Farfield AnaLight range

You are here:HomeLaboratory productsAnaLight range Bibliography

Bibliography

Jump to...
Surface Science Research Papers | Biophysics Research Papers

Surface Science Research Papers

2007
Authors: A W Sonesson, TH Callisen, H Brismar and U M Elofsson
Title: A comparison between Dual Polarisation Interferometry (DPI) and Surface Plasmon Resonance (SPR) for protein adsorption studies.
Journal: Colloids and Surfaces B: Biointerfaces 54 236-240 (2007).
Abstract: This work was performed with the aim of comparing protein adsorption results obtained from the recently developed dual polarization interferometry (DPI) with the well-established surface plasmon resonance (SPR) technique. Both techniques use an evanescent field as the sensing element but completely different methods to calculate the adsorbed mass. As a test system we used adsorption of the lipase from Thermomyces lanuginosus (TLL) on C18 surfaces. The adsorbed amount calculated with both techniques is in good agreement, with both adsorption isotherms saturating at 1.30–1.35 mg/m2 at TLL concentrations of 1000nM and above. Therefore, this supports the use of both SPR and DPI as tools for studying protein adsorption, which is very important when comparing adsorption data obtained from the use different techniques. Due to the spot sensing in SPR, this technique is recommended for initial kinetic studies, whereas DPI is more accurate when the refractive index and thickness of the adsorbed layer is of more interest.


2006
Authors: Xiaonong Chen and Robert Pelton
Title: Pre-Adsorption of Amphiphilic Polymers on Synthetic Surfaces for Biofouling Retardation.
Journal: Adv. Materials Research 11 363-366 (2006).
Abstract: Polystyrene (PS), polyethylene (PE), polypropylene (PP), glass and stainless steel were exposed to aqueous solutions of a series of amphiphilic polymers at room temperature, including N-isopropylacrylamide (NIPAM)-based polymers, polyvinylpyrrolidone (PVP), polypropylene oxide (PPO)-polyethylene oxide (PEO) block copolymers and PEO. Dynamic contact angle measurements of the material surfaces before and after the treatment indicate that only NIPAM-based polymers can adsorb on both hydrophobic and hydrophilic surfaces. The surface morphologies of the materials before and after polymer adsorption were investigated by profilometry. Protein adsorption on the surfaces pre-adsorbed NIPAM-based polymers was investigated by dual polarisation interferometry (DPI) and profilometry using lysozyme as the model protein. The results obtained indicate that NIPAM-based polymers can significantly improve the biofouling resistance of synthetic surfaces.

Authors: T Halthur, P Claessen and U Elofsson
Title: Immobilization of Enamel Matrix Derivate Protein onto Polypeptide Multilayers: Comparative in Situ Measurements Using Ellipsometry, Quartz Crystal Microbalance with Dissipation, and Dual-Polarization Interferometry.
Journal: Langmuir 22 11065-71 (2006)
Abstract: The buildup of biodegradable poly(L-glutamic acid) (PGA) and poly(L-lysine) (PLL) multilayers on silica and titanium surfaces and the immobilization of enamel matrix derivate (EMD) protein was followed by utilizing in situ ellipsometry, quartz crystal microbalance with dissipation, and dual-polarization interferometry (DPI). The use of the relatively new DPI technique validated earlier published ellipsometry measurements of the PLL-PGA polypeptide films. The hydrophobic aggregating EMD protein was successfully immobilized both on top of and within the multilayer structures at pH 5.0. DPI measurements further indicated that the immobilization of EMD is influenced by the flow pattern during adsorption. The formed polypeptide-EMD multilayer films are of interest since it is known that EMD is able to trigger cell response and induce biomineralization. The multilayer films thus have potential to be useful as bioactive and biodegradable coatings for future dental implants.

Authors: E Yates, C Terry, C Rees, T Rudd, L Duchesne, M Skidmore, R Levy, N Thanh, R Nichols, D Clarke and D Fernig
Title: Protein-GAG Interactions: New Surface-based Techniques, Spectroscopies and Nanotechnology Probes.
Journal: Biochem. Soc. Trans. 34 427-430 (2006)
Abstract:New approaches, rooted in the physical sciences, have been developed to gain a more fundamental understanding of protein–GAG (glycosaminoglycan) interactions. DPI (dual polarization interferometry) is an optical technique, which measures real-time changes in the mass of molecules bound at a surface and the geometry of the bound molecules. QCM-D (quartz crystal microbalance-dissipation), an acoustic technique, measures the mass and the viscoelastic properties of adsorbates. The FTIR (Fourier-transform IR) amide bands I, II and III, resulting from the peptide bond, provide insight into protein secondary structure. Synchrotron radiation CD goes to much shorter wavelengths than laboratory CD, allowing access to chromophores that provide insights into the conformation of the GAG chain and of β-strand structures of proteins. To tackle the diversity of GAG structure, we are developing noble metal nanoparticle probes, which can be detected at the level of single particles and so enable single molecule biochemistry and analytical chemistry. These new approaches are enabling new insights into structure–function relationships in GAGs and together they will resolve many of the outstanding problems in this field.

