Can supervise: YES
Hossain, KR, Turkewitz, DR, Holt, SA, Herson, L, Brown, LJ, Cornell, BA, Curmi, PMG & Valenzuela, SM 2019, 'A conserved GXXXG motif in the transmembrane domain of CLIC proteins is essential for their cholesterol-dependant membrane interaction.', Biochimica et biophysica acta. General subjects, vol. 1863, no. 8, pp. 1243-1253.View/Download from: Publisher's site
BACKGROUND:Sterols have been reported to modulate conformation and hence the function of several membrane proteins. One such group is the Chloride Intracellular Ion Channel (CLIC) family of proteins. The CLIC protein family consists of six evolutionarily conserved protein members in vertebrates. These proteins exist as both monomeric soluble proteins and as membrane bound proteins. To date, the structure of their membrane-bound form remains unknown. In addition to several studies indicating cellular redox environment and pH as facilitators of CLIC1 insertion into membranes, we have also demonstrated that the spontaneous membrane insertion of CLIC1 is regulated by membrane cholesterol. METHOD:We have performed Langmuir-film, Impedance Spectroscopy and Molecular Docking Simulations to study the role of this GXXXG motif in CLIC1 interaction with cholesterol. RESULTS:Unlike CLIC1-wild-type protein, the G18A and G22A mutants, that form part of the GXXXG motif, showed much slower initial kinetics and lower ion channel activity compared to the native protein. This difference can be attributed to the significantly reduced membrane interaction and insertion rate of the mutant proteins and/or slower formation of the final membrane configuration of the mutant proteins once in the membrane. CONCLUSION:In this study, our findings uncover the identification of a GXXXG motif in CLIC1, which likely serves as the cholesterol-binding domain, that facilitates the protein's membrane interaction and insertion. Furthermore, we were able to postulate a model by which CLIC1 can autonomously insert into membranes to form functional ion channels. GENERAL SIGNIFICANCE:Members of the CLIC family of proteins demonstrate unusual structural and dual functional properties - as ion channels and enzymes. Elucidating how the CLIC proteins' interact with membranes, thus allowing them to switch between their soluble and membrane form, will provide key information as to a mechanism of moonlighting ac...
Berry, T, Dutta, D, Chen, R, Leong, A, Wang, H, Donald, WA, Parviz, M, Cornell, B, Willcox, M, Kumar, N & Cranfield, CG 2018, 'Lipid Membrane Interactions of the Cationic Antimicrobial Peptide Chimeras Melimine and Cys-Melimine.', Langmuir : the ACS journal of surfaces and colloids, vol. 34, no. 38, pp. 11586-11592.View/Download from: Publisher's site
Melimine and its derivatives are synthetic chimeric antimicrobial agents based on protamine and melittin. The binding of solubilized melimine and its derivative, with a cysteine on N-terminus, (cys-melimine) on tethered bilayer lipid membranes (tBLMs) was examined using ac electrical impedance spectroscopy. The addition of melimine and cys-melimine initially increased membrane conduction, which subsequently falls over time. The results were obtained for tBLMs comprising zwitterionic phosphatidylcholine, anionic phosphatidylglycerol, or tBLMs made using purified lipids from Escherichia coli. The effect on conduction is more marked with the cysteine variant than the noncysteine variant. The variation in membrane conduction most probably arises from individual melimines inducing increased ionic permeability, which is then reduced as the melimines aggregate and phase-separate within the membrane. The actions of these antimicrobials are modeled in terms of altering the critical packing parameter (CPP) of the membranes. The variations in the peptide length of cys-melimine were compared with a truncated version of the peptide, cys-mel4. The results suggest that the smaller molecule impacts the membrane by a mechanism that increases the average CPP, reducing membrane conduction. Alternatively, an uncharged alanine-replacement version of melimine still produced an increase in membrane conduction, further supporting the CPP model of geometry-induced toroidal pore alterations. All the data were then compared to their antimicrobial effectiveness for the Gram-positive and Gram-negative strains of bacteria, and their fusogenic properties were examined using dynamic light scattering in 1-oleoyl-2-hydroxy- sn-glycero-3-phosphocholine lipid spheroids. We conclude that a degree of correlation exists between the antimicrobial effectiveness of the peptides studied here and their modulation of membrane conductivity.
