MRES 2015

MRes RESEARCH in the DEPARTMENT OF CHEMISTRY

& BIOMOLECULAR SCIENCES

This booklet outlines research projects available with staff of the Department of Chemistry & Biomolecular Sciences, Macquarie University for 2015. The booklet will introduce you to the Department and help you identify research projects that interest you. In the MRes program CBMS students will work under the supervision of one or more academic staff, as they progress towards completing their degree. In year one of the program CBMS students will undertake two separate five week research experience projects to gain familiarity with research areas of interest. In year two they will select a topic and conduct research and report these results in the form of a written thesis.

You are free to choose to select any project on offer in this booklet, provided that facilities and research supervisor are available. Clearly, the outlines here are very brief and general, so you should contact staff offering projects that interest you in and discuss your options further. There is often scope for modifying a project to take advantage of your particular skills and interests.

Members of the MRes Committee are always available to assist students, particularly those from other institutions, in finding a suitable project amongst CBMS research activities.

Members of the CBMS MRes Committee for 2015: Dr Louise Brown Assoc. Professor Bridget Mabbutt Assoc. Professor Mark Molloy Assoc. Professor Andrew Try

Department of Chemistry and Biomolecular Sciences

Dr Louise Brown Louise.Brown@mq.edu.au F7B335 Ph 9850 8294

Structural Biology Many key physiological processes are controlled at a molecular level by large multi-protein complexes. These complexes are also often prone to disease-producing mutations. Our research group is interested in two systems (i) The switch for turning on muscle contraction – Troponin & (ii) an unusual auto-inserting ion channel family – the CLICs. Due to the large size and the dynamic nature of both protein complexes, their structures are difficult to determine by conventional biophysical methods. We therefore rely on spectroscopic methods and in particular chemical reporter group techniques to study these challenging protein systems.

Single probe spectroscopy The techniques of Electron Paramagnetic Resonance Spectroscopy (EPR) & Fluorescence Spectroscopy require the introduction of small chemical labels onto a protein’s backbone. We do this by using site-directed mutagenesis methods to selectively place a spectroscopic label onto a protein

Figure 1: “Spin Labeling” - the attachment of a spin label to the side chain of a cysteine residue that has been introduced onto the protein by site-directed mutagenesis.

molecule (Fig. 1). The label then reports on the local environment of the protein to provide us with dynamic structural information from which we can build up and test models for how the protein system works. We are also working on an exciting new, cross-disciplinary research field which involves the use of brightly fluorescing nanodiamonds as biomarkers.5 We develop methodology to modify surface properties to obtain bright nanodiamonds to be used as biolabels which we can ultimately use to track and image single molecules.

SDSL-EPR – ‘Site-Directed-Spin-Labeling Electron Paramagnetic Resonance Spectroscopy’ : For this novel method of determining the structure of large 'difficult' proteins, we attach a small paramagnetic ‘spin’ label to the protein. Then, like other atomic-resolution structural techniques, EPR is then used to provide information on the secondary, tertiary and quaternary structure of proteins. We can also measure conformational changes that accompany the function of the protein.4,6

FRET - Fluorescence Spectroscopy: We can measure distances between two ‘fluorescent’ labels attached to a protein using ‘FRET’ – Fluorescence Resonance Energy Transfer. Similar to SDSL-EPR, we can determine protein structure and conformational changes.2 We have also used our nanodiamonds as FRET probes.

Paramagnetic Resonance Enhancement Nuclear Magnetic Resonance spectroscopy (PRE-NMR). This novel NMR application allows us to use the EPR paramagnetic ‘spin’ label, as described above, to measure long range distances (up to 30Å) within the protein of interest. We obtain many distances that allow us to determine protein folds and dynamics1. The NMR is performed in-house on the 600MHz NMR within the Dept.

The two protein systems for which we apply these above techniques are described on the following page. Projects in our lab would suit students with backgrounds in any of the following: molecular biology, biochemistry, protein chemistry, physical chemistry (spectroscopy), organic chemistry or computational chemistry.

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1) Structure of Chloride Intracellular Ion channels - CLICs Chloride ion channels are involved in diverse physiological processes and channel malfunction can lead to severe diseases. This project examines the structure and conformational changes of a unique chloride ion channel, called ‘CLIC’ (Chloride Intracellular Ion Channel). It is highly unusual in that it can transit between a soluble and active membrane channel form (Fig 2). We have x-ray crystal structures of several members of this family and are using EPR, fluorescence spectroscopy and EM methods to determine the structure of the CLICs in the membrane bilayer.2,3 Our approach allows us to address questions including (i) what conditions allow for insertion of CLICs into the membrane; (ii) what keeps the CLICs inserted in the membrane; (iii) what structure is adapted for the channel upon insertion; and (iv) can we correlate the structure of the ion channels with channel gating?

Figure 2: Our current model for the Insertion of CLIC into the membrane bilayer. Structural studies, using single label probe studies, will help us understand this unusual ion channel family.2

2) The control of muscle contraction by the Troponin Complex There are many debilitating and even fatal cardiovascular and skeletal diseases that arise from defects in musle proteins. This project aims to understand the molecular basis for some of these disorders in the muscle protein ‘Troponin’. There are now more than 60 mutations in Troponin linked to heart disease (Figure 3). We are determining the structure of Troponin using EPR6 and NMR1 methods which will allow us to understand (i) how this large protein complex can switch on muscle contraction, and (ii) why mutations lead to disease states. Figure 3: Our current model of the Troponin

complex. Locations of mutations that cause Cardiomyopathy are shown as spheres.

Selected Publications 1 Cordina NM, Liew CK, Gell DA, Fajer PG, Mackay JP, Brown LJ. “Interdomain orientation of cardiac

Troponin C characterized by paramagnetic relaxation enhancement NMR reveals a compact state” Protein Science 2012 21:1376-1387

2 Goodchild SC, Angstmann CN, Breit SN, Curmi PM, Brown LJ. “Transmembrane Extension and Oligomerization of the CLIC1 Chloride Intracellular Channel Protein upon Membrane Interaction” Biochemistry 2011 50:10887-97

3 Goodchild, Curmi & Brown “Structural Gymnastics of Multifunctional Metamorphic Proteins” Biophysical Reviews, 2011, 3,143-153.

4 Cooke J & Brown L “Distance measurements by Continuous Wave EPR spectroscopy to monitor protein folding. Methods in Molecular Biology: Protein folding and disease” 2011, ISBN 1603272216.

5 Bradac, Gaebel, Naidoo, Sellars, Twamley, Brown, Barnard, Plakhotnik, Zvyagin & Rabeau “Observation and control of blinking nitrogen vacancy centres in discrete nanodiamonds” Nature Nanotechnology, 2010, 5, 345-349.

6 Brown, Sale, Hills, Rouvierre, Song, Zhang & Fajer “Structure of the Inhibitory region of Troponin by Site Directed Spin Labeling Electron Paramagnetic Resonance” Proc. Natl Acad. Sci. USA., 2002, 99, 12765-12770.

http://www.cbms.mq.edu.au/~biophysical/

Department of Chemistry and Biomolecular Sciences

Professor Paul A. Haynes paul.haynes@mq.edu.au Room F7B 331 Ph: 9850 6258

Environmental Proteomics Research in our laboratory focusses on environmental proteomics. We aim to understand what happens at the molecular level when an organism is exposed to changes in its external environment. We work in different systems, including plants and animals. We have two mass spectrometers in our laboratory, and we are constantly refining the analysis approach we use, in terms of both protein chemistry and bioinformatics. In recent years we have published a number of studies on the effects of temperature stress on rice cells and seedlings, and drought stress on rice plants. We are currently working on a project involving analysis of temperature stress on grape cells and drought stress on grape vines as part of a field experiment trial, in collaboration with colleagues at the University of Nevada, Reno. We also work on marine organisms, and have published studies on the effects of heavy metal contamination on Sydney rock Oysters, and are following this up with a study of how marine amphipods are affected by heavy metal contaminated sediments. The latter work is performed in collaboration with Professor David Raftos in Biological Sciences at MQ and is supported by funding from the Australian Research Council.