Authors: B Lillis, M Manning, H Berney, E Hurley, A Mathewson and M Sheehan
Title: Dual Polarisation Interferometry Characterisation of DNA Immobilisation and Hybridisation on a Silanised Support.
Journal: Biosensors & Bioelectronics 21 1459-1467 (2006)
Abstract:Dual polarisation interferometry is an analytical technique that allows the simultaneous determination of thickness, density and mass of a biological layer on a sensing waveguide surface in real time. We evaluated, for the first time, the ability of this technique to characterize the covalent immobilisation of single stranded probe DNA and the selective detection of target DNA hybridisation on a silanised support. Two immobilisation strategies have been evaluated: direct attachment of the probe molecule and a more complex chemistry employing a 1,2 homobifunctional crosslinker molecule. With this technique we demonstrate it was possible to determine probe orientation and measure probe coverage at different stages of the immobilisation process in real time and in a single experiment. In addition, by measuring simultaneously changes in thickness and density of the probe layer upon hybridisation of target DNA, it was possible to directly elucidate the impact that probe mobility had on hybridisation efficiency. Direct covalent attachment of an amine modified 19mer resulted in a thickness change of 0.68 nm that was consistent with multipoint attachment of the probe molecule to the surface. Blocking with BSA formed a dense layer of protein molecules that absorbed between the probe molecules on the surface. The observed hybridisation efficiency to target DNA was ∼35%. No further significant reorientation of the probe molecule occurred upon hybridisation. The initial thickness of the probe layer upon attachment to the crosslinker molecule was 0.5 nm. Significant reorientation of the probe molecule surface normal occurred upon hybridisation to target DNA. This indicated that the probe molecule had greater mobility to hybridise to target DNA. The observed hybridisation efficiency for target DNA was ∼85%. The results show that a probe molecule attached to the surface via a crosslinker group is better able to hybridise to target DNA due to its greater mobility.

Authors: Shiming Lin, Chih-Kung Lee, Yu-Ming Wang, Long-Sun Huang, Yin-Hang Lin, Shih-Yuan Lee, Bor-Ching Sheu, Su-Ming Hsu
Title: Measurement of dimensions of pentagonal doughnut-shaped C-reactive protein using an atomic force microscope and a dual polarisation interferometric biosensor.
Journal: Biosensors & Bioelectronics 22 323-7 (2006)
Abstract:In order to develop the C-reactive protein (CRP) sensor chips for clinical detection of atherosclerosis and coronary heart disease, we used an atomic force microscope (AFM) and a dual polarization interferometric (DPI) biosensor to probe the surface ultrastructure and to measure the dimensions of CRP. A single pentagonal structure was directly visualized by AFM, and quantitative measurements of the dimensions of the protein were provided. The average height calculated for each pentagonal CRP particle was approximately 3.03±0.37 nm, which basically corresponds to that (36 °A in protomer diameter) previously obtained from the structure of CRP determined by X-ray crystallography. Moreover, a experiment using dual polarization interferometric (DPI) as a biosensor was then performed, and the average monolayer thickness value (3.18±0.43 nm) that was calculated basically corresponds to that obtained from the experimental value (3.03±0.37 nm) of the height measured by an AFM method for CRP. Further investigations will be performed to study the surface ultrastructure of a single pentagonal CRP molecule, and for this purpose a CRP sample (at low concentration) was scanned in vacuum by AFM. The higher-resolution images clearly revealed the presence of doughnut-shaped CRP molecules. In addition, phase images of CRP molecules were captured simultaneously with their height images, and the lateral dimensions of the doughnut-shaped CRP molecules were then measured. It was found that the average values calculated for the outer diameter (11.13±1.47 nm) and pore diameter (3.52±0.42 nm) are respectively close to those (102 °A in outer diameter and 30 °A in pore diameter) previously obtained from the structure of CRP determined by X-ray crystallography. This study represents the first direct characterization of the surface ultrastructure and dimensional measurement of the CRP molecule on the sensor chip.