Kuppusamy, R, Yasir, M, Berry, T, Cranfield, CG, Nizalapur, S, Yee, E, Kimyon, O, Taunk, A, Ho, KKK, Cornell, B, Manefield, M, Willcox, M, Black, DS & Kumar, N 2018, 'Design and synthesis of short amphiphilic cationic peptidomimetics based on biphenyl backbone as antibacterial agents.', European journal of medicinal chemistry, vol. 143, pp. 1702-1722.View/Download from: Publisher's site
Antimicrobial peptides (AMPs) and their synthetic mimics have received recent interest as new alternatives to traditional antibiotics in attempts to overcome the rise of antibiotic resistance in many microbes. AMPs are part of the natural defenses of most living organisms and they also have a unique mechanism of action against bacteria. Herein, a new series of short amphiphilic cationic peptidomimetics were synthesized by incorporating the 3'-amino-[1,1'-biphenyl]-3-carboxylic acid backbone to mimic the essential properties of natural AMPs. By altering hydrophobicity and charge, we identified the most potent analogue 25g that was active against both Gram-positive Staphylococcus aureus (MIC = 15.6 μM) and Gram-negative Escherichia coli (MIC = 7.8 μM) bacteria. Cytoplasmic permeability assay results revealed that 25g acts primarily by depolarization of lipids in cytoplasmic membranes. The active compounds were also investigated for their cytotoxicity to human cells, lysis of lipid bilayers using tethered bilayer lipid membranes (tBLMs) and their activity against established biofilms of S. aureus and E. coli.
Deplazes, E, Poger, D, Cornell, B & Cranfield, CG 2018, 'The effect of H3O+ on the membrane morphology and hydrogen bonding of a phospholipid bilayer', Biophysical Reviews, vol. 10, no. 5, pp. 1371-1376.View/Download from: Publisher's site
© 2018, International Union for Pure and Applied Biophysics (IUPAB) and Springer-Verlag GmbH Germany, part of Springer Nature. At the 2017 meeting of the Australian Society for Biophysics, we presented the combined results from two recent studies showing how hydronium ions (H3O+) modulate the structure and ion permeability of phospholipid bilayers. In the first study, the impact of H3O+ on lipid packing had been identified using tethered bilayer lipid membranes in conjunction with electrical impedance spectroscopy and neutron reflectometry. The increased presence of H3O+ (i.e. lower pH) led to a significant reduction in membrane conductivity and increased membrane thickness. A first-order explanation for the effect was assigned to alterations in the steric packing of the membrane lipids. Changes in packing were described by a critical packing parameter (CPP) related to the interfacial area and volume and shape of the membrane lipids. We proposed that increasing the concentraton of H3O+ resulted in stronger hydrogen bonding between the phosphate oxygens at the water–lipid interface leading to a reduced area per lipid and slightly increased membrane thickness. At the meeting, a molecular model for these pH effects based on the result of our second study was presented. Multiple μs-long, unrestrained molecular dynamic (MD) simulations of a phosphatidylcholine lipid bilayer were carried out and showed a concentration dependent reduction in the area per lipid and an increase in bilayer thickness, in agreement with experimental data. Further, H3O+ preferentially accumulated at the water–lipid interface, suggesting the localised pH at the membrane surface is much lower than the bulk bathing solution. Another significant finding was that the hydrogen bonds formed by H3O+ ions with lipid headgroup oxygens are, on average, shorter in length and longer-lived than the ones formed in bulk water. In addition, the H3O+ ions resided for longer periods in association with the carb...
