Social and Behavioral Sciences Commons^™

A Novel Zinc-Binding Fold In The Helicase Interaction Domain Of The Bacillus Subtilis Dnal Helicase Loader, Karin V. Loscha, Kristaps Jaudzems, Charikleia Ioannou, Xun-Cheng Su, Flynn R. Hill, Gottfried Otting, Nicholas E. Dixon, Edvards Liepinsh

Faculty of Science - Papers (Archive)

The helicase loader protein DnaI (the Bacillus subtilis homologue of Escherichia coli DnaC) is required to load the hexameric helicase DnaC (the B. subtilis homologue of E. coli DnaB) onto DNA at the start of replication. While the C-terminal domain of DnaI belongs to the structurally well-characterized AAA+ family of ATPases, the structure of the N-terminal domain, DnaI-N, has no homology to a known structure. Three-dimensional structure determination by nuclear magnetic resonance (NMR) spectroscopy shows that DnaI presents a novel fold containing a structurally important zinc ion. Surface plasmon resonance experiments indicate that DnaI-N is largely responsible for binding of …

Real-Time Single-Molecule Observation Of Rolling-Circle Dna Replication, Nathan A. Tanner, Joseph J. Loparo, Samir M. Hamdan, Slobodan Jergic, Nicholas E. Dixon, Antoine M. Van Oijen

Faculty of Science - Papers (Archive)

We present a simple technique for visualizing replication of individual DNA molecules in real time. By attaching a rolling-circle substrate to a TIRF microscope-mounted flow chamber, we are able to monitor the progression of single-DNA synthesis events and accurately measure rates and processivities of single T7 and Escherichia coli replisomes as they replicate DNA. This method allows for rapid and precise characterization of the kinetics of DNA synthesis and the effects of replication inhibitors.

The Major Toxin From The Australian Common Brown Snake Is A Hexamer With Unusual Gas-Phase Dissociation Properties, Andrew Aquilina

Faculty of Science - Papers (Archive)

Asymmetric dissociation of multiply charged proteins assemblies has been frequently reported. This phenomenon, which relies on the dissociation of one or more highly charged monomers, has been shown to provide insights into the structure and organization of large monodisperse and polydisperse assemblies. Here, the process of asymmetric dissociation is investigated using the multi-subunit protein, textilotoxin, which has unusually high structural constraints on its monomers due to multiple disulfide linkages. Initially, it is shown that, contrary to previous reports, textilotoxin is made up of 6, rather than 5 subunits. Furthermore, the hexamer exists as two isoforms, one of which is substantially …

Synthesis Of Novel N-Protected Hydrophobic Phenylalanines And Their Application In Potential Antibacterials, Timothy P. Boyle, J. B. Bremner, Jonathan A. Coates, John Deadman, Paul A. Keller, Stephen G. Pyne, Kittiya Somphol

Faculty of Science - Papers (Archive)

An efficient synthesis of two new N-acetyl-4’-arylphenylalanines is described together with their incorporation in to a number of cationic peptoid antibacterial agents, one of which had an MIC of 7.8 μg/mL against Staphylococcus aureus.

The Synthesis Of Carbon Linked Bis-Benzylisoquinolines Using Mizoroki–Heck And Sonagashira Coupling Reactions, Uta Batenburg-Nguyen, Alison T. Ung, Stephen G. Pyne

Faculty of Science - Papers (Archive)

Novel laudanosine dimers in which two laudanosine units are linked at C-2′ via a two or three-carbon linker (alkane, alkene or alkyne) have been prepared using palladium-catalysed cross-coupling reactions (Mizoroki–Heck and Sonagashira reactions). In one example, a second three-carbon linker between the two isoquinoline N-atoms was also present leading to a novel macrocyclic ring system.

