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Manchester, United Kingdom

Coan K.E.D.,Novartis | Swann M.J.,Farfield | Ottl J.,Novartis
Analytical Chemistry | Year: 2012

In early drug discovery, knowledge about ligand-induced conformational changes and their influence on protein activity greatly aids the identification of lead candidates for medicinal chemistry efforts. Efficiently acquiring such information remains a challenge in the initial stages of lead finding. Here we investigated the application of dual polarization interferometry (DPI) as a method for the real-time characterization of low molecular weight (LMW) ligands that induce conformational changes. As a model system we chose calmodulin (CaM), which undergoes large and distinct structural rearrangements in response to calcium ion and small molecule inhibitors such as trifluoperazine (TFP). We measured concentration-dependent mass, thickness, and density responses of an immobilized CaM protein layer, which correlated directly with binding and conformational events. Calcium ion binding to CaM induced an increase in thickness (≤0.05 nm) and decrease in density (0.03 g/cm 3) whereas TFP induced an increase in both thickness (0.05 nm) and density (0.01 g/cm 3). The layer measurements reported here show how DPI can be used to assess and differentiate ligands with distinct structural modes of action. © 2011 American Chemical Society. Source


Hirst D.J.,Monash University | Lee T.-H.,Monash University | Swann M.J.,Farfield | Aguilar M.-I.,Monash University
Analytical Chemistry | Year: 2013

Kinetic analysis of peptide-membrane interactions generally involves a curve fitting process with no information about what the different curves may physically correspond to. Given the multistep process of peptide-membrane interactions, a computational method that utilizes physical parameters that relate to both peptide binding and membrane structure would provide new insight into this complex process. In this study, kinetic models accounting for two-state and three-state mechanisms were fitted to our previously reported simultaneous real-time measurements of mass and birefringence during the binding and dissociation of the peptide HPA3 (Hirst, D.; Lee, T.-H.; Swann, M.; Unabia, S.; Park, Y.; Hahm, K.-S.; Aguilar, M. Eur. Biophys. J. 2011, 40, 503-514); significantly, the mass and birefringence are constrained by the same set of kinetic constants, allowing the unification of peptide binding patterns with membrane structure changes. For the saturated phospholipid dimyristoyl- phosphatidylcholine (DMPC) the two-state model was sufficient to account for the observed changes in mass and birefringence, whereas for the unsaturated phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) the two-state model was found to be inadequate and a three-state model gave a significantly better fit. The third state of interaction for POPC was found to disrupt the bilayer much more than the previous two states. We propose a hypothesis for the mechanism of membrane permeabilization based on the results featuring a loosely bound first state, a tightly bound second state, and a highly membrane-disrupting third state. The results demonstrate the importance of the difference in membrane fluidity between the gel phase DMPC and the liquid crystal phase POPC for peptide-membrane interactions and establish the combination of DPI and kinetic modeling as a powerful tool for revealing features of peptide-membrane interaction mechanisms, including intermediate states between initial binding and full membrane disruption. © 2013 American Chemical Society. Source


Groftehauge M.K.,Durham University | Hajizadeh N.R.,Durham University | Swann M.J.,Farfield | Pohl E.,Durham University
Acta Crystallographica Section D: Biological Crystallography | Year: 2015

Over the last decades, a wide range of biophysical techniques investigating protein-ligand interactions have become indispensable tools to complement high-resolution crystal structure determinations. Current approaches in solution range from high-throughput-capable methods such as thermal shift assays (TSA) to highly accurate techniques including microscale thermophoresis (MST) and isothermal titration calorimetry (ITC) that can provide a full thermodynamic description of binding events. Surface-based methods such as surface plasmon resonance (SPR) and dual polarization interferometry (DPI) allow real-time measurements and can provide kinetic parameters as well as binding constants. DPI provides additional spatial information about the binding event. Here, an account is presented of new developments and recent applications of TSA and DPI connected to crystallography. © 2015. Source


Lee T.-H.,Monash University | Heng C.,Monash University | Swann M.J.,Farfield | Gehman J.D.,University of Melbourne | And 2 more authors.
Biochimica et Biophysica Acta - Biomembranes | Year: 2010

Effective antimicrobial peptides (AMPs) distinguish between the host and microbial cells, show selective antimicrobial activity and exhibit a fast killing mechanism. Although understanding the structure-function characteristics of AMPs is important, the impact of the peptides on the architecture of membranes with different lipid compositions is also critical in understanding the molecular mechanism and specificity of membrane destabilisation. In this study, the destabilisation of supported lipid bilayers (SLBs) by the AMP aurein 1.2 was quantitatively analysed by dual polarisation interferometry. The lipid bilayers were formed on a planar silicon oxynitride chip, and composed of mixed synthetic lipids, or Escherichia coli lipid extract. The molecular events leading sequentially from peptide adsorption to membrane lysis were examined in real time by changes in bilayer birefringence (lipid molecular ordering) as a function of membrane-bound peptide mass. Aurein 1.2 bound weakly without any change in membrane ordering at low peptide concentration (5 μM), indicating a surface-associated state without significant perturbation in membrane structure. At 10 μM peptide, marked reversible changes in molecular ordering were observed for all membranes except DMPE/DMPG. However, at 20 μM aurein 1.2, removal of lipid molecules, as determined by mass loss with a concomitant decrease in birefringence during the association phase, was observed for DMPC and DMPC/DMPG SLBs, which indicates membrane lysis by aurein. The membrane destabilisation induced by aurein 1.2 showed cooperativity at a particular peptide/lipid ratio with a critical mass/molecular ordering value. Furthermore, the extent of membrane lysis for DMPC/DMPG was nearly double that for DMPC. However, no lysis was observed for DMPC/DMPG/cholesterol, DMPE/DMPG and E. coli SLBs. The extent of birefringence changes with peptide mass suggested that aurein 1.2 binds to the membrane without inserting through the bilayer and membrane lysis occurs through detergent-like micellisation above a critical P/L ratio. Real-time quantitative analysis of the structural properties of membrane organisation has allowed the membrane destabilisation process to be resolved into multiple steps and provides comprehensive information to determine the molecular mechanism of aurein 1.2 action. © 2010. Source


Baumann M.K.,ETH Zurich | Swann M.J.,Farfield | Textor M.,ETH Zurich | Reimhult E.,ETH Zurich | Reimhult E.,University of Natural Resources and Life Sciences, Vienna
Analytical Chemistry | Year: 2011

We have determined the kinetics and affinity of binding of PH-PLCδ 1 to the PIP 2 headgroup lipids using an optical surface-sensitive technique in a time-resolved manner. The use of dual polarization interferometry to probe supported lipid bilayers (SLBs) of different compositions allowed determination of accurate affinity constants and a layer structure of the peptide binding to the model membrane platform. In addition, the platform enabled us to monitor the detailed adsorption kinetics characterized by a strong initial electrostatic attraction of the peptide to the SLB surface followed by rearrangement and loss of possibly clustered peptides upon specific binding to the phosphoinositide headgroup. These kinetics differed substantially from adsorption kinetics for nonspecific binding to similarly charged control SLBs. © 2011 American Chemical Society. Source

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