Deplazes, E, Poger, D, Cornell, B & Cranfield, CG 2018, 'The effect of hydronium ions on the structure of phospholipid membranes.', Physical Chemistry Chemical Physics, vol. 20, no. 1, pp. 357-366.View/Download from: Publisher's site
This work seeks to identify the mechanisms by which hydronium ions (H3O+) modulate the structure of phospholipid bilayers by studying the interactions of H3O+ with phospholipids at the molecular level. For this, we carried out multiple microsecond-long unrestrained molecular dynamics (MD) simulations of a POPC bilayer at different H3O+ concentrations. The results show that H3O+ accumulates at the membrane surface where it displaces water and forms strong and long-lived hydrogen bonds with the phosphate and carbonyl oxygens in phospholipids. This results in a concentration-dependent reduction of the area per lipid and an increase in bilayer thickness. This study provides an important molecular-level insight into the mechanism of how H3O+ modulates the structure of biological membranes and is a critical step towards a better understanding of the effect of low pH on mammalian and bacterial membranes.
Cranfield, CG, Henriques, ST, Martinac, B, Duckworth, P, Craik, DJ & Cornell, B 2017, 'Kalata B1 and Kalata B2 Have a Surfactant-Like Activity in Phosphatidylethanolomine-Containing Lipid Membranes.', Langmuir: the ACS journal of surfaces and colloids, vol. 33, no. 26, pp. 6630-6637.View/Download from: Publisher's site
Cyclotides are cyclic disulfide-rich peptides that are chemically and thermally stable and possess pharmaceutical and insecticidal properties. The activities reported for cyclotides correlate with their ability to target phosphatidylethanolamine (PE)-phospholipids and disrupt cell membranes. However, the mechanism by which this disruption occurs remains unclear. In the current study we examine the effect of the prototypic cyclotides, kalata B1 (kB1) and kalata B2 (kB2), on tethered lipid bilayer membranes (tBLMs) using swept frequency electrical impedance spectroscopy. We confirmed that kB1 and kB2 bind to bilayers only if they contain PE-phospholipids. We hypothesize that the increase in membrane conduction and capacitance observed upon addition of kB1 or kB2 is unlikely to result from ion channel like pores but is consistent with the formation of lipidic toroidal pores. This hypothesis is supported by the concentration dependence of effects of kB1 and kB2 being suggestive of a critical micelle concentration event rather than a progressive increase in conduction arising from increased channel insertion. Additionally, conduction behavior is readily reversible when the peptide is rinsed from the bilayer. Our results support a mechanism by which kB1 and kB2 bind to and disrupt PE-containing membranes by decreasing the overall membrane critical packing parameter, as would a surfactant, which then opens or increases the size of existing membrane defects. The cyclotides need not participate directly in the conductive pore but might exert their effect indirectly through altering membrane packing constraints and inducing purely lipidic conductive pores.
Hoiles, W, Gupta, R, Cornell, B, Cranfield, C & Krishnamurthy, V 2016, 'The Effect of Tethers on Artificial Cell Membranes: A Coarse-Grained Molecular Dynamics Study', PLOS ONE, vol. 11, no. 10.View/Download from: Publisher's site
Al Khamici, H, Hossain, KR, Cornell, BA & Valenzuela, SM 2016, 'Investigating Sterol and Redox Regulation of the Ion Channel Activity of CLIC1 Using Tethered Bilayer Membranes.', Membranes, vol. 6, no. 4, pp. 1-13.View/Download from: Publisher's site
The Chloride Intracellular Ion Channel (CLIC) family consists of six conserved proteins in humans. These are a group of enigmatic proteins, which adopt both a soluble and membrane bound form. CLIC1 was found to be a metamorphic protein, where under specific environmental triggers it adopts more than one stable reversible soluble structural conformation. CLIC1 was found to spontaneously insert into cell membranes and form chloride ion channels. However, factors that control the structural transition of CLIC1 from being an aqueous soluble protein into a membrane bound protein have yet to be adequately described. Using tethered bilayer lipid membranes and electrical impedance spectroscopy system, herein we demonstrate that CLIC1 ion channel activity is dependent on the type and concentration of sterols in bilayer membranes. These findings suggest that membrane sterols play an essential role in CLIC1's acrobatic switching from a globular soluble form to an integral membrane form, promoting greater ion channel conductance in membranes. What remains unclear is the precise nature of this regulation involving membrane sterols and ultimately determining CLIC1's membrane structure and function as an ion channel. Furthermore, our impedance spectroscopy results obtained using CLIC1 mutants, suggest that the residue Cys24 is not essential for CLIC1's ion channel function. However Cys24 does appear important for optimal ion channel activity. We also observe differences in conductance between CLIC1 reduced and oxidized forms when added to our tethered membranes. Therefore, we conclude that both membrane sterols and redox play a role in the ion channel activity of CLIC1.