Intermolecular Addition Reactions Of N-Acyliminium Ions (Part I), Arife Yazici, Stephen G. Pyne

Faculty of Science - Papers (Archive)

This review highlights the advances in the literature up to July 2008 on the intermolecular reactions of acyclic and cyclic N-acyliminium ions. This is an update of an earlier review in 2000 on this topic and does not include intramolecular addition reactions to N-acyliminium ions which was recently reviewed. This review is presented in two parts, with the first part dealing with acyclic and pyrrolidinone-based N-acyliminium ions. Part II continues with other five-membered heterocyclic derivatives and higher systems.

Apolipoprotein-E Forms Dimers In Human Frontal Cortex And Hippocampus, David A. Elliott, Glenda M. Halliday, Brett Garner

Faculty of Science - Papers (Archive)

Background: Apolipoprotein-E (apoE) plays important roles in neurobiology and the apoE4 isoform increases risk for Alzheimer’s disease (AD). ApoE3 and apoE2 are known to form disulphide-linked dimers in plasma and cerebrospinal fluid whereas apoE4 cannot form these dimers as it lacks a cysteine residue. Previous in vitro research indicates dimerisation of apoE3 has a significant impact on its functions related to cholesterol homeostasis and amyloid-beta peptide degradation. The possible occurrence of apoE dimers in cortical tissues has not been examined and was therefore assessed. Human frontal cortex and hippocampus from control and AD post-mortem samples were homogenised and analysed …

From Powder To Solution: Hydration Dependence Of Human Hemoglobin Dynamics Correlated To Body Temperature, A. M. Stadler, I. Digel, J. P. Embs, T. Unruh, Moeava Tehei, G. Zaccai, G. Büldt, G. M. Artmann

Faculty of Science - Papers (Archive)

A transition in hemoglobin (Hb), involving partial unfolding and aggregation, has been shown previously by various biophysical methods. The correlation between the transition temperature and body temperature for Hb from different species, suggested that it might be significant for biological function. In order to focus on such biologically relevant human Hb dynamics, we studied the protein internal picosecond motions as a response to hydration, by elastic and quasielastic neutron scattering. Rates of fast diffusive motions were found to be significantly enhanced with increasing hydration from fully hydrated powder to concentrated Hb solution. In concentrated protein solution, the data revealed that …

Synthesis And Applications Of Covalent Protein-Dna Conjugates, Patrick M. Schaeffer, Nicholas E. Dixon

Faculty of Science - Papers (Archive)

Synthetic protein-DNA conjugates are valuable tools with applications in fields including nanobiotechnology, bioanalytical chemistry, and molecular diagnostics, and various synthetic methods for their production have been developed during the past three decades. The present article reviews current methodologies for the synthesis of covalent protein-DNA conjugates with particular focus on the regiospecificity and stoichiometry of these reactions.

Solid-State And Solution-Phase Conformations Of Pseudoproline-Containing Dipeptides, James R. Cochrane, Nima Sayyadi, Danielle Skropeta, Peter Turner, Jack K. Clegg, Katrina A. Jolliffe

Faculty of Science - Papers (Archive)

The conformations of 14 threonine-derived pseudoproline-containing dipeptides (including four d-allo-Thr derivatives) have been investigated by NMR. In solution, the major conformer observed for all dipeptides is that in which the amide bond between the pseudoproline and the preceding amino acid is cis. For dipeptides in which the N-terminus is protected, the ratio of cis- to trans-conformers does not depend significantly on the side chain of the N-terminal amino acid, or the stereochemistry of the Thr residue. However, for dipeptides bearing a free N-terminus, there are significant differences in the ratios of cis- to trans-conformers depending on the side chain present. …

Thermal Attributes Of Chrysomya Species, James F. Wallman, Leigh Nelson, Mark P. Dowton

Faculty of Science - Papers (Archive)

The correct identification of forensically important arthropods for post-mortem interval estimation is crucial, as the rate of larval development can vary substantially between species. The identification of forensically important blowflies of the genus Chrysomya (Diptera: Calliphoridae) may be hampered by their close morphological similarities, especially as immatures. The aim of this study was to establish whether genetically closely related blowfly species would share similar developmental profiles. This could permit the application of developmental data to a number of closely related species, including those for which thermodevelopmental studies are lacking. If Australian Chrysomya were found to share developmental profiles, identification of …