Analysis of temperature and drought stress in rice plants The figure to the right is a heat map generated from label-free quantitative shotgun proteomic analysis of rice cells exposed to five different temperatures. The cluster on the right corresponds to cells subjected to 3 days at 44 C, and is clearly the most different to the others. This is a summary of the identification and quantification of more than 2500 proteins, generated from more than two million spectra of raw mass spectrometric data. We also developed our own software to enable quantification of those proteins which are differentially expressed between different environmental conditions.

Analysis of temperature and drought stress in grapevines Drought stress affects plants severely and is a real problem facing the wine industry in the face of future climate change. The figure to the right shows rice plants from a previous study involving analysis of drought signalling. We were able to show using split-rooted pots that the molecular signal for drought stress is communicated from droughted roots to well-watered roots, but not the other way around.

Department of Chemistry and Biomolecular Sciences

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We are now undertaking a similar approach to analysis of temperature and drought stress in grapevines.

Selected Publications 1. K.A. Neilson, M. Mariani and P.A. Haynes. Quantitative proteomic analysis of cold-responsive

proteins in rice. Proteomics 2011, 11(9):1696-706. 2. K.A. Neilson, N.A. Ali, S. Muralidharan, M. Mirzaei, M. Mariani, G. Assadourian, A. Lee, S. Van Sluyter

and P.A. Haynes. Less label, more free: Approaches in label-free quantitative mass spectrometry. Proteomics, 2011, ISSN 1615-9853, 11(4):535-53.

3. C.G. Gammulla, D. Pascovici, B.J. Atwell and P.A. Haynes. Differential proteome response of cultured rice (Oryza sativa) leaves exposed to high- and low- temperature stress. Proteomics 2011, 11(14):2839-50.

4. E.L. Thompson, D.A. Taylor, S.V. Nair, G. Birch, P.A. Haynes and D.A.Raftos. Proteomic discovery of biomarkers of metal contamination in Sydney Rock oysters (Saccostrea glomerata). A q u a t i c T o x i c o l o g y , 2011, Mar;109:202-12.

5. M. Mirzaei, N. Soltani, E. Sarhadi, D. Pascovici, T. Keighley, G.H. Salekdeh, P.A. Haynes and B.J. Atwell. Shotgun Proteomic Analysis of Long-distance Drought Signaling in Rice Roots. J. Proteome Res. 2012, 11(1):348-58.

6. D. Pascovici, T. Keighley, M. Mirzaei, P.A. Haynes P.A. and B. Cooke. PloGO: Plotting Gene Ontology annotation and abundance in multi-condition proteomics experiments. Proteomics, 2012, Feb;12(3):406-10. 7. S. Muralidharan, E.L. Thompson, D.A. Raftos, G. Birch and P.A. Haynes. Quantitative proteomics of heavy metal stress responses in Sydney Rock oysters. Proteomics, 2012, Mar;12(6):906-21. 8. G.R. Cramer, S. Van Sluyter, D.W. Hopper, D. Pascovici, T. Keighley and P.A. Haynes. Proteomic analysis indicates massive changes in metabolism prior to the inhibition of growth and photosynthesis of grapevine (Vitis vinifera L.) in response to water deficit. BMC Plant Biol. 2013 Mar 21;13:49. doi: 10.1186/1471-2229-13-49. 9. K.A. Neilson, A.P. Scafaro, J.M. Chick, I.S. George, S.C. Van Sluyter, S.P. Gygi, B.J. Atwell and P.A. Haynes. The influence of signals from chilled roots on the proteome of shoot tissues in rice seedlings. Proteomics. 2013 Jun;13(12-13):1922-33. doi: 10.1002/pmic.201200475. 10. M. Mirzaei, N. Soltani, E. Sarhadi, I.S. George, K.A. Neilson, D. Pascovici, S. Shahbazian, P.A. Haynes, B.J. Atwell and G.H. Salekdeh. Manipulating root water supply elicits major shifts in the shoot proteome. J Proteome Res. 2014 Feb 7;13(2):517-26.

http://www.cbms.mq.edu.au/academics/phaynes.html

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the presence of useful compounds, stage of plant development and the maturation state of fruit. The relatively new technique of Solid-Phase Microextraction (SPME) offers a route to convenient in situ sampling. SPME combines in one-step sampling and preconcentration, prior to GC or GC-MS analysis. Our research activity aims at developing methods of in situ SPME-GC analysis, and to develop a database of VOC emissions from Australian native vegetation. We are also interested in the ways that plants and animals use VOCs for signalling and deception purposes. Maths Anxiety in Chemistry Students

Maths anxiety is described in a number of ways, but the common theme is that a sufferer feels, to greater or lesser extent, panic, helplessness, paralysis, and mental disorganization. This may mean that the student stops him- or herself from starting on a task, even if capable of doing it. Students may be caught in a cycle of maths avoidance when, in the past, the student

has suffered a bad experience relating to maths. It is of interest to measure the extent of maths anxiety amongst the student cohort, and to develop mechanisms for identifying these people early in their studies, so that appropriate support for them can be provided. Chemical Misconceptions and Constructivism “Constructivism” refers to the theory that the process of learning is not one of simple acceptance and remembrance of facts, but one where the learner must incorporate them into an already constructed world-view. It is necessary for teachers to understand the ways in which students incorporate knowledge into these existing knowledge frameworks, which may include preconceptions and/or misconceptions. Misconceptions in chemistry are extremely persistent and are likely to still be present in tertiary level students, right through to those studying for their Ph.D.’s. It is important that teachers are aware of the range of preconceptions and misconceptions that students bring with them, and put in place appropriate teaching methods that adequately address these issues. Selected Publications 1. M. Azzia, S.J. White, D.E. Angove and I.M. Jamie, “Modelling the Photooxidation of ULP, E5 and E10

in the CSIRO Smog Chamber”, Atmospheric Environment, 2010, 44, 5375-5382. 2. M. Azzi, S.J. White, D.E. Angove, I.M. Jamie and A. Kaduewela, “Evaluation of the SAPRC-07

mechanism against CSIRO smog chamber data”, Atmospheric Environment, 2010, 44, 1707-1713. 3. A. George, M. Buntine, J. Read, S. Barrie, R. Bucat, G. Crisp, I. Jamie and S. Kable (2009) “What makes

a good laboratory learning exercise? Student feedback from the ACELL project” in Chemistry Education in the ICT Age, Gupta-Bhowon, M.; Jhaumeer-Laulloo, S.; Li Kam Wah, H.; Ramasami, P. (Eds.) Springer 2009, 363-376.

4. M. Camenzuli and I. Jamie, “The Effect of Elevated CO 2 on the Emission of Biogenic Volatile Organic Compounds” Proceedings of the 19th Clean Air and Environment Conference, Perth, 6-9 Sep 2009.

5. S. White, M. Azzi, D. Angove, I. Jamie and L. Singh-Peterson, “Evaluation of Isoprene Photooxidation Using Smog Chamber Data and Chemical Mechanisms” Proceedings of the 19th Clean Air and Environment Conference, Perth, 6-9 Sep 2009.