Authors: C J. Terry, J F. Popplewell, M J. Swann, N J. Freeman and D G. Fernig
Title: Characterisation of Membrane Mimetics on a Dual Polarisation Interferometer.
Journal: Biosensors & Bioelectronics 22 627-32 (2006)
Abstract: Dual polarisation interferometry (DPI) has been used to characterise the formation of hybrid bilayer membranes (HBM) on a silicon-oxynitride surface. This technique allows the simultaneous determination of multiple physical properties of an HBM, as the HBM is being formed in a single experiment: mass, thickness in the z-direction (normal to the surface), tilt angle of the first layer and refractive index. Decanoic acid was covalently attached to an amine modified silicon-oxynitride sensor chip surface via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride condensation reaction. The decanoic acid layer was 0.92±0.12 nm thick, indicating a tilt angle of 57◦ from surface normal, and possessed a mass of 1.05±0.10 ngmm−2 and a refractive index (RI) of 1.450±0.020. Phospholipid vesicles made from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) were injected over the fatty acid surface to form an HBM. The DPPC HBM was 4.32±0.68 nm thick, with a total mass of 3.18±0.60 ngmm−2 and a RI of 1.404±0.007. The DMPC HBM was 2.12±0.34 nm thick, with a total mass of 2.25±0.51 ngmm−2, and a RI of 1.435±0.007. DPI thus provides an insight into HBM formation and differences between the structural organisation of HBMs of different composition.
Authors: Megan S. Lord, Martina H. Stenzel, Anne Simmons, Bruce K. Milthorpe
Title: Lysozyme Interaction with Poly(HEMA)-based Hydrogel
Journal: Biomaterials 27 1341-1345 (2006)
Abstract: Lysozyme interaction with an acrylic-based hydrogel, poly(2-hydroxyethyl methacrylate) co-methacrylic acid (P(HEMA-MAA)), was investigated using a combination of quartz crystal microbalance with dissipation (QCM-D), surface plasmon resonance (SPR) and dual polarisation interferometry (DPI). This combination of techniques demonstrated that lysozyme initially absorbed into the hydrogel matrix and displaced water from the hydrogel while subsequent lysozyme additions were adsorbed onto the surface of the hydrogel material. QCM-D, being sensitive to bound water, showed an overall decrease in mass and stiffening of the layer after lysozyme addition. SPR, a water insensitive technique, showed a net mass increase after addition of lysozyme and buffer rinses. DPI showed that the first exposure of lysozyme to P(HEMA-MAA) was consistent with lysozyme absorption while subsequent lysozyme exposures were consistent with lysozyme adsorption.


2005
Authors: Helen Berney, Karen Oliver
Title: Dual Polarization Interferometry size and density characterisation of DNA Immobilisation and hybridisation
Journal: Biosensors & Bioelectronics 21 618-26 (2005)
Abstract: Investigation of nucleic acid interactions was performed using dual polarization interferometry, a novel approach to elucidating molecular interactions. This paper presents a preliminary study of adsorption of single stranded DNA onto functionalised silicon oxynitride, compared with covalent linkage, and avidin–biotin immobilisation. The effect of probe concentration on hybridisation efficiency was also examined. We found that increasing the electrolyte concentration resulted in a decrease of adsorbed DNA and that capture of a biotinylated duplex DNA on an adsorbed avidin layer resulted in four times fewer molecules per cm2 than for duplex DNA covalently bound via an amine end terminal. The rate of thickness increase of a biotin probe layer on an adsorbed avidin capture layer increased 10-fold when the probe concentration was increased from 0.1 _M to 1_M. The close grafting density of the higher concentration probe meant that the immobilised probes were unavailable for hybridisation.



2004
Authors: NJ Freeman, LL Peel, MJ Swann, GH Cross, A Reeves, S Brand and JR Lu
Title: Real Time, High Resolution Studies of Protein Adsorption and Structure at the Solid-Liquid Interface Using Dual Polarisation Interferometry
Journal: J. Phys. Condens. Matter 16 S2493-S2496 (2004)
Abstract: A novel method for the analysis of thin biological films, called dual polarization interferometry (DPI), is described. This high resolution (<1 Å), laboratory based technique allows the thickness and refractive index (density) of biological molecules adsorbing or reacting at the solid–liquid interface to be measured in real time (up to 10 measurements per second). Results from the adsorption of bovine serum albumin (BSA) on to a silicon oxynitride chip surface are presented to demonstrate how time dependent molecular behaviour can be examined using DPI. Mechanistic and structural information relating to the adsorption process is obtained as a function of the solution pH.

Authors: J Armstrong, HJ Salacinski, Q Mu, AM Seifalian, L Peel, N Freeman, CM Holt, JR Lu
Title: Interfacial Adsorption of Fibrinogen and its Inhibition by RGD Peptide: A Combined Physical Study
Journal: J. Phys. Condens. Matter 16 S2483-S2491 (2004)
Abstract: The Arg–Gly–Asp (RGD) peptide sequence is known as a cell recognition site for numerous adhesive proteins present in the extracellular matrix (ECM) and in blood. Whilst surface immobilized RGD groups enhance cell attachment, RGD components present in solution can effectively inhibit cell attachment by competing with endogenous ligands for the same recognition site. In contrast to the widely reported binding to cell integrin, this study demonstrates a new RGD feature: its inhibitive effect on fibrinogen adsorption. Through a combined analysis of spectroscopic ellipsometry, neutron reflection and dual polarization interferometry, we show that the kinetic process of fibrinogen adsorption as a model pro-coagulant at the silica/solution interface and in the absence of any cells can be substantially reduced by the addition of RGD in solution and that the extent of the reduction is dependent on the relative concentration of RGD.