Cranfield, CG, Berry, T, Holt, SA, Hossain, KR, Le Brun, AP, Carne, S, Al Khamici, H, Coster, H, Valenzuela, SM & Cornell, B 2016, 'Evidence of the Key Role of H3O+ in Phospholipid Membrane Morphology', LANGMUIR, vol. 32, no. 41, pp. 10725-10734.View/Download from: Publisher's site
Yeoh, GH, Gu, X, Timchenko, V, Valenzuela, SM & Cornell, BA 2016, 'High order accurate dual-phase-lag numerical model for microscopic heating in multiple domains', INTERNATIONAL COMMUNICATIONS IN HEAT AND MASS TRANSFER, vol. 78, pp. 21-28.View/Download from: Publisher's site
Cranfield, CG, Bettler, T & Cornell, B 2015, 'Nanoscale Ion Sequestration To Determine the Polarity Selectivity of Ion Conductance in Carriers and Channels', LANGMUIR, vol. 31, no. 1, pp. 292-298.View/Download from: Publisher's site
Al Khamici, H, Brown, LJ, Hossain, KR, Hudson, AL, Sinclair-Burton, AA, Jane, PMN, Daniel, EL, Hare, JE, Cornell, BA, Curmi, PMG, Davey, MW & Valenzuela, SM 2015, 'Members of the Chloride Intracellular Ion Channel Protein Family Demonstrate Glutaredoxin-Like Enzymatic Activity', PLOS ONE, vol. 10, no. 1.View/Download from: Publisher's site
Cranfield, CG, Cornell, BA, Grage, SL, Duckworth, P, Carne, S, Ulrich, AS & Martinac, B 2014, 'Transient potential gradients and impedance measures of tethered bilayer lipid membranes: pore-forming peptide insertion and the effect of electroporation.', Biophysical Journal, vol. 106, no. 1, pp. 182-189.View/Download from: Publisher's site
In this work, we present experimental data, supported by a quantitative model, on the generation and effect of potential gradients across a tethered bilayer lipid membrane (tBLM) with, to the best of our knowledge, novel architecture. A challenge to generating potential gradients across tBLMs arises from the tethering coordination chemistry requiring an inert metal such as gold, resulting in any externally applied voltage source being capacitively coupled to the tBLM. This in turn causes any potential across the tBLM assembly to decay to zero in milliseconds to seconds, depending on the level of membrane conductance. Transient voltages applied to tBLMs by pulsed or ramped direct-current amperometry can, however, provide current-voltage (I/V) data that may be used to measure the voltage dependency of the membrane conductance. We show that potential gradients >~150 mV induce membrane defects that permit the insertion of pore-forming peptides. Further, we report here the novel (to our knowledge) use of real-time modeling of conventional low-voltage alternating-current impedance spectroscopy to identify whether the conduction arising from the insertion of a polypeptide is uniform or heterogeneous on scales of nanometers to micrometers across the membrane. The utility of this tBLM architecture and these techniques is demonstrated by characterizing the resulting conduction properties of the antimicrobial peptide PGLa.