Identification Of Abundant Alkyl Ether Glycerophospholipids In The Human Lens By Tandem Mass Spectrometry Techniques, Roger J. Truscott, Jane Deeley, Todd W. Mitchell, Michael Thomas, Stephen J. Blanksby

Faculty of Science - Papers (Archive)

Previous studies have shown that the human lens contains glycerophospholipids with ether linkages. These lipids differ from conventional glycerophospholipids in that the sn-1 substituent is attached to the glycerol backbone via an 1-O-alkyl or an 1-O-alk-1'-enyl ether rather than an ester bond. The present investigation employed a combination of collision-induced dissociation (CID) and ozone-induced dissociation (OzID) to unambiguously distinguish such 1-O-alkyl and 1-O-alk-1'-enyl ethers. Using these methodologies the human lens was found to contain several abundant 1-O-alkyl glycerophos-phoethanolamines, including GPEtn(16:0e/9Z-18:1), GPEtn(11Z-18:1e/9Z-18:1), and GPEtn(18:0e/9Z-18:1), as well as a related series of unusual 1-O-alkyl glycerophosphoserines, including GPSer(16:0e/9Z-18:1), GPSer(11Z-18:1e/9Z-18:1), GPSer(18:0e/9Z-18:1) that to our …

Defining The Structural Basis Of Human Plasminogen Binding By Streptococcal Surface Enolase, Amanda J. Cork, Slobodan Jergic, Sven Hammerschmidt, Bostjan Kobe, Vijay Pancholi, Justin L.P. Benesch, Carol V, Robinson, Nicholas E. Dixon, J Andrew Aquilina, Mark J. Walker

Faculty of Science - Papers (Archive)

The flesh-eating bacterium group A Streptococcus (GAS) binds and activates human plasminogen, promoting invasive disease. Streptococcal surface enolase (SEN), a glycolytic pathway enzyme, is an identified plasminogen receptor of GAS. Here we used mass spectrometry (MS) to confirm that GAS SEN is octameric, thereby validating in silico modeling based on the crystal structure of S. pneumoniae -enolase. Site-directed mutagenesis of surface-located lysine residues (SENK252+255A, SENK304A, SENK334A, SENK344E, SENK435L and SEN434-435) was used to examine their roles in maintaining structural integrity, enzymatic function and plasminogen binding. Structural integrity of the GAS SEN octamer was retained for all mutants except SENK344E, as …

On The Utilization Of Energy Minimization To The Study Of Ion Selectivity, Haibo Yu, Benoit Roux

Faculty of Science - Papers (Archive)

The major pitfalls in studying ion selectivity in binding site models using energy minimization based methods are examined and discussed. It is shown that the properties derived from energy minimization are strongly configuration-dependent and that the results should be interpreted with caution. It is concluded that computational studies of ion selectivity must include thermal fluctuations and entropic effects.

Crystallin Proteins And Amyloid Fibrils, Heath Ecroyd, John A. Carver

Faculty of Science - Papers (Archive)

Improper protein folding (misfolding) can lead to the formation of disordered (amorphous) or ordered (amyloid fibril) aggregates. The major lens protein, alpha-crystallin, is a member of the small heat-shock protein (sHsp) family of intracellular molecular chaperone proteins that prevent protein aggregation. Whilst the chaperone activity of sHsps against amorphously aggregating proteins has been well studied, its action against fibril-forming proteins has received less attention despite the presence of sHsps in deposits found in fibril-associated diseases (e.g. Alzheimer's and Parkinson's). In this review, the literature on the interaction of alpha B-crystallin and other sHsps with fibril-forming proteins is summarized. In particular, …

(-)-Epigallocatechin-3-Gallate (Egcg) Maintains K-Casein In Its Pre-Fibrillar State Without Redirecting Its Aggregation Pathway, Sean A. Hudson, Heath Ecroyd, Francis C. Dehle, Ian F. Musgrave, John Carver