Authors: JR Lu, MJ Swann, LL Peel and NJ Freeman
Title: Lysozyme Adsorption Studies at the Silica-Water Interface using Dual Polarisation Interferometry
Journal: Langmuir 20 1827-1832 (2004)
Abstract: Lysozyme adsorption at the silica/water interface has been studied using a new analytical technique called dual polarization interferometry. This laboratory-based technique allows the build up or removal of molecular layers adsorbing or reacting on a lightly doped silicon dioxide (silica) surface to be measured in terms of thickness and refractive index changes with time. Lysozyme adsorption was studied at a range of concentrations from 0.03 to 4.0 g dm -3 and at both pH 4 and pH 7. Adsorbed layers ranging from 14 to 43 ± 1 Å in thickness and 0.21 to 2.36 ( 0.05 mg m -2 in mass coverage were observed at pH 4 with increasing lysozyme concentration, indicating a strong deformation of the monolayer over the low concentration range and the formation of an almost complete sideways-on bilayer toward the high concentration of 4 g dm-3. At pH 7, the thickness of adsorbed layers varied from 16 to 54 ±1 Å with significantly higher surface coverage (0.74 to 3.29±0.05 mgm -2), again indicating structural deformation during the initial monolayer formation, followed by a gradual transition to bilayer adsorption over the high concentration end. The pH recycling performed at a fixed lysozyme concentration of 1.0 g dm-3 indicated a broadly reversible adsorption regardless of whether the pH was cycled from pH 7 to pH 4 and back again or vice versa. These observations are in good agreement with earlier studies undertaken using neutron reflection although the fine details of molecular orientations in the layers differ subtly.

Authors: GH Cross, A Reeves, S Brand, MJ Swann, LL Peel, NJ Freeman, JR Lu
Title: The Metrics of Surface Adsorbed Small Molecules on the Young’s Fringe Dual-Slab Waveguide Interferometer
Journal: J. Phys. D Appl. Phys. 37 74-80 (2004)
Abstract: A method for analysing thin films using a dual-waveguide interferometric technique is described. Alternate dual polarization addressing of the interferometer sensor using a ferroelectric liquid crystal polarization switch allowed the opto-geometrical properties (density and thickness) of adsorbed layers at a solid–liquid interface to be determined. Differences in the waveguide mode dispersion between the transverse electric and transverse magnetic modes allowed unique combinations of layer thickness and refractive index to be determined at all stages of the layer formation process. The technique has been verified by comparing the analysis of the surface adsorption of surfactants with data obtained using neutron scattering techniques, observing their behaviour on trimethylsilane coated silicon oxynitride surfaces. The data obtained were found to be in excellent agreement with analogous neutron scattering experiments and the precision of the measurements taken to be of the order of 40 pm with respect to adsorbed layer thicknesses. The study was extended to a series of surfactants whose layer morphology could be correlated with their hydrophilicity/lipophilicity balance. Those in the series with longer alkyl chains were observed to form thinner, denser layers at the hydrophobic solid/aqueous liquid interface and the degree of order attained at sub-critical micelle concentrations to be correlated with molecular fluidity. The technique is expected to find utility with those interested in thin film analysis. An important and growing area of application is within the life sciences, especially in the field of protein structure and function.

Biophysics Research Papers

2007
Authors: Conor Horgan, Arkadiusz Oleksy, Alexander Zhdanov, Patrick Lall, Ian White, Amir Khan, Clare Futter, John McCaffrey and Mary McCaffrey.
Title: Rab11-FIP3 is critical for the structural integrity of the endosomal-recycling compartment
Journal: doi:10.1111/j.1600-0854.2006.00543.x
Abstract: Rab11-FIP3 is an endosomal-recycling compartment (ERC) protein that is implicated in the process of membrane delivery from the ERC to sites of membrane insertion during cell division. Here we report that Rab11-FIP3 is critical for the structural integrity of the ERC during interphase. We demonstrate that knockdown of Rab11-FIP3 and expression of a mutant of Rab11-FIP3 that is Rab11-binding deficient causes loss of all ERC-marker protein staining from the pericentrosomal region of A431 cells. Furthermore, we find that fluorophore-labelled transferrin cannot access the pericentrosomal region of cells in which Rab11-FIP3 function has been perturbed. We find that this Rab11-FIP3 function appears to be specific, since expression of the equivalent Rab11-binding deficient mutant of RCP does not perturb ERC morphology. In addition, we find that other organelles such as sorting and late endosomes are unaffected by loss of Rab11-FIP3 function. Finally, we demonstrate the presence of an extensive coiled-coil region between residues 463 and 692 of Rab11-FIP3, which exists as a dimer in solution and is critical to support its function on the ERC. Together, these data indicate that Rab11-FIP3 is necessary for the structural integrity of the pericentrosomal ERC.

Authors: K Karim, J.D. Taylor, D.C. Cullen, M.J. Swann and N. J. Freeman.
Title: Measurement of Conformational Changes in the Structure of Transglutaminase on Binding Calcium Ions using Optical Evanescent Dual Polarisation Interferometry
Journal: Anal. Chem. 79 3023-3031 (2007)
Abstract: The conformational changes occurring when the protein transglutaminase binds calcium ions have been studied using the optical evanescent technique of dual polarization interferometry (DPI) implemented via a dual slab waveguide structure. Immobilized transglutaminase layers of 4-5 nm in thickness were obtained, which when challenged with calcium ions underwent a contraction of approximately 0.5 nm (depending on the concentration of calcium) and an increase in refractive index of approximately 1 x 10-2. The affinity constant for the calcium binding was found to be in the range of 0.95 +/- 0.2 mM. The results reported are in good agreement with those found in the literature obtained by other techniques. It has also been shown that the structural changes occurring during the binding event are considerably larger than the mass changes that take place; thus, DPI offers a potentially valuable method to study real-time structural changes occurring to proteins when they bind metal ions.