6. D.W.T. Griffith, I.M. Jamie, M. Esler, S.R. Wilson, S.D. Parkes, C. Waring and G.W. Bryant, “Real Time Field Measurements of Stable Isotopes in Water and CO2 By Fourier Transform Infrared Spectrometry”, Isotopes in Environmental and Health Studies, 2006, 42, 9-20.

http://www.cbms.mq.edu.au/~ijamie

Department of Chemistry and Biomolecular Science

Associate Professor Joanne Jamie joanne.jamie@mq.edu.au F7B231 Ph 9850 8283

Bio-Organic & Medicinal Chemistry & Science Outreach Current research is focussed on studies aimed at understanding medicinally important human enzymes and developing potent inhibitors of them; collaborative partnerships with Indigenous communities for documentation, biological screening and isolation of bioactive compounds from traditional medicines; and studies on isolation and synthesis of fruit fly attractants and analysis of their effectiveness. Projects on development of educational resources for a science engagement program and/or evaluation of the effectiveness of the program, are also available. Human Indoleamine 2,3-Dioxygenase (Willows) Indoleamine 2,3-dioxygenase (IDO) catalyses the cleavage of L-tryptophan to N-formylkynurenine. This is the first and rate limiting step of the kynurenine pathway - the major metabolic pathway for tryptophan breakdown (see below). Under a variety of pathological conditions IDO is over-expressed. This leads to increased levels of the neurotoxins quinolinic acid and 3-hydroxykynurenine, and these have been linked to neurological disorders, including AIDS dementia complex, cerebral malaria and Alzheimer's disease. Additionally, various tumour cells are known to express IDO and IDO inhibition has been shown to be an anti-cancer immunotherapeutic strategy.

Research projects on investigating IDO’s active site and the design, synthesis and biological testing of IDO inhibitors are available. An understanding of IDO’s substrate active site is critical for the rational design of inhibitors. Mutants of recombinant human IDO will be produced and their enzyme kinetics, confirmation and stability compared to wild type IDO to confirm the importance of specific regions of the active site for normal IDO activity and their role in inhibitor binding. In related studies, we have identified promising IDO inhibitors, with novel structural features, from in silico screening, synthesis and from traditional medicines. Further investigations into these molecules will be directed towards confirmation of their inhibitory kinetics, as well as structure-activity relationship studies to identify the features important for their inhibitory activity. These studies will assist in the development of potent and selective IDO inhibitors. IDO projects can be customised to combine synthesis, biological testing, molecular modelling and mutant studies or can be focussed on a particular aspect based on interest.

Attractant and Pheromone Compounds of Fruit Flies (lead by Ian Jamie, CBMS and Phil Taylor, Biology)

Bactrocera fruit flies – a genus of more than 500 species – include some of the world’s most devastating insect pests of horticulture. Air-borne pheromones are used by these insects to communicate, and in synthetic form also have potential as tools for control. Attractants are used to monitor and control fruit fly populations. Projects within this field may focus on one or the other category of compounds, but in all cases would involve a

combination of collection and identification of compounds, analysis of dispersal and transformation in the atmosphere, and synthesis of novel and reference compounds.

Department of Chemistry and Biomolecular Sciences

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Dr. Dayong Jin (MQ Vice-Chancellor's Innovation Fellow) dayong.jin@mq.edu.au Room F7B 332 Ph 9850 4168

Nanotechnology

Our research is highly multidisciplinary spanning the photonics devices, luminescence materials, bio-/nano- chemistry and analytical biotechnology. Collaborating with international and industry partners, we are working at a highly practical level to create new

technologies for early diagnosis of disease and anti-counterfeiting industry.

Molecules that are altered as a result of a pathological condition are generally present in very low abundance, and pose a “needle-in-a-haystack” problem. At the nanoscale – scales less than a billionth of a metre wide, our research is to develop the new generation of luminescent probes (SuperDots) to understand and detect diseases. The probes are ultrabright, low background interference, and have large-scale multiplexing capacity. The probes can be attached to DNAs and antibodies that can detect pathogens and cancer cells, and shine brightly like a light bulb in nanoscale. The new cytometry technologies can therefore rapidly inspect cell by cell so every brightly-shining disease cell or molecules are detected.

Super-sensitive probes will also impact other significant applications spanning the fields of immunofluorescence imaging, flow cytometry, security printing, and super-resolution nanoscopy. SuperDots research opens viable avenues to cope with the complexity challenges in the life sciences, medicine and data storage and security.

The Advanced Cytometry Labs @ Macquarie is a young energetic team of PhD students and Postdoctoral research fellows from Chemistry, Physics, and Advanced Medicine. We train our young researchers to be future leaders in science research and technology development. The new suite of technologies has been internationally patented, and the breakthrough results have been published on Nature Nanotechnology (Sep. 2013). A scientific collaboration between Macquarie University in Sydney and industry partners has been awarded a $617,000 grant to support the development of new highly sensitive, non-invasive cancer diagnostic kits. Other research progress and achievements can be followed at my website.

Department of Chemistry and Biomolecular Sciences

SuperDots are nanocrystals embedding thousands of light emitters

The molecular probes, SuperDots are also found useful to covert features as luminescent nano-inks for security printing

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Chemical Biology Our research interests lie in the application of small molecules to biological systems, which involves new and exciting multidisciplinary approaches incorporating organic synthesis, analytical chemistry, NMR spectroscopy, molecular modelling, ecology, molecular biology and biochemistry to solving medicinally relevant problems. We are particularly interested in marine natural products and fluorescent molecules, their biological activity, ecological roles, biosynthesis and most importantly, their modes of action as drugs and uses in biotechnology. HO H O

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activity than plants, animals or microorganisms. In this project, we use bioassay directed isolation to discover new compounds from marine sponges with antibiotic7, herbicidal and anticancer activity. In this area, I can offer a number of projects that range from collection and isolation to the structure elucidation of new natural products from marine animals, medicinal plants from Africa and Iraq or microbes (in collaboration with MST) and the discovery of new fluorophores. Please see me for further details.

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Analytical Chemistry Recent discoveries in my group5,6 have resulted in the commercialisation of a fluorescent natural product from a fungus and we have discovered other new highly-fluorescent natural products from marine sponges. We are also interested in other fluorescent probes for use in biotechnology such as analogues of the fluorescent (anticancer) marine natural product ageladine A3 and latent fluorophores such as sulforhodamine trimethyl lock and

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Isozyme Specificity: Targeting Conformational Flexibility in Drug Discovery Enzymes are major drug targets, and their conformational flexibility is known to be a

major factor in designing drug leads with isozyme specificity (Nature Review Drug Discovery. 2003, 527). This is exemplified by the discovery of Gleevec as the first CML drug that specifically induces a particular conformational state of the Bcr-Abl kinase domain, which is

the molecular driver behind chronic myelogenous leukemia (CML). Protein flexibility is difficult to predict or model thus presenting considerable challenges in rational drug design. Our group has used a semi-targeted approach, using natural products as leads, to access derivatives that can induce specific conformational change in the protein target for achieving isozyme specific recognition. In particular, we are using nucleotide mimics such as the K252 family and balanol to construct libraries of compounds using a fragment based approach (Journal of Organic Chemistry 2009,

254; Organic Letter 2007, 195). Subsite targeting motifs and conformational tuners are assembled using diversity-oriented synthesis. Activity- and binding- based SAR profiles are then established to guide the next cycle of library synthesis to improve specificity and potency.

Activity-based Proteomics: Finding the Molecular Signature of Cancer

The long history of research in covalent modification of enzymes serves as a rich source for developing activity probes for functional proteomics (Annu. Rev. Biochem., 1984, 53, 493-535). We have developed highly specific labeling techniques for tagging protein active sites (Journal of the American Chemical Society 2004, 7754. Highlighted by the Chemistry & Engineering News of the American Chemical Society). These techniques have allowed analysis of protein function and their mechanistic role in a complex environment.

One of the most important applications of ABPs is in drug discovery (Curr. Opin. Chem. Biol., 2011,57). In addition, this approach has been successfully used to clarify molecular mechanisms that drive disease onset and progression (Mol. Cell. Proteomics, 2008, 1887- 2006). Our group, in collaboration with the Australian Proteomics Analysis Facility (APAF), is investigating the role of signaling

Activity-based proteomics

enzymes in cancer by designing and synthesizing activity probes for membrane signaling enzymes (Proteomics, 2011, 2683). The elucidation of signaling networks and mechanisms responsible for cancer progression will lead to identification of more effective drug targets.