Faculty of Science - Papers (Archive)

The polyphenol (-)-epigallocatechin-3-gallate (EGCG) has recently attracted much research interest in the field of protein-misfolding diseases because of its potent anti-amyloid activity against amyloid-beta, alpha-synuclein and huntingtin, the amyloid-fibril-forming proteins involved in Alzheimer's, Parkinson's and Huntington's diseases, respectively. EGCG redirects the aggregation of these polypeptides to a disordered off-folding pathway that results in the formation of non-toxic amorphous aggregates. whether this anti-fibril activity is specific to these disease-related target proteins or ismore generic remains to be established. In addition, the mechanism by which EGCG exerts its effects, as with all anti-amyloidogenic polyphenols, remains unclear. To address these aspects, we have …

The Thioflavin T Fluorescence Assay For Amyloid Fibril Detection Can Be Biased By The Presence Of Exogenous Compounds, Sean A. Hudson, Heath Ecroyd, Tak W. Kee, John A. Carver

Faculty of Science - Papers (Archive)

Thioflavin T (ThT) dye fluorescence is used regularly to quantify the formation and inhibition of amyloid fibrils in the presence of anti-amyloidogenic compounds such as polyphenols. However, in this study, it was shown, using three polyphenolics (curcumin, quercetin and resveratrol), that ThT fluorescence should be used with caution in the presence of such exogenous compounds. The strong absorptive and fluorescent properties of quercetin and curcumin were found to significantly bias the ThT fluorescence readings in both in situ real-time ThT assays and single time-point dilution ThT-type assays. The presence of curcumin at concentrations as low as 0.01 and 1 uM …

Structural Characterization Of Clusterin-Chaperone Client Protein Complexes, Amy R. Wyatt, Justin J. Yerbury, Mark R. Wilson

Faculty of Science - Papers (Archive)

Clusterin (CLU) is a potent extracellular chaperone that inhibits protein aggregation and precipitation otherwise caused by physical or chemical stresses (e.g. heat, reduction). This action involves CLU forming soluble high molecular weight (HMW) complexes with the client protein. Other than their unquantified large size, the physical characteristics of these complexes were previously unknown. In this study, HMW CLU-citrate synthase (CS), HMW CLU-fibrinogen (FGN), and HMW CLU-glutathione S-transferase (GST) complexes were generated in vitro, and their structures studied using size exclusion chromatography (SEC), ELISA, SDS-PAGE, dynamic light scattering (DLS), bisANS fluorescence, and circular dichroism spectrophotometry (CD). Densitometry of …

Alkaloids From The Roots Of Stemona Aphylla, Pitchaya Mungkornasawakul, Sukanda Chaiyong, Thanapat Sastraruji, Araya Jatisatienr, Chaiwat Jatisatienr, Stephen G. Pyne, Alison T. Ung, Jurgen Korth, Wilford Lie

Faculty of Science - Papers (Archive)

Three known compounds, stemofoline, (2'S)-hydroxystemofoline, and (11Z)-1 ',2'-didehydrostemofoline, along with two new alkaloids, stemaphylline and stemaphylline-N-oxide, have been isolated from a root extract of Stemona aphylla. The structures of these alkaloids were determined on the basis of their spectroscopic data. The analysis of the crude dichloromethane extract by GC-MS in the ElMS mode showed the presence of alkaloids 1-4, the alkaloid 11, and stilbostemin R. The crude dichloromethane extract and 4 were tested for their comparative biological activities. The results of their acetylcholinesterase (AChE) inhibitory activities showed that the crude extract had higher activity than that of 4. The insecticidal …

Structural Revision Of Stemoburkilline From An E-Alkene To A Z-Alkene, Kwankamol Sastraruji, Stephen G. Pyne, Alison T. Ung, Pitchaya Mungkornawawakul, Wilford Lie, Araya Jatisatienr

Faculty of Science - Papers (Archive)

