedA member YghB is responsible for alkaline sensitivity. Overall, this research concludes that YqjA is a form of efflux protein located in the membrane and plays an important role in the export of different biocides. This has provided a greater insight into the possible role of DedA in E. coli.
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