Authors: J Popplewell, M Swann, N Freeman, C McDonnell and R Ford
Title: Quantifying of the Effects of Melittin on Liposomes.
Journal: Biochimica et Biophysica Acta 1768 13-20 (2007)
Abstract: Melittin, the soluble peptide of bee venom, has been demonstrated to induce lysis of phospholipid liposomes. We have investigated the dependence of the lytic activity of melittin on lipid composition. The lysis of liposomes, measured by following their mass and dimensions when immobilised on a solid substrate, was close to zero when the negatively charged lipids phosphatidyl glycerol or phosphatidyl serine were used as the phospholipid component of the liposome. Whilst there was significant binding of melittin to the liposomes, there was little net change in their diameter with melittin binding reversed upon salt injection. For the zwitterionic phosphatidyl choline the lytic ability of melittin is dependent on the degree of acyl chain unsaturation, with melittin able to induce lysis of liposomes in the liquid crystalline state, whilst those in the gel state showed strong resistance to lysis. By directly measuring the dimensions and mass changes of liposomes on exposure to melittin using Dual Polarisation Interferometry, rather than following the florescence of entrapped dyes we attained further information about the initial stages of melittin binding to liposomes.


2006
Authors: Shiro Suetsugu, Kazutaka Murayama, Ayako Sakamoto, Kyoko Hanawa-Suetsugu, Azusa Seto, Tsukasa Oikawa, Chiemi Mishima, Mikako Shirouzu, Tadaomi Takenawa and Shigeyuki Yokoyama.
Title: The RAC Binding Domain/IRSp53-MIM Homology Domain of IRSp53 Induces RAC-dependent Membrane Deformation.
Journal: J. Biol. Chem 281 35347-35358 (2006)
Abstract: The concave surface of the crescent-shaped Bin-amphiphysin- Rvs (BAR) domain is postulated to bind to the cell membrane to induce membrane deformation of a specific curvature. The Rac binding (RCB) domain/IRSp53-MIM homology domain (IMD) has a dimeric structure that is similar to the structure of the BAR domain; however, the RCB domain/IMD has a "zeppelin-shaped" dimer. Interestingly, the RCB domain/IMD of IRSp53 possesses Rac binding, membrane binding, and actin filament binding abilities. Here we report that the RCB domain/IMD of IRSp53 induces membrane deformation independent of the actin filaments in a Rac-dependent manner. In contrast to the BAR domain, the RCB domain/IMD did not cause long tubulation of the artificial liposomes; however, the Rac binding domain caused the formation of small buds on the liposomal surface. When expressed in cells, the Rac binding domain induced outward protrusion of the plasma membrane in a direction opposite to that induced by the BAR domain. Mapping of the amino acids responsible for membrane deformation suggests that the convex surface of the Rac binding domain binds to the membrane in a Rac-dependent manner, which may explain the mechanism of the membrane deformation induced by the RCB domain/IMD.

Authors: S Suetsugu S Kurisu, T Oikawa, D Yamazaki, A Oda and T Takenawa
Title: Optimization of WAVE2 complex-induced actin polymerization by membrane-bound IRSp53, PIP3, and Rac
Journal: Journal of Cell Biology 173 571-585 (2006)
Abstract: WAVE2 activates the actin-related protein (Arp) 2/3 complex for Rac-induced actin polymerization during lamellipodium formation and exists as a large WAVE2 protein complex with Sra1/ PIR121, Nap1, Abi1, and HSPC300. IRSp53 binds to both Rac and Cdc42 and is proposed to link Rac to WAVE2. We found that the knockdown of IRSp53 by RNA interference decreased lamellipodium formation without a decrease in the amount of WAVE2 complex. Localization of WAVE2 at the cell periphery was retained in IRSp53 knockdown cells. Moreover, activated Cdc42 but not Rac weakened the association between WAVE2 and IRSp53. When we measured Arp2/3 activation in vitro, the WAVE2 complex isolated from the membrane fraction of cells was fully active in an IRSp53-dependent manner but WAVE2 isolated from the cytosol was not. Purified WAVE2 and purified WAVE2 complex were activated by IRSp53 in a Rac-dependent manner with PIP3-containing liposomes. Therefore, IRSp53 optimizes the activity of the WAVE2 complex in the presence of activated Rac and PIP3.