Hoiles, W, Krishnamurthy, V, Cranfield, CG & Cornell, B 2014, 'An Engineered Membrane to Measure Electroporation: Effect of Tethers and Bioelectronic Interface', BIOPHYSICAL JOURNAL, vol. 107, no. 6, pp. 1339-1351.View/Download from: Publisher's site
Martinac, B, Nomura, T, Chi, G, Petrov, E, Rohde, PR, Battle, AR, Foo, A, Constantine, M, Rothnagel, R, Carne, S, Deplazes, E, Cornell, B, Cranfield, CG, Hankamer, B & Landsberg, MJ 2014, 'Bacterial mechanosensitive channels: models for studying mechanosensory transduction.', Antioxidants and Redox Signaling, vol. 20, no. 6, pp. 952-969.View/Download from: Publisher's site
SIGNIFICANCE: Sensations of touch and hearing are manifestations of mechanical contact and air pressure acting on touch receptors and hair cells of the inner ear, respectively. In bacteria, osmotic pressure exerts a significant mechanical force on their cellular membrane. Bacteria have evolved mechanosensitive (MS) channels to cope with excessive turgor pressure resulting from a hypo-osmotic shock. MS channel opening allows the expulsion of osmolytes and water, thereby restoring normal cellular turgor and preventing cell lysis. RECENT ADVANCES: As biological force-sensing systems, MS channels have been identified as the best examples of membrane proteins coupling molecular dynamics to cellular mechanics. The bacterial MS channel of large conductance (MscL) and MS channel of small conductance (MscS) have been subjected to extensive biophysical, biochemical, genetic, and structural analyses. These studies have established MscL and MscS as model systems for mechanosensory transduction. CRITICAL ISSUES: In recent years, MS ion channels in mammalian cells have moved into focus of mechanotransduction research, accompanied by an increased awareness of the role they may play in the pathophysiology of diseases, including cardiac hypertrophy, muscular dystrophy, or Xerocytosis. FUTURE DIRECTIONS: A recent exciting development includes the molecular identification of Piezo proteins, which function as nonselective cation channels in mechanosensory transduction associated with senses of touch and pain. Since research on Piezo channels is very young, applying lessons learned from studies of bacterial MS channels to establishing the mechanism by which the Piezo channels are mechanically activated remains one of the future challenges toward a better understanding of the role that MS channels play in mechanobiology.
Valenzuela, S, Alkhamici, H, Brown, LJ, Almond, OC, Goodchild, SC, Carne, S, Curmi, PM, Holt, S & Cornell, BA 2013, 'Regulation Of The Membrane Insertion And Conductance Activity Of The Metamorphic Chloride Intracellular Channel Protein CLIC1 By Cholesterol', Plos One, vol. 8, no. 2, pp. 1-8.View/Download from: Publisher's site
The Chloride Intracellular ion channel protein CLIC1 has the ability to spontaneously insert into lipid membranes from a soluble, globular state. The precise mechanism of how this occurs and what regulates this insertion is still largely unknown, although factors such as pH and redox environment are known contributors. In the current study, we demonstrate that the presence and concentration of cholesterol in the membrane regulates the spontaneous insertion of CLIC1 into the membrane as well as its ion channel activity. The study employed pressure versus area change measurements of Langmuir lipid monolayer films; and impedance spectroscopy measurements using tethered bilayer membranes to monitor membrane conductance during and following the addition of CLIC1 protein. The observed cholesterol dependent behaviour of CLIC1 is highly reminiscent of the cholesterol-dependent-cytolysin family of bacterial pore-forming proteins, suggesting common regulatory mechanisms for spontaneous protein insertion into the membrane bilayer.