Selected Recent Publications 1. Dolai, S.; Xu, Q.; Liu, F.; Molloy, M. (2011) Proteomics “Quantitative chemical proteomics in small-scale

culture of phorbol ester stimulated basal breast cancer cells”, 2683. 2. Garnier, J-M.; Anstiss, C.; Liu, F.* (2009) Advanced Synthesis & Catalysis. “Enantioselective

Trifunctional Organocatalysts for Rate-Enhanced Aza-Morita-Baylis-Hillman Reactions at Room Temperature”, 331.

3. Stephens, B. E.; Liu, F.* (2009) Journal of Organic Chemistry. “A Regio- and Diastereoselective Intramolecular Nitrone Cycloaddition for Practical 3- and 2,3-Substituted Piperidine Synthesis from gamma-Butyrolactone”, 254.

4. Yang, M.; Liu, F.* (2008) Journal of Organic Chemistry. “An Ullmann Coupling of Aryl Iodides and Amines Using an Air-Stable Diazaphospholane Ligand”, 8969.

5. Patil, SN, Liu, F.* (2007) Organic Letters. “Base-assisted regio- and diastereoselective conversion of functionalized furans to butenolides using singlet oxygen”, 195.

6. Yin, J.; Liu F.,; Li, X.; Walsh, C. T. (2004) Journal of the American Chemical Society. “Labeling proteins with small molecules by site-specific posttranslational modification”, 7754-5.

Associate Professor Bridget Mabbutt bridget.mabbutt@mq.edu.au F7B338 Ph 9850 8282

Protein Structure The structure and form of any protein shapes its unique function, as well as driving its interactions with biological partners. Structural biology and structural genomics are vibrant research activities worldwide, providing detailed molecular maps of large biomolecules and even larger protein complexes. Today, 3D structures of proteins and protein complexes are being solved as part of projects ranging across medicine, biochemistry and nanomaterial design. Our Protein Structure group offers significant expertise in recombinant expression, protein engineering, structure methods, and protein reactivity. Our work is very collaborative; current collaborators include New Zealand researchers, as well as fellow members of the Biomolecular Frontiers Centre at Macquarie.

Synthetic biology- fabrication of ring-shaped nanotubules The self assembly of proteins occurs naturally in the cell, forming well-defined structures and supramolecular assemblies. These biological structures are now attracting attention in nanotechnology, which aims to exploit ‘bottom-up’ construction to develop novel molecular scaffolds.

Our team is one of the world’s few labs studying structures of the Lsm family of RNA-binding proteins, implicated in autoimmune disease and some cancer types [1-3]. This project takes a new direction, in which we use the remarkable self-assembling property of Lsm proteins as building blocks to control-build artificial protein rings and tubules in the 5-10 nm range. These relatively large rings expand the repertoire and complexity of molecular forms available, and our ultimate aim is to fabricate new RNA sensing materials or delivery capsules.

You will engineer recombinant Lsm proteins into ordered ring assemblies, chemically modify them into molecular conjugates, and utilize biophysical techniques (chromatography, electron microscopy, AFM, etc) to examine how they organise. This project has attracted US funding, in partnership with the Biomolecular Interactions Centre, University of Canterbury (NZ).

Department of Chemistry and Biomolecular Sciences

Genomic islands and pathogenicity in Acinetobacter baumanii (with Ian Paulsen) Acinetobacter baumannii is poses a serious global health threat due its emerging multidrug resistant forms. Genome sequencing of A. baumannii isolates, including Australian strains, is revealing their extraordinary genetic plasticity [4]. We have pursued structural genomics [5] to discover some of the highly novel proteins found within genomic islands of Australian strains. Of the proteins for which we have obtained crystal structures, several are ready for functional testing prior to publication. They appear to be enzymes involved in biosynthesis of cell wall polysaccharide. Recombinant enzymes will be screened for specific functions (e.g. saccharide binding) and/or relevant bioassays will be conducted on original bacterial strains. Alternatively, you can be involved in learning crystallisation techniques to grow and optimise high-quality protein crystals in conjunction with relevant cofactors and substrates. More highly refined structures will provide atomistic detail to the the catalytic mechanism of these new proteins, a necessary step for developing new antibiotics.

Structure/function relationships of medically-important proteins (with ASAM) Researchers Ian Blair and Roger Chung have recently transferred their research teams to the neurobiology division of ASAM. We are instigating new projects to investigate structure/function links for several proteins of relevance to their research into neurological disorders. Projects will cover RNA-binding systems, as well as proteins with amyloid propensities. You will be learning how to prepare and probe the strcutre of proteins in both laboratory and cellular contexts.

Selected Publications 1. Naidoo, Harrop, Sobti, Haynes, Szymczyna, Williamson, Curmi, & Mabbutt “Crystal structure of Lsm3 octamer

from Saccharomyces cerevisiae: Implications for Lsm ring organisation and recruitment”, J. Mol. Biol., 2008, 377, 1357-1371.

2. Sobti, Cubeddu, Haynes & Mabbutt “Engineered Rings of Yeast Lsm Proteins Show Differential Interactions with Translation Factors and U-Rich RNA”, Biochemistry, 2010, 49, 2335-2345.

3. Moll, Sobti & Mabbutt “The Lsm Proteins: Ring Architectures for RNA Capture”, in "RNA Processing", 2011, Intech. ISBN: 978-953-307-332-3.

4. Farrugia, Elbourne, Hassan, Eijkelkamp, Tetu, Brown, Shah, Peleg, Mabbutt & Paulsen. “The complete genome and phenome of a community-acquired Acinetobacter baumannii.”, PLoS One, 2013, 8(3):e58628.

5. Robinson, Guilfoyle, Sureshan, Howell, Harrop, Boucher, Stokes, Curmi & Mabbutt “Structural Genomics of the Bacterial Mobile Metagenome: An Overview”, Meth. Mol. Biol., 2008, 426, 589-595, Humana Press, NJ.

http://www.cbms.mq.edu.au/~proteins

Department of Chemistry and Biomolecular Sciences

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Dr Christopher McRae christopher.mcrae@mq.edu.au F7B328 Ph 9850 8288

Analytical & Environmental Chemistry Research in the Analytical Geochemistry laboratory focuses on both the development of new analytical methodology and instrumentation, and their application to the understanding of environmental and geochemical problems. The problems we address are often extremely diverse, employing almost the complete gambit of analytical instrumentation; all modes of chromatography, flow injection analysis, mass spectrometry, NMR and FTIR spectroscopy, and thermal analysis. The skills you gain in this field of research will vastly improve your employability in the Australian chemical and biochemical job market. The following projects are just a few examples of projects available.

Rescuing Alumina from Humic Substances

Alumina is made from bauxite at refineries in the Northern Territory, Queensland and Western Australia. Around two and a half tonnes of bauxite are needed to produce one tonne of alumina. Australia is the world's leading producer of alumina, producing 30% of global output. More than 90% of the world's alumina production is used to make aluminium. The majority of the world's alumina is produced in a process known as the “Bayer Process” wherein ground bauxite is mixed

Bauxite (left), Alumina (top), Aluminium (right) with concentrated NaOH to form a slurry. The amphoteric nature of alumina (Al2 O3 ) means

it dissolves out of the bauxite (forming aluminate) and leaving behind the other components of bauxite (Fe2 O3 , TiO2 & SiO2 ). The dissolved aluminate is filtered, cooled and “seeded” with crystals of aluminium hydroxide to precipitate alumina hydrate from the supersaturated aluminate liquor. The precipitated hydrate is filtered off, washed and passed through rotating calcinating kilns operating at high temperatures to produce the white powder known as alumina. The problem is, natural organic material (humic substances) in the bauxite interfere with the precipation step, preventing recrystalisation and resulting in an estimated production loss of 20% per annum. In context, this equates to a loss of $2.94 billion export dollars at current alumina prices. This project will involve the structural characterisation of the humic substances isolated from Bayer Liquor with a view to developing a strategy for their removal.