Authors: K Tsujita, S Suetsugu, N Sasaki, M Furutani, T Oikawa, and T Takenawa
Title: Co-ordination between the actin cytoskeleton and membrane deformation by a novel membrane tubulation domain of PCH proteins is involved in endocytosis
Journal: Journal of Cell Biology 172 269-279 (2006)
Abstract:The conserved FER-CIP4 homology (FCH) domain is found in the pombe Cdc15 homology (PCH) protein family members, including formin-binding protein 17 (FBP17). However, the amino acid sequence homology extends beyond the FCH domain. We have termed this region the extended FC (EFC) domain. We found that FBP17 coordinated membrane deformation with actin cytoskeleton reorganization during endocytosis. The EFC domains of FBP17, CIP4, and other PCH protein family members show weak homology to the Bin-amphiphysin-Rvs (BAR) domain. The EFC domains bound strongly to phosphatidylserine and phosphatidylinositol 4,5-bisphosphate and deformed the plasma membrane and liposomes into narrow tubules. Most PCH proteins possess an SH3 domain that is known to bind to dynamin and that recruited and activated neural Wiskott-Aldrich syndrome protein (N-WASP) at the plasma membrane. FBP17 and/or CIP4 contributed to the formation of the protein complex, including N-WASP and dynamin-2, in the early stage of endocytosis. Furthermore, knockdown of endogenous FBP17 and CIP4 impaired endocytosis. Our data indicate that PCH protein family members couple membrane deformation to actin cytoskeleton reorganization in various cellular processes.

Authors: G Thibault, J Yudin, P Wong, V Tsitrin, R Sprangers, R Zhao and W A Houry
Title: Specificity in substrate and cofactor recognition by the N-terminal domain of the chaperone ClpX.
Journal: Proc Natl Acad Sci USA 103 17724-9 (2006)
Abstract:Clp ATPases are a unique group of ATP-dependent chaperones supporting targeted protein unfolding and degradation in concert with their respective proteases. ClpX is a representative member of these ATPases; it consists of two domains, a zinc-binding domain (ZBD) that forms dimers and a AAA+ ATP-binding domain that arranges into a hexamer. Analysis of the binding preferences of these two domains in ClpX revealed that both domains preferentially bind to hydrophobic residues but have different sequence preferences, with the AAA+ domain preferentially recognizing a wider range of specific sequences than ZBD. As part of this analysis, the binding site of the ClpX dimeric cofactor, SspB2, on ZBD in ClpX was determined by NMR and mutational analysis. The SspB C terminus was found to interact with a hydrophobic patch on the surface of ZBD. The affinity of SspB2 toward ZBD2 and the geometry of the SspB2-ZBD2 complex were investigated by using the newly developed quantitative optical biosensor method of dual polarization interferometry. The data suggest a model for the interaction between SspB2 and the ClpX hexamer.

Authors: E Yates, C Terry, C Rees, T Rudd, L Duchesne, M Skidmore, R Levy, N Thanh, R Nichols, D Clarke and D Fernig
Title: Protein-GAG Interactions: New Surface-based Techniques, Spectroscopies and Nanotechnology Probes.
Journal: Biochem. Soc. Trans. 34 427-430 (2006)
Abstract: New approaches, rooted in the physical sciences, have been developed to gain a more fundamental understanding of protein–GAG (glycosaminoglycan) interactions. DPI (dual polarization interferometry) is an optical technique, which measures real-time changes in the mass of molecules bound at a surface and the geometry of the bound molecules. QCM-D (quartz crystal microbalance-dissipation), an acoustic technique, measures the mass and the viscoelastic properties of adsorbates. The FTIR (Fourier-transform IR) amide bands I, II and III, resulting from the peptide bond, provide insight into protein secondary structure. Synchrotron radiation CD goes to much shorter wavelengths than laboratory CD, allowing access to chromophores that provide insights into the conformation of the GAG chain and of β-strand structures of proteins. To tackle the diversity of GAG structure, we are developing noble metal nanoparticle probes, which can be detected at the level of single particles and so enable single molecule biochemistry and analytical chemistry. These new approaches are enabling new insights into structure–function relationships in GAGs and together they will resolve many of the outstanding problems in this field.