Semisynthesis studies starting from (11Z)-I',2'-didehydrostemofoline indicated that the known Stemona alkaloid stemoburkilline is the Z-isomer and not the E-isomer as initially reported. The semisynthesis involved conversion of (11Z)-1',2' -didehydrostemofoline to II(S), 12(S)-dihydrostemofoline followed by a stereoselective base-catalyzed ring-opening reaction to give (Z)-stemoburkilline. The same product was obtained using a similar synthetic protocol stariing from isostemofoline via a based-catalyzed ring-opening reaction of II (S), 12(R)-dihydrostemofoline. A re-examination of the crude root extracts of Stemona burkillii Prain and further NOE studies established stemoburkilline as the Z-isomer.

The Cd-Loop Of Pai-2 (Serpinb2) Is Redundant In The Targeting, Inhibition And Clearance Of Cell Surface Upa Activity, Blake J. Cochran, Lakshitha P. Gunawardhana, Kara L. Perrow, Jodi A. Lee, Sergei A. Lobov, Marie Ranson

Faculty of Science - Papers (Archive)

Background: Plasminogen activator inhibitor type-2 (PAI-2, SERPINB2) is an irreversible, specific inhibitor of the urokinase plasminogen activator (uPA). Since overexpression of uPA at the surface of cancer cells is linked to malignancy, targeting of uPA by exogenous recombinant PAI-2 has been proposed as the basis of potential cancer therapies. To this end, reproducible yields of high purity protein that maintains this targeting ability is required. Herein we validate the use in vitro of recombinant 6 × His-tagged-PAI-2 lacking the intrahelical loop between C and D alpha-helices (PAI- 2 ΔCD-loop) for these purposes.

Results: We show that PAI-2 ΔCD-loop …

Testicular Descent, Sperm Maturation And Capacitation. Lessons From Our Most Distant Relatives, The Monotremes, Russell C. Jones, Heath Ecroyd, Jean-Louis Dacheux, Brett Nixon

Faculty of Science - Papers (Archive)

The present review examines whether monotremes may help to resolve three questions relating to sperm production in mammals: why the testes descend into a scrotum in most mammals, why spermatozoa are infertile when they leave the testes and require a period of maturation in the specific milieu provided by the epididymides, and why ejaculated spermatozoa cannot immediately fertilise an ovum until they undergo capacitation within the female reproductive tract. Comparisons of monotremes with other mammals indicate that there is a need for considerable work on monotremes. It is hypothesised that testicular descent should be related to epididymal differentiation. Spermatozoa and …

Model For Amorphous Aggregation Processes, Samual Stranks, Heath Ecroyd, Steve Van Sluyter, Elizabeth J. Waters, John A. Carver, Lorenz Von Smekal

Faculty of Science - Papers (Archive)

The amorphous aggregation of proteins is associated with many phenomena, ranging from the formation of protein wine haze to the development of cataract in the eye lens and the precipitation of recombinant proteins during their expression and purification. While much literature exists describing models for linear protein aggregation, such as amyloid fibril formation, there are few reports of models which address amorphous aggregation. Here, we propose a model to describe the amorphous aggregation of proteins which is also more widely applicable to other situations where a similar process occurs, such as in the formation of colloids and nanoclusters. As first …

Online Ozonolysis Methods For The Determination Of Double Bond Position In Unsaturated Lipids, Michael Thomas, Todd W. Mitchell, Stephen J. Blanksby

Faculty of Science - Papers (Archive)

Modern lipidomics relies heavily on mass spectrometry for the structural characterization and quantification of lipids of biological origins. Structural information is gained by tandem mass spectrometry (MS/MS) whereby lipid ions are fragmented to elucidate lipid class, fatty acid chain length, and degree of unsaturation. Unfortunately, however, in most cases double bond position cannot be assigned based on MS/MS data alone and thus significant structural diversity is hidden from such analyses. For this reason, we have developed two online methods for determining double bond position within unsaturated lipids; ozone electrospray ionization mass spectrometry (OzESI-MS) and ozone-induced dissociation (OzID). Both techniques utilize …