Development of a Solid Phase, Silane Based Reducing Agent

Silanes are established reducing agents. Indeed, we developed a novel silane based reagent, n-butylsilane [3], which enabled the complete and specific reduction of carboxylic acid, alcohol, aldehyde, and ketone functions to their hydrocarbon backbone in high yield, and in a one-pot reaction. A feat has yet to be replicated or improved on by any other reducing agent. We have used this reagent extensively in our studies of humic substances, however in that role the reagent has one major flaw. A by-product of the

reduction is the formation of butyl siloxanes. Whilst these by-products are readily identified during chromatographic/mass spectrometric analysis, their presence could

Department of Chemistry and Biomolecular Sciences

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potentially be masking important analytes. Thus the ideal situation would be for these siloxane by-products to be eliminated from the reaction products altogether. The immobilisation of the reducing agent on a solid support would allow the ready removal of the siloxane byproducts by simple filtration. The aim of this project is thus the immobilisation of an n-butyl silane analogue on a solid support and to test the efectiveness of this novel solid phase reducing agent.

Studying The Effect of Low Power Microwave Energy on Gas Chromatographic Separations

An electromagnetic wave is an oscillating electrical field and interacts only with molecules that can undergo a change in dipole moment.

A molecule’s rotational energy can be changed by exposure to microwave energy. The degree of the effect is dependent on the molecule’s dipole. In gas chromatography (GC), movement through the system can only occur while the molecule is in the gas phase. Microwave energy can be used to excite certain classes of compound over others providing a separation parameter not otherwise available in GC separations. The application of microwave energy for the purpose of adjusting chromatographic behaviour is entirely novel with the potential to greatly enhance the analytical potential of GC. As such, your aim for this

project will be to study and quantify the effect of microwave energy on GC separations using and refining a heavily modified gas chromatograph fitted with a 1000W microwave generator.

Selected Publications

1. McIntyre, C. P; Wressnig, A. M; McRae, C. R. “Fish gut content analysis by thermochemolysis with tetramethylammonium hydroxide (TMAH) and gas chromatography–mass spectrometry (GC–MS)” Journal of Analytical and Applied Pyrolysis, 2007, 80 (1), 6-15.

2. Nimmagadda, R. D.; McRae, C. “Characterisation of the backbone structures of several fulvic acids using a novel selective chemical reduction method” Organic Geochemistry, 2007, 38, 1061-1072.

3. Nimmagadda, R. D.; McRae, C. “A novel reduction of polycarboxylic acids into their corresponding alkanes using n-butylsilane or diethylsilane as the reducing agent” Tetrahedron Letters, 2006, 47, 3505- 3508.

4. Nimmagadda, R. D.; McRae, C. “A novel reduction reaction for the conversion of aldehydes, ketones and primary, secondary and tertiary alcohols into their corresponding alkanes” Tetrahedron Letters, 2006, 47, 5755-5758.

5. McIntyre, C.; McRae, C. “Proposed guidelines for sample preparation and ESI-MS analysis of humic substances to avoid self-esterification” Organic Geochemistry, 2005, 36(4), 543-553.

6. Simeoni, M. A.; Batts, B. D.; McRae, C. “Effect of groundwater fulvic acid on the adsorption of arsenate by ferrihydrite and gibbsite” Applied Geochemistry, 2003, 18(10), 1507-1515.

7. McIntyre, C.; McRae C.; Jardine, D.; Batts, B. D. “Identification of compound classes in soil and peat fulvic acids as observed by electrospray ionisation tandem mass spectrometry” Rapid Communications in Mass Spectrometry, 2002, 16, 1604-1609.

8. Whitelaw, M. J.; Batts, B. D.; Murray-Wallace, C.V.; McRae C. R. “Diagenesis of the organic matrix in Anadara trapezia during the Late Quaternary: Preliminary findings” Proceedings of the Linnean Society of New South Wales, 2001, 123, 225-234.

http://www.cbms.mq.edu.au/~geochem

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The separation of homogeneous catalysts from products or substrates continues to be a challenge. To overcome this, we are attaching catalysts already developed by our group onto a variety of robust structures and surfaces. The new anchored catalyst systems can be readily separated from reaction mixtures. This will not only allow easy catalyst/product separation, but will also provide a greater control over the nature of catalyst reactivity. The supports themselves can use the electrochemical properties of the catalysts to promote reactivity, or induce high enantioselectivity in asymmetric transformations.

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1. Andrey A. Tregubov, Khuong Q. Vuong, Erwann Luais, J. Justin Gooding, and Barbara A. Messerle* Rh(I) Complexes Bearing N,N and N,P Ligands Anchored on Glassy Carbon Electrodes: towards Recyclable Hydroamination Catalysts, Journal of the American Chemical Society, 2013, 135 (44), 16429–16437

2. Sandra W. S. Choy, Michael J. Page, Mohan Bhadbhade and Barbara A. Messerle*, Cooperative Catalysis: Large Rate Enhancements with Bimetallic Rhodium Complexes, Organometallics, 2013, 32 (17), 4726–4729.

3. Chin-Min Wong, Khuong Q. Vuong, Mark R. D. Gatus, Carol Hua, Mohan Bhadbhade and Barbara A. Messerle*, Catalysed Tandem C-N/C-C Bond Formation for the Synthesis of Tricyclic Indoles using Ir(III) Pyrazolyl-1,2,3-Triazolyl Complexes, Organometallics, 2012, 31 (21), 7500–7510.

4. Joanne Hui Hui Ho, Sandra Choy, Stuart Macgregor, Barbara A. Messerle* “Cooperativity in Bimetallic Dihydroalkoxylation Catalysts built on Aromatic Scaffolds: Significant Rate Enhancements with a Rigid Anthracene Scaffold”, Organometallics, 2011, 30(21), 5978–5984.

5. Danielle F. Kennedy, Ainara Nova, Anthony C. Willis, Odile Eisenstein* and Barbara A. Messerle,* “The mechanism of N-vinylindole formation via tandem imine formation and cycloisomerisation of o-ethynylanilines”, Dalton Trans, 2009, 10296-10304

6. Serin L. Dabb, Barbara A. Messerle,* Jörg Wagler, “Formation of Metallacyclobutene Complexes via the Addition of Hydrazines to Ruthenium Vinylidene Complexes”, Organometallics,27(18), 4657-4665, 2008

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Professor Helena Nevalainen Helena.Nevalainen@mq.edu.au Room E8C302, Ph 9850 8531

MOLECULAR BIOTECHNOLOGY Filamentous fungi are the world-champions of protein secretion and loving it! The research projects available within the group contribute to the development of new technologies for high level expression of genes in filamentous fungi and understanding protein secretion in the fungal cell factories. We are also expanding our research to medical fungi. Our group forms a part of the EDGE laboratory (Enzyme Development and Gene Expression group) which houses HDR students, postdoctoral research fellows and overseas visitors. We also have direct links to biotechnology industry and thereby conduct a number of industry-oriented research projects.

Helena is a founding scientist in the Biomolecular Frontiers Research Centre (BMFRC) at Macquarie University. She collaborates with Prof Nicki Packer in the novel area of Microbial Glycopathogenesis, Prof Ian Paulsen (microbial genomics) and Prof Paul Haynes (proteomics), all featured in this booklet.

If you are interested in working with fungi but the two projects/project areas outlined below are not what you are looking for, please come and talk to us to canvass some alternative research options!

Making products in a fungal cell factory We have a number of exciting new fungal recombinant strains that produce efficiently various thermophilic enzymes that have industrial applications.

A New Brunswick Scientific BioFlo III Batch/Continuous Benchtop Fermentor. Fermentors are computer-controlled instruments for scaling up the production of industrially-important gene products. Many different types of microorganisms can be grown in these fermentors. We are specialising in boosting up enzyme production in filamentous fungi. Our bench-top fermentors are equipped with the latest technology and software.