Authors: S Ricard-Blum, L Peel, F Ruggerio and N Freeman
Title: Dual Polarization Interferometry Characterization of Carbohydrate-Protein Interactions
Journal: Anal. Biochem. 352 252-259 (2006)
Abstract: Dual polarization interferometry (DPI) is an analytical technique that allows the simultaneous determination of thickness, density, and mass of a biological layer on a sensing waveguide surface in real time. The technique was applied to the analysis of carbohydrate– protein interactions. The selected system involved a 12-kDa recombinant fragment of collagen V (HepV) and heparin, a complex polysaccharide. Here we report on the analysis of thickness, density, and mass of surface structures obtained during the binding of HepV to heparin, which is a useful model compound for the sulfated, protein-binding regions of heparan sulfate. This system, which was initially studied for its biological relevance, displayed anomalous behavior in kinetic studies using surface plasmon resonance (SPR) assays that has been attributed to putative conformational changes. It was this putative conformational change that prompted us to investigate the binding using an alternative analytical approach. While using DPI to monitor binding events, a streptavidin layer (surface coverage 2.105 ng mm -2) was bound to the sensor surface (92% coverage), which captured 0.105 ng mm -2 of biotinylated heparin (a stoichiometric ratio of 1:6 heparin–streptavidin). The heparin inserted into the streptavidin layer but was still found to be capable of binding 0.154 ng mm -2 of HepV, which was also observed to insert into the streptavidin layer. This allowed the reliable calculation of the stoichiometric ratio for the HepV–heparin complex (_1.7:1.0), which has proved to be difficult to evaluate by SPR assays. Furthermore, real-time analysis of the heparin–HepV interaction by DPI suggested that there was some surface loss (probably of streptavidin) while the binding was occurring rather than the putative conformational change that has been suggested on the basis of kinetic data alone. This gives further insight into the binding mechanism of HepV to heparin.
Authors: S Lin, C-K Lee, Y-H Lin, S-Y Lee, B-C Sheu, J-C Tsai and S-M Hsu
Title: Homopolyvalent Antibody-Antigen Interaction Kinetic Studies with the Use of a Dual Polarisation Interferometric Biosensor
Journal: Biosensors & Bioelectronics 22 715-721 (2006)
Abstract: We used dual-polarization interferometry (DPI) to study the interaction kinetics between a ‘homopolyvalent’ antigen (Ag) and a monoclonal antibody (Ab). A model system, which uses a monoclonal Ab against a homopentameric Ag, C-reactive protein (CRP), is presented with principle and experiments for the study of the interactions between an Ab and an Ag that has multiple identical epitopes. This allows evaluation of the dissociation constant (KD) and of the binding stoichiometry by DPI based on measurements of phase changes of Ab–Ag complexes in the transverse magnetic (TM) and transverse electric (TE) polarization modes. The average experimental value of KD found by the DPI technique for anti-CRP Ab was shown to be in close agreement with the value obtained by an indirect competition-enzyme-linked immunosorbent assay (ELISA). Moreover, the total number of Ab combining sites on the DPI sensor chip was calculated, and the binding stoichiometry of the surface Ag–Ab complex was obtained. This study illustrates the advantages of the DPI method in biosensing in its capacity for simultaneous evaluation of the thickness and refractive index (density, mass) of adsorbed layers. This allowed a comprehensive analysis of affinity reactions between an Ab having two binding sites and a multi-sited Ag.


2005
Authors: J. Popplewell, N. Freeman, S. Carrington, G. Ronan, C. McDonnell, R.C. Ford
Title: Quantification of the effects of melittin on liposome structure
Journal: Biochem. Soc. Trans. 3 931-933 (2005)
Abstract: An optical technique, dual-polarization interferometry, has been used to examine lipid structures at the solid/liquid interface. Changes in the lipid structures, in real time, were examined as a consequence of challenging them with a peptide (melittin) that is known to induce liposome rupture. This work suggests that it should be possible to obtain a better understanding of the detail of the melittin rupture process.
Authors: D West, C Rees, L Duchesne, S Patey, C Terry, J Turnbull, M Delehedde, C Heegaard, F Allain, C Vanpouille, D Ron, and D Fernig
Title: Interactions of Multiple Heparin Binding Growth Factors with Neuropilin-1 and Potentiation of the Activity of Fibroblast Growth Factor-2
Journal: J. Biol. Chem. 280 13457-13464 (2005)
Abstract: The hypothesis that neuropilin-1 (Npn-1) may interact with heparin-binding proteins other than vascular endothelial growth factor has been tested using an optical biosensor-based binding assay. The results show that fibroblast growth factor (FGF) 1, 2, 4, and 7, FGF receptor 1, hepatocyte growth factor/scatter factor (HGF/SF), FGF binding protein, normal protease sensitive form of prion protein, antithrombin III, and Npn-1 itself are all able to interact with Npn-1 immobilized on the sensor surface. FGF-2, FGF-4, and HGF/SF are also shown to interact with Npn-1 in a solution assay. Moreover, these protein-protein interactions are dependent on the ionic strength of the medium and are inhibited by heparin, and the kinetics of binding of FGF-2, FGF-4 and HGF/SF to Npn-1 are characterized by fast association rate constants (270,000– 1,600,000 M - 1 s - 1 ). These results suggest that Npn-1 possesses a “heparin” mimetic site that is able to interact at least in part through ionic bonding with the heparin binding site on many of the proteins studied. Npn-1 was also found to potentiate the growth stimulatory activity of FGF-2 on human umbilical vein endothelial cells, indicating that Npn-1 may not just bind but also regulate the activity of heparin-binding proteins.


2004
Authors: Biehle, J Carrozzella, R Shukla, JF Popplewell, MJ Swann, NJ Freeman and JF Clark
Title: Apolipoprotein E Isoprotein Specific Interactions with Tissue Plasminogen Activator
Journal: Biochimica et Biophysica Acta 1689 244-251 (2004)
Abstract: : Apolipoprotein E (Apo E) is an important genetic risk factor for multiple neurological, vascular and cardiovascular diseases. Previously, we reported Apo E isoprotein-specific modulation of tissue plasminogen activator (tPA) using an in vitro blood-clotting assay. Here, we studied the conformational changes of Apo E2, E3 and E4 in the presence of tPA and vice versa using circular dichroism (CD) and dual polarization interferometry (DPI). We report isoprotein and state-specific intermolecular interactions between the Apo E isoforms and tPA. Apo E2 interaction with immobilized tPA leads to significant conformational changes which are not observed with Apo E3 or E4. Additionally, tPA induces changes in helicity of lipidated Apo E2 whereas no detectible changes were observed in Apo E3 or E4. The Tukey’s test for interaction indicated a significant ( P < 0.001) interaction between tPA and Apo E2 in the lipidated environment. These results may be important regarding the mechanism by which Apo E has isoprotein-specific effects on many biological processes and diseases involving blood clotting, proteolysis and perfusion.