Battle, AR, Valenzuela, S, Mechler, A, Nichols, R, Praporski, S, Di Maio, I, Islam, H, Girard-Egrot, AP, Cornell, BA, Prashar, J, Caruso, F, Martin, LL & Martin, DK 2009, 'Novel engineered ion channel provides controllable ion permability for polyelectrolyte microcapsules coated with a lipid membrane', Advanced Functional Materials, vol. 19, pp. 201-208.View/Download from: Publisher's site
The development of nanostructured microcapsules based on a biomimetic lipid bilayer membrane (BLM) coating of poly(SODIUM STYRENESUFONATE) (pss) /POLY(ALLYLAMINE HYDROCHLORIDE) (pah) POLYELECTROLYTE HOLLOW MICROCAPSULES IS REPORTED
Titanium nitride thin films are widely used in biomedical implants because of thier biocompatibility, good mechanical properties and high corrosion resistance. Titanium nitride (TiN) thin films on silicon and glass substrates were prepared using a dc megnetron sputtering system under condition of systematically varying the nitrogen pressure and titanium megnetron power.
Krishnamurthy, V, Luk, KY, Cornell, BA, Prashar, J, Di Maio, I, Islam, H, Battle, AR, Valenzuela, S & Martin, DK 2007, 'Gramicidin Ion Channel-Based Nano-Biosensors: Construction, Stochastic Dynamical Models and Statistical Detection Algorithms', IEEE Sensors Journal, vol. 7, no. 9, pp. 1281-1288.View/Download from: Publisher's site
This paper deals with the experimental construction, stochastic modeling, and statistical signal processing of a novel, artificially constructed biosensor comprised of biological ion channels. Such nanoscale biosensors have been built by incorporating dimeric gramicidin A (bis-gA) ion channels into bilayer membranes of giant unilamellar liposomes, and then excising small patches of the membrane loaded with ion channels. We present a stochastic model for the response of the biosensor and present statistical model validation tests to verify the adequacy of the model., We show that in the presence of specific target molecules, the statistics of the gating mechanisms of the gA channels are altered. By capturing the change in real time, we devise a maximum-likelihood detector to detect the presence of target molecules. To test the sensitivity of this model, we conducted patch-clamp experiments with two compounds known to inhibit conduction of the gA channels. We found experimentally that the real-time detection algorithm was able to accurately identify the addition of the compounds even when the alterations in the patch-clamp recordings were very small. This algorithm provides the sensitive detection system for ongoing development of lipid-based nanosensors.
Krishna, G, Schulte, J, Cornell, BA, Pace, RJ & Osman, PD 2003, 'Tethered bilayer membranes containing ionic reservoirs: Selectivity and conductance', Langmuir, vol. 19, no. 6, pp. 2294-2305.View/Download from: Publisher's site
Ion channels, such as gramicidin A, selectively facilitate the transport of ions across biological and synthetic membranes. The conductance properties of ion channels are frequently characterized in synthetic bilayer lipid membranes (BLMs). The instability of BLMs has seriously limited the range of applications for these structures, and tethered bilayer lipid membranes (tBLMs) have addressed the problem through tethering many of the membrane components to a solid surface. In the present study, thin gold substrates have been used to tether thiol- and disulfide-terminated membrane components to form a tBLM electrode to provide a reservoir for ions. This study reports on the ion selectivity and apparent permeability of gramicidin channels in such tethered bilayer membranes. The investigations using electrical impedance spectroscopy indicated that the magnitude of ionic conductance varies substantially in reservoirs with different chemical structures.
Cranfield, C, Carne, S, Martinac, B & Cornell, B 2015, 'The assembly and use of tethered bilayer lipid membranes (tBLMs).', Humana Press (Springer Imprint), pp. 45-53.View/Download from: Publisher's site
Because they are firmly held in place, tethered bilayer lipid membranes (tBLMs) are considerably more robust than supported lipid bilayers such as black lipid membranes (BLMs) (Cornell et al. Nature 387(6633): 580-583, 1997). Here we describe the procedures required to assemble and test tethered lipid bilayers that can incorporate various lipid species, peptides, and ion channel proteins.