A considerable task after molecular constrution of a production strain is to optimise the cultivation so that the production potential of the fungus will be utilised to its maximum. This involves testing different growth media, pH conditions, aeration rates etc. in a computer-controlled laboratory fermenter where such parameters can be adjusted. The work may also involve ‘molecular mapping’ of the production where the messenger RNA levels will be assessed and ‘production proteomes’ created to follow the progress of fermentation. We invite interested Masters students to contact us for more information on the available projects. This research area suits well for a biotechnology- oriented person who would like to develop practical skills in product fermentation, well appreciated by potential employers. Fermentation projects will be co-supervised by Dr Junior Te’o.

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Tracking secretion of the fluorescent DsRed1 protein in fungal hyphae Recombinant foreign proteins (such as DsRed1) produced in surrogate hosts are often cleared from the secretory pathway by cellular protein quality control mechanisms. We are currently developing methods to track secretion of proteins in fugal hyphae and map their location in relation to the resident cell organelles with a view of identifying potential secretion roadblocks. This work will involve confocal fluorescence microscopy and collaboration with Ms Debra Birch, manager of the Microscopy facility.

Co-localisation of the DsRed 1 protein with endoplasmic reticulum (ER). From left to right: ER staining, DsRed1, merged image (co-localisation shown in yellow).

Selected Publications 1. Kautto, Grinyer, Paulsen, Tetu, Pillai, Pardiwalla, Sezerman, Akcapinar, Bergquist, Te’o and Nevalainen (2012). Cellular effects caused by expression of a mutant cellobiohydrolase I in Trichoderma reesei and effects of proteasome inhibition on stress response. New Biotechnology, 2012. 2. Peterson and Nevalainen (2012). Trichoderma reesei RUT-C30- 30 years of strain improvement. Microbiology 158: 59-68. 3. Peterson, Grinyer, Joss, Khan and Nevalainen (2009). Fungal proteins with mannanase activity identified directly from a Congo Red stained zymogram by mass spectrometry. Journal of Microbiological Methods 79: 374-377. 4. Deshpande, Wilkins, Packer and Nevalainen (2008). Protein glycosylation pathways in filamentous fungi. Glycobiology 18: 626-637. 5. Grinyer, Kautto, Traini, Te’o, Bergquist and Nevalainen (2007). Proteome mapping of the Trichoderma reesei 20S proteasome. Current Genetics 51: 79-88. 6. Nevalainen, Te’o and Bergquist (2005). Heterologous Protein Expression in Filamentous Fungi. Trends in Biotechnology 23:468-474.

http://www.chem.mq.edu.au/academics/hnevalainen.html

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Microbial Genomics

The research interests of my group are focused on applying high throughput genomic approaches to understand bacteria. Potential projects include:

Using Pseudomonas bacteria to protect plants from disease Australia is home to a number of serious plant diseases, which, if left unchecked, could devastate our multibillion dollar agricultural industry. In modern agriculture diseases are typically controlled mainly through the use of agrochemicals, which are expensive and environmentally damaging. We are currently investigating a group of natural plant-associated bacteria that are able to act as biocontrol organisms suppressing infections from a range of important fungal, bacterial, viral and insect pests. This well-funded project will apply a combination of next- generation transcriptomic and proteomic technologies, as well as innovative genome-wide transposon mutagenesis methods to identify the key genes and gene clusters involved in biocontrol mediated by Pseudomonas bacteria.

Factors influencing the success of the pathogen Acinetobacter baumannii The hospital intensive care unit should be a place of healing and care for the most vulnerable. Nonetheless, several microbial pathogens continue to plague this environment, causing serious infections in the immunocompromised patients that pose ever more challenging problems for clinicians. Acinetobacter baumannii has recently emerged as one of the most problematic hospital acquired pathogens worldwide due to its highly drug resistant nature. This project aims to define the key mechanisms of drug resistance operating in clinical A. baumannii isolates using a combination of cutting-edge next-generation transcriptomics and proteomics, and essential resistance genes identified by saturation mutagenesis methods. An alternative project in collaboration with A/Prof. Bridget Mabbutt uses structural and functional genomics to characterize laterally-acquired genes in A. baumannii that might help it flourish in clinical settings.

Molecular ecology of an ancient symbiosis between sponges and bacteria Marine sponges are crucial members of marine ecosystems that are often overlooked despite being a dominant and ubiquitous component of the sea bed. Research involving sponges is linked to various scientific aspects from environmental and evolutionary studies to biotechnological and medical applications, with anti-cancer drugs and anti-HIV products derived from sponges. A large proportion of species contains sponge-specific photosynthetic symbionts related to free-living cyanobacteria, which are abundant and key primary producers of marine environments. This projects aims to elucidate the molecular basis of the stable symbiosis of these two modern day "fossils", using a combination of traditional and next-generation genomics and transcriptomics.

Genomics and Ecology of Marine Cyanobacteria in Australian Waters Tiny single-celled marine cyanobacteria constitute up to two thirds of all marine productivity. As the base of the marine food-web, the activity of these organisms impacts on all marine life. Using a rapid molecular diagnostic we will perform the

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first survey of the environmental distribution of marine Synechococcus cyanobacteria along ecosystem gradients of the Australian Coast. Representative isolates will be selected for further study to identify key genes and proteins involved in adaptation to tropical and temperate habitats. This is a multidisciplinary project that will combine elements of fieldwork with the latest generation molecular techniques to understand the spatial and seasonal distribution of locally adapted ‘ecotypes’. Understanding the environmental factors that affect the abundance and activity of these organisms is fundamental to predicting the impacts of climate change on our local marine resources.

Coal: prokaryotic pioneers (with CSIRO) Using natural gas, rather than coal for electricity generation provides a means of reducing CO2 emissions and combating climate change. In coal seams where moisture and sufficient nutrition is available, natural gas is produced from microbial activity. To date, we have identified the types of microbes that inhabit coal, but have not identified those microbial pioneers whose metabolic degradation of the coal, not only underpins the microbial community but also facilitates the production of natural gas. Using culturing, sequencing and bioinformatics techniques and an established coal-degrading microbial consortia, the project aims to identify these early pioneers and how they degrade coal.

Life in a gluey sticky mess (with Packer, Super Science team) The thick mucus that forms in the lungs of cystic fibrosis (CF) patients provides a breeding ground for chronic bacterial infection. Pseudomonas aeruginosa, an opportunistic human pathogen is one of the main and most successful colonisers in CF lungs. As a part of the multidisciplinary ARC Super Science project, this study will be aimed at identifying specific adaptations developed by P.

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aeruginosa during chronic CF infections, including its ability to form biofilms. Whole genome transcriptomic analysis using RNA-Seq to sequence the entire bacterial transcriptome will be applied to identify genes important for adaptation and virulence in CF lung mucus. Selected Publications

1. Loper et al. (2012) Comparative genomics of plant-associated Pseudomonas spp: insights into diversity and inheritance of traits involved in multitrophic interactions. PLoS Genetics 8: e1002784.

2. Hassan et al. (2011) Roles of DHA2 Family Transporters in Drug Resistance and Iron Homeostasis in Acinetobacter spp. J Mol Microbiol Biotechnol. 20: 116-24.

3. Palenik et al. (2009) Coastal Synechococcus metagenome reveals major roles for horizontal gene transfer and plasmids in population diversity. Env. Micro.11: 349-59.

4. Myers et al. (2007) Genome sequence and identification of candidate vaccine antigens from the animal pathogen Dichelobacter nodosus. Nature Biotechnology 25:569-575.

5. Palenik et al. (2006) Genome sequence of Synechococcus CC9311: Insights into adaptation to a coastal environment. Proc. Natl. Acad. Sci., USA 103: 13555-13559.

http://www.chem.mq.edu.au/academics/ipaulsen.html

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Professor Shoba Ranganathan shoba.ranganathan@mq.edu.au F7B121 Ph 9850 6262

Bioinformatics and Computational Biosciences Our research area is Bioinformatics, which is the application of computational approaches to understand how biological systems function. Bioinformatics addresses key problems in biomolecular, biomedical and chemical sciences, using computational approaches. Our group focusses on comparative genome sequence analysis, computational structural biology and biodiversity analysis.