Development And Application Of A System For The Analysis Of Atmospheric, Water And Sediment Nitrogen And Carbon, Ann Stavert, Stephen R. Wilson, Dianne F. Jolley

Faculty of Science - Papers (Archive)

Eutrophication and climate change, key environmental concerns, are both linked to the carbon and nitrogen cycles hence the improved understanding of these cycles is essential. Currently, there is no system that simultaneously measures the fluxes of the three key gas phase products of nitrogen and carbon cycling (CO2, CH4 and N2O) in submerged ecosystems with hourly time resolution. A “Lake-in-a-box” (mesocosm) was developed in the laboratory which allowed the monitoring of key components of the carbon and nitrogen cycles within the air, water and sediments. The approach is automated, simple and time efficient and novel in its ability to examine …

Macroglobulin And Haptoglobin Suppress Amyloid Formation By Interacting With Prefibrillar Protein Species, Justin J. Yerbury, Janet R. Kumita, Sarah Meehan, Christopher M. Dobson, Mark R. Wilson

Faculty of Science - Papers (Archive)

α₂-Macroglobulin (α₂M) and haptoglobin (Hp) are both abundant secreted glycoproteins that are best known for their protease trapping and hemoglobin binding activities, respectively. Like the small heat shock proteins, both these glycoproteins have in common the ability to protect a range of proteins from stress-induced amorphous aggregation and have been described as extracellular chaperones. Using an array of biophysical techniques, this study establishes that in vitro at substoichiometric levels and under physiological conditions α₂M and Hp both inhibit the formation of amyloid fibrils from a range of proteins. We also provide evidence that both …

Therapeutic Targets In Extracellular Protein Deposition Diseases, Amy R. Wyatt, Justin J. Yerbury, Stephen Poon, Mark R. Wilson

Faculty of Science - Papers (Archive)

Many litres of fluids are found outside cells in the human body. These fluids are rich in dissolved proteins that each have a characteristic three dimensional shape, necessary for normal function, which has been attained by the correct folding of their polypeptide chain(s). The structure of these extracellular proteins can be damaged by a variety of environmental stresses (e. g. heat and oxidation) leading to their partial unfolding and aggregation. This in turn can produce toxic soluble aggregates and/or large insoluble protein deposits, either of which can disrupt normal body function (e. g. in Alzheimer's disease and the systemic amyloidoses). …

Stemofoline Ethyl Acetate Solvate, Pitchaya Mungkornasawakul, Stephen G. Pyne, Alison T. Ung, Araya Jatisatienr, Anthony C. Willis

Faculty of Science - Papers (Archive)

Crystals of the title compound, C2zH29NOs,C4HsOz, ([ systematic name: (2R,3R,5R,5aS,6R,8aR,9S)-(5Z)-5-[3-butyltetrah ydro-6-me thyl- 2,5-me thano-4,3 ,8a -[1] propan yl[3]ylidenefuro[ 3,2-j] [1,4]oxazepin-7( 5H)-ylidene ]-4-methoxy-3- methylfuran-2(5H)-one ethyl acetate solvate) were isolated from the root extracts of Stemona aphylla (Stemonaceae). The structure closely resembles those of stemofoline derivatives which have previously been reported. Intermolecular contacts are observed between some C-bonded H atoms and nearby 0 atoms, perhaps indicating weak interactions which could influence the packing of species within the unit cell.

Intermolecular Addition Reactions Of N-Acyliminium Ions (Part Ii), Arife Yazici, Stephen G. Pyne

Faculty of Science - Papers (Archive)

This review highlights the advances in the literature up to July 2008 on the intermolecular reactions of acyclic and cyclic N-acyliminium ions. This is an update of an earlier review in 2000 oil this topic and does not include intramolecular addition reactions to N-acyliminium ions which was recently reviewed. This review is presented in two parts, with the first part having dealt with acyclic and pyrrolidinone-based N-acyliminium ions. Part II continues with other five-membered heterocyclic derivatives and higher systems.