Authors: MJ Swann, LL Peel, S Carrington and NJ Freeman
Title: Dual Polarisation Interferometry: An Analytical Technique to Measure Changes in Protein Structure in Real Time, to Determine the Stoichiometry of Binding Events and to Differentiate Between Specific and Non-Specific Interactions
Journal: Analytical Biochemistry 329 190-198 (2004)
Abstract: The study of solution-phase interactions between small molecules and immobilized proteins is of intense interest, especially to the pharmaceutical industry. An optical sensing technique, dual polarization interferometry, has been employed for the detailed study of a model protein system, namely, D-biotin interactions with streptavidin immobilized on a solid surface. Changes in thickness and density of an immobilized streptavidin layer as a result of the binding of D-biotin have been directly measured in solution and in real time. The results obtained from this approach are in excellent agreement with X-ray crystallographic data for the structural changes expected in the streptavidin-D-biotin system. The mass changes measured on binding D-biotin also agree closely with anticipated binding capacity values. Determination of the density changes occurring in the protein adlayer provides a means for differentiation between specific and nonspecific interactions.
Authors: T Oikawa, H Yamaguchi, T Itoh, M Kato, T Ijuin, D Yamazaki, S Suetsugu &T Takenawa
Title: PtdIns(3,4,5) P3 Binding is Necessary for WAVE2-induced Formation of Lamellipodia
Journal: Nature Cell Biology 6 420-426 (2004)
Abstract: Polarized cell movement is triggered by the development of a PtdIns(3,4,5)P3 gradient at the membrane, which is followed by rearrangement of the actin cytoskeleton. The WASP family verprolin homologous protein (WAVE) is essential for lamellipodium formation at the leading edge by activating the Arp2/3 complex downstream of Rac GTPase. Here, we report that WAVE2 binds to PtdIns(3,4,5)P3 through its basic domain. The amino-terminal portion of WAVE2, which includes the PtdIns(3,4,5)P3-binding sequence, was localized at the leading edge of lamellipodia induced by an active form of Rac (RacDA) or by treatment with platelet derived growth factor (PDGF). Production of PtdIns(3,4,5)P3 at the cell membrane by myristoylated phosphatidylinositol- 3-OH kinase (PI(3)K) is sufficient to recruit WAVE2 in the presence of dominant-negative Rac and latrunculin, demonstrating that PtdIns(3,4,5)P3 alone is able to recruit WAVE2. Expression of a full-length mutant of WAVE2 that lacks the lipid-binding activity inhibited proper formation of lamellipodia induced by RacDA. These results suggest that one of the products of PI(3)K, PtdIns(3,4,5)P3, recruits WAVE2 to the polarized membrane and that this recruitment is essential for lamellipodium formation at the leading edge.


2003
Authors: MJ Swann, NJ Freeman, S Carrington, G Ronan and P Barrett
Title: Quantifying Structural Changes and Stoichiometry of Protein Interactions Using Size and Density Profiling
Journal: Letters in Peptide Science 10 487-494 (2003)
Authors: GH Cross, A Reeves, S Brand, JF Popplewell, LL Peel, MJ Swann and NJ Freeman
Title: A New Quantitative Optical Biosensor for Protein Characterisation
Journal: Biosensors and Bioelectronics 19 383-390 (2003)
Abstract: A new optical biosensor is described based on a dual waveguide interferometric technique. By addressing the waveguide structure with alternate polarisations the optogeometrical properties (density and thickness) of adsorbed protein layers at the sensor (solid)-liquid interface have been determined. Differences in the waveguide mode dispersion between the transverse electric (TE) and transverse magnetic (TM) modes allow unique solutions for adlayer thickness and refractive index to be determined at all stages during the formation process. The technique has been verified using standard protein systems and by comparing the data with published work using X-ray crystallography and neutron reflection techniques. The data obtained was found to be in excellent agreement with previously reported X-ray experiments given that typical film thicknesses for streptavidin layers were in the range 5.5_/6.5 nm compared with the short axis crystal structure of between 4.8 and 5.6 nm. The precision of the measurements taken was of the order of 40 pm with respect to adsorbed adlayer thicknesses. This biosensor approach provides measurements of both thickness and density of adlayers to a high precision, simultaneously and in real time enabling detail of the structure and function of proteins to be elucidated. From such data it is possible to obtain information on the orientation, distortion and efficiency of immobilisation procedures as well as the interaction event of interest. The technique is expected to find utility with those interested in protein structure and function. This is an area of growing importance within the life sciences as the demand for quantitative analytical techniques increases with the growth in 'proteomics'.

 

 

Email a colleague about our NIR Chemical Imaging analyzers