Alternative Splicing and Human Diseases Alternative pre-mRNA splicing is an important mechanism for controlling gene expression in higher eukaryotes. A single gene produces several functionally diverse proteins by alternative usage of exons or introns within pre-mRNA transcripts. These gene products can be specific to tissue, developmental stage, and disease state. We have pioneered the use of graph theory for genome-wide analysis of alternative splicing in the fruitfly, chicken compared to mouse and human (1) and more recently, the cow.

The major alternative splicing events involved in human diseases are shown below, in the splicing graph formalism:

1. exon skipping (cassette exon) and 2. intron retention.

Why are some exons skipped and some introns ignored? A detailed genome-wide analysis of the information content of the regions surrounding the splice sites for all “normal” exons and “disease-related” alternatively spliced exons could provide the answer.

Secretome Database of Helminth Parasites Excretory-secretory (ES) proteins are an important class of proteins in many organisms, spanning from bacteria to human beings, and are potential drug targets for several diseases. ES proteins consitute the secretome of any organism and are particularly relevant for parasitic organisms. Helminth parasites are responsible for a range of neglected tropical diseases, such as ancylostomatosis, necatoriasis, lymphatic filariasis, onchocerciasis, ascariasis and strongyloidiasis in humans and others can cause massive production or economic losses to farmers as well as to animal and plant industries.

Recent transcriptomic and proteomic analysis (2) has shown that parasites adopt non- classical pathways to generate ES products. To identify novel genes for parasite intervention, the secretome of helminth parasites needs to be compiled. This project is aimed at developing a searchable helminth parasite secretome database with experimentally identified ES proteins.

Mapping disease gene mutations to protein structure for genome -phenome correlations Mapping disease mutations to the structure of the protein can help in understanding the functional consequences of these mutations and thus indirectly, the finer aspects of the pathology and clinical manifestations of the disease, including phenotypic severity as a function of the genotype. Recently, we studied mutations in the gene (MAN2B1),

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Dr Anwar Sunna anwar.sunna@mq.edu.au Room E8C207, Ph 9850 4220

MOLECULAR BIOTECHNOLOGY The research interest in my laboratory is protein biochemistry and application of biotechnological relevant proteins. Currently we are focusing on immobilization of industrial enzymes and proteins to different inorganic solid matrices. The immobilization is mediated through affinity of synthetic peptides and selected through phage display screening. A second aspect of our research is development of cell-free biocatalytic modules for biotechnology and enzyme-based processes by incorporating the principles of in vitro synthetic biology.

Projects

New generation of protease resistant synthetic peptide linkers Most of the methods available for immobilizing proteins onto solid supports traditionally have relied on non-specific adsorption or on the reaction of naturally occurring chemical groups within proteins with appropriate reactive groups on the matrix. In both cases, the corresponding proteins are attached to the surface in a random orientation that may cause the reduction or loss of the protein’s biological activity. We have developed synthetic peptide linkers with high binding affinity to a large range of commercially available and inexpensive silica-containing materials. This project is aimed at producing a new generation of protease resistant peptides for immobilization of proteins in industrial–scale applications using combinatorial display technologies like phage-display and cell surface display. The industrial host organisms for this application is Trichoderma reesei, a filamentous fungus used extensively by industry for the production for autologous and heterologous proteins.

Fig. 1 Diagrammatic representation of enzyme recycling using peptide linker technology and solid matrix immobilization. Lower panel represents a real time assay performed at 60ºC.

Department of Chemistry and Biomolecular Sciences

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Development of cell-free biocatalytic modules The aim of this research project is to incorporate the principles of in vitro synthetic biology to design cell-free biocatalytic modules. This principle is based on the development of stable building blocks (individual immobilized enzymes) with defined functions and catalytic properties. Assembly of both natural and non-natural pathways for especial applications is achieved by combination of all pathway enzymes (basic building blocks) into complex biocatalytic modules. This “mix & match” approach combines several aspect of enzyme engineering and enzyme immobilization. The result is increasing engineering flexibility and decreasing dependence on cells and physiological constrains.

Fig. 2 Cell-free biocatalytic modules for biomanufacturing

Selected Publications

1. Lu, Zhao, Zhang, Liu, Liu, Goldys, Yang, Xi, Sunna, Lu, Shi, Leif, Huo, Shen, Piper, Robinson and Jin (2014). Tunable lifetime multiplexing using luminescent nanocrystals. Nature Photonics 8:32-36.

2. Care, Chi, Bergquist and Sunna (2014). Biofunctionalization of silica-coated magnetic particles mediated by a peptide linker. Journal of Nanoparticle Research 16:2543.

3. Care, Nevalainen, Bergquist and Sunna (2014). Effect of Trichoderma reesei proteinases on the affinity of an inorganic-binding peptide. Applied Biochemistry and Biotechnology 173:2225-2240.

4. Sunna, Chi and Bergquist. (2013). A linker peptide with high affinity towards silica-containing materials. New Biotechnology 30:485-492.

5. Sunna, Chi and Bergquist. (2013). Efficient capture of pathogens with a zeolite matrix. Parasitology Research 112:2441-2452.

6. Laczka, Skillman, Ditcham, Hamdorf, Wong, Bergquist and Sunna. (2013). Application of an ELISA-type screen printed electrode-based potentiometric assay to the detection of Cryptosporidium parvum oocysts. Journal of Microbiological Methods 95:182-185.

7. Sunna. (2010). Modular organisation and functional analysis of dissected modular β-mannanase CsMan26 from Caldicellulosiruptor Rt8B.4. Applied Microbiology and Biotechnology 86:189-200.

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Proteomics of cereals

The cereal research is associated with Grain Growers Pty Ltd looking at proteomics of barley and wheat grains. We are interested in determining the extent to which environmental and genetic variation determines protein composition in wheat, rice and barley. The protein profile between varieties grown at the same location and varieties grown at different locations is being determined. Initial trial scale analyses have found numerous differences between the varieties of barley grain grown at the same time and location. The reason for these variations may shed light on how cereals cope with environmental factors such as nutrient availability, drought, salinity and temperature as well as biological stresses. In addition many cereal proteins have distinct impacts on quality; taste and performance in baking and malting are key examples. Understanding these factors are very important for optimizing quality in the food industry.

Selected Publications 1. Schliep M., Crossett B., Willows R. D., Chen M. “18O-Labelling of chlorophyll d in

Acaryochloris marina reveal chlorophyll a and molecular oxygen are precursors” J. Biol. Chem. 2010, 285 (37), 28450-28456.

2. Chen, Min, Schliep, Martin, Willows, Robert D, Cai, Zheng-Li, Neilan, Brett A, Scheer, Hugo “A Red- Shifted Chlorophyll” Science, 2010, 329 (5997), 1318-1319.

3. Lundqvist J., Elmlund H., Peterson Wulff R., Berglund L., Elmlund D., Emanuelsson C., Hebert H., Willows R.D., Hansson M., Lindahl M. and Al-Karadaghi S. “ATP-induced conformational dynamics in the AAA+ motor unit of magnesium chelatase” Structure, 2010, 18, 354-365.

4. Meinecke L., Alawady A., Schroda M., Willows R. D., Kobayashi M.C., Niyogi K.K., Grimm B., and Beck C. F. “Chlorophyll-deficient mutants of Chlamydomonas reinhardtii that accumulate magnesium protoporphyrin IX” Plant Molecular Biology, 2010, 72 (6), 643-658.

5. Jerkovic A., Kriegel A. M., Bradner J. R., Atwell B. J., Roberts T. H., Willows R. D. “Strategic distribution of protective proteins within bran layers of wheat (Triticum aestivum L.) protects the nutrient-rich endosperm” Plant Physiol., 2010, 152, 1459-1470.

6. Zhou, S., Sawicki, A., Willows, R.D., Luo, M. (2012) C-terminal residues of Oryza sativa GUN4 are required for the activation of the ChlH subunit of magnesium chelatase in chlorophyll synthesis. FEBS Letters 586, 205-210

7. Li, L., Scales, N., Blankenship, R.E., Willows, R.D., Chen, M. (2012) Extinction coefficient for re-shifted chlorophylls: chlorophyll d and chlorophyll f. Biochimica et Biophysica Acta - Bioenergetics 1817 1292–1298

8. Chen M, Li Y., Birch D. & Willows R.D. (2012) A cyanobacterium that contains chlorophyll f - a red-absorbing photopigment. FEBS Letters, 586(19), 3249-3254

9. Robert D. Willows, Yaqiong Li , Hugo Scheer , and Min Chen (2013) Structure of Chlorophyll f Org. Lett., 2013, 15 (7), pp 1588–1590

10. Müller, A. H., Sawicki, A., Zhou, S., Tabrizi, S. T., Luo, M., Hansson, M., & Willows, R. D. (2014). Inducing the oxidative stress response in Escherichia coli improves the quality of a recombinant protein: Magnesium chelatase ChlH. Protein Expression and Purification. Protein Expression and Purification, 101(C), 61–67

Department of Chemistry and Biomolecular Sciences

Department of Chemistry and Biomolecular Sciences

Dr Danny Wong danny.wong@mq.edu.au F7B235 Ph 9850 8300

Biological, Environmental and Medical Analytical Chemistry Dr Wong and his research group are particularly interested in the development and applications of (i) electrochemical sensors and/or (ii) electroanalytical techniques to biological, environmental or medical analyses. The following projects offer an opportunity to students with interest in some of these areas. As such, almost all of our research projects are interdisciplinary and they couple electroanalytical chemistry to a diverse area including immunology, biochemistry, neuroscience, medical science, biotechnology, polymer chemistry, environmental science, method validation and quality assurance. Each of these projects will engage students in acquiring hands-on experience in a range of analytical techniques. There is also opportunity to tailor make a project of mutual interest as well. New Electrochemical Microsensor Design for Biological, Medical and Psychological Diagnostics Dr Wong’s electroanalytical chemistry laboratory is internationally recognised for designing structurally small electrochemical sensors. Indeed, increasing biological, medical and psychological diagnostics are relying on electroanalytical techniques because of their unique capability in performing real-time measurements. In Dr Wong’s laboratory, electrodes with small physical dimensions (≤1 µm in tip diameter) are routinely manufactured by pyrolysing hydrocarbon gases inside and outside pulled capillaries. The carbon produced is then deposited at the tip and on the shank of the capillaries. In recent years, we have perfected the technique to produce a large carbon surface area to obtain amplified detection signal. We are particularly interested in exploiting these electrodes in detecting neurotransmitters in mammalian brain systems. Such a study enables a better understanding of the central neural pathways that stimulate dopamine neurons to burst fire in various neural processes. In this project, we aim at employing sensors with physical dimensions down to 1 µm to detect the release of dopamine in targeted regions in the brain. Such a study will aid in identifying the chemical pathways involved in mediating dopamine neuron burst firing and forebrain dopamine release in the central nervous system. This project provides an excellent opportunity for students to gain research experience in both a chemistry laboratory and a medically oriented laboratory. A Versatile Molecular Architecture for an Electrochemical Immunosensor or an Electrochemical DNA Biosensor In this research area, we are keen to fabricate and characterise biological sensors based on the principles of immunology and DNA hybridisation. In the former, the interactions between an antibody and an antigen are known to be very specific chemical reactions. Such a specific molecular recognition of antigens by antibodies has been exploited in immunoassays to develop highly selective detection methods in many clinical analyses and medical diagnostics as well as for environmental monitoring. Similarly, in the latter, owing to specific recognitions, a DNA probe will only hybridise with its complementary DNA target. Many detection tools used in forensic identifications, medical diagnoses, drug discovery are based on DNA hybridisation. Electrochemical detection is particularly well suited for immunoassays and DNA hybridisation detection owing to its ease and sensitivity. Currently, a lot of work is being focused on the development of rapid, simple, sensitive, automated, and on-site electrochemical immunoassays. In this project, we are interested in fabricating a simple electrochemical immunosensor using a range of chemical and biological reagents. Compared to other methodologies used, our design has a distinct capability in aligning an antibody or a DNA probe in an optimum orientation for interaction with an antigen analyte or a DNA target, respectively. This is a significant factor in maximising the detection sensitivity of the immunosensor. We will also explore the application of nanoparticles or graphene to the development of an immunosensor or a DNA biosensor to further enhance their sensitivity.

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In immunosensor development, we will apply it to the detection of a real-life analyte (e.g. cortisol, tumour marker), while the DNA biosensor will be used to study the interaction between DNA and selected drugs of medical significance. Graduates with familiarity in analytical techniques are of demand in the current employment market. Note that a background in biology is not required but willingness to acquire new bioanalytical skills will be essential. Probing Environmental Chemistry using Electroanalytical Techniques Determination of copper speciation in natural waters or manganese speciation in drinking waters Copper is a highly toxic trace metal present at elevated concentration in many natural waters. The toxicity of copper is dependent on its physicochemical form (speciation) with inorganic species such as ionic copper being the most toxic. Copper speciation can be determined using electrochemical methods including different forms of stripping voltammetry. However, there have been surprisingly few direct comparisons of these methods on natural water samples and it is difficult to determine the best method for use in metal bioavailability studies. In this project, various electrochemical procedures for the determination of copper speciation will be set up and compared on a range of copper-contaminated natural water samples. The copper toxicity of these samples will also be assessed using a highly sensitive bacterial bioassay. The relationship between copper speciation and bacterial toxicity will be investigated. In this project, a speciation study of manganese using electrochemical methods will be conducted to address concerns about the taste of some drinking water samples collected from Canberra suspected to have arisen from the presence of manganese species in the water samples. Note these projects will be of interest to students with an interest in trace metal bioavailability / environmental chemistry. Determination of Cr(VI) in natural waters Chromium is a toxic trace element present at part per billion (ppb) concentrations in natural waters. The toxicity of chromium varies with its oxidation state. For example, Cr(VI) is known to be far more toxic than Cr(III). In order to effectively protect aquatic ecosystems from the effects of anthropogenically-derived chromium inputs, it is therefore necessary to not only determine total chromium concentrations, but also the oxidation states. The determination of Cr(VI) at low ppb concentrations is surprisingly challenging. Most methods require the preconcentration of Cr(VI) through coprecipitation with iron hydroxide before measurement by colorimetry or some form of atomic spectroscopy. In this project, we will examine the potential of several electrochemical methods for the determination of Cr(VI) at low ppb concentrations. Methods will include square wave voltammetry and cathodic stripping voltammetry. The developed method will be compared against existing non- electrochemical methods for Cr(VI) determination on a range of natural water samples. This project will be of interest to students with an interest in method development and the monitoring of toxic trace metals in natural waters. Electro-remediation of polluted textile effluents Azo dyes are commonly used in the textile and carpet dyeing industries. Very often, enormous quantities of dye containing wastewaters are being released into effluent streams. Such dyes are harmful to aquatic fauna and flora as well as humans. In this project, electrochemical removal and/or treatment of azo dyes in textile effluents will be explored. This will be achieved using the conducting polymer, polypyrrole, or its derivatives. A distinct advantage of this method is that dye molecules are entrapped in the polymer film for removal, rather than being chemically treated that generates even more harmful products as exhibited by many other treatment methods. Apart from electrochemistry, students will also engage in polymer chemistry, materials chemistry and environmental chemistry in this project. These projects will provide an opportunity for students to gain experience with a range of analytical techniques, as well as that in method validation and quality assurance. Graduates equipped with all these skills are always of demand in the current employment market.

www.cbms.mq.edu.au/academics/wong.html

Department of Chemistry and Biomolecular Sciences