Analysis of the diverse antigenic landscape of the malaria protein RH5 identifies a potent vaccine-induced human public antibody clonotype.

The highly conserved and essential Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) has emerged as the leading target for vaccines against the disease-causing blood stage of malaria. However, the features of the human vaccine-induced antibody response that confer highly potent inhibition of malaria parasite invasion into red blood cells are not well defined. Here, we characterize 236 human IgG monoclonal antibodies, derived from 15 donors, induced by the most advanced PfRH5 vaccine. We define the antigenic landscape of this molecule and establish that epitope specificity, antibody association rate, and intra-PfRH5 antibody interactions are key determinants of functional anti-parasitic potency. In addition, we identify a germline IgG gene combination that results in an exceptionally potent class of antibody and demonstrate its prophylactic potential to protect against P. falciparum parasite challenge in vivo. This comprehensive dataset provides a framework to guide rational design of next-generation vaccines and prophylactic antibodies to protect against blood-stage malaria.

Use of surface plasmon resonance in the binding study of vitamin D, metabolites and analogues with vitamin D binding protein.

Vitamin D3 and its metabolites are lipophilic molecules with low aqueous solubility and must be transported bound to plasma carrier proteins, primarily to vitamin D binding protein (DBP). The biological functions of vitamin D3 metabolites are intimately dependent on the protein, hence the importance of determining their affinity for DBP. In this study, we developed a novel procedure for measuring the kinetic and equilibrium constants of human-DBP with vitamin D3 and three metabolites: 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], 25-hydroxyvitamin D3 (25OHD3) and 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] by surface plasmon resonance (SPR). At the same time, five different analogues, synthetized in our laboratory, were evaluated in order to compare the affinity values with the metabolites. Real-time SPR measurements showed that 25OHD3 and 24,25(OH)2D3 had higher affinity (0.3 µM) than 1,25(OH)2D3 (5 µM), with the higher affinity of 25OHD3 and 24,25(OH)2D3 due to dissociation constants 1 order of magnitude slower. In the case of the analogues, the affinity values were lower, with 1-hydroxy-25-nitro-vitamin D3 (NO2-446), structurally closer to 1,25(OH)2D3, showing the highest value with a K D of 50 µM. (24R)-1,25-dihydroxyvitamin-24-buthyl-28-norvitamin D2 (Bu-471) and (24R)-1,25-dihydroxyvitamin-24-phenyl-28-norvitamin D2 (Ph-491), structurally similar, had affinities of 180 and 170 µM, respectively. (22R,23E)-1-hydroxy-22-ethenyl-25-methoxy-23-dehydrovitamin D3 (MeO-455) and 1-hydroxy-20(R)-[5(S)-(2,2-dimethyltetrahydropyran-5-yl)]-22,23-dinor vitamin D3 (Oxan-429) had affinities of 310 and 100 µM, respectively. The binding of the metabolites and analogues was reversible allowing the rapid capture of data for replicates. The kinetic and equilibrium data for both the metabolites and the analogues fitted to the Langmuir model describing a 1:1 interaction. Graphical Abstract Label-free, real time binding study between vitamin D binding protein immmobilized on the surface of a SPR sensor chip and the vitamin D, metabolites and analogues passed over it as analytes.

A quantitative binding study of fibrinogen and human serum albumin to metal oxide nanoparticles by surface plasmon resonance.

The interaction of plasma proteins with metal oxide nanoparticles (NPs) is important due to the potential biomedical application of these NPs. In this study, new approaches were applied to measure quantitatively the kinetics and affinities of fibrinogen and human serum albumin (HSA) for TiO2, CeO2, Al2O3 and ZnO NPs immobilized on a sensor chip. Real-time surface plasmon resonance (SPR) measurements showed that fibrinogen interacted with TiO2 and CeO2 NPs with high affinity (135 and 40 pM, respectively) and to Al2O3 NPs with moderate affinity (15 nM). The data fitted well to the Langmuir model describing a 1:1 interaction. In contrast, HSA interacted with TiO2, CeO2 and Al2O3 NPs with lower affinity (80 nM, 37 nM and 2 µM, respectively) with the data fitting better to the conformational change model. TiO2 and CeO2 NPs had fast association rate constants with fibrinogen (1×10(6) M(-1) s(-1)) and Al2O3 NPs had a slower association rate constant (1×10(4) M(-1) s(-1)). By contrast, HSA had markedly slower association rate constants (1×10(3)-1×10(4) M(-1) s(-1)). The binding of the proteins was reversible, thus allowing the rapid capture of data for replicates. The occurrence of matrix effects was evaluated by using surfaces with different chemistries to capture the NPs, namely alginate, NeutrAvidin and bare gold. The affinity values determined for the NP-protein interactions were largely independent of the underlying surface used to capture the NPs.

Fabrication of carbohydrate surfaces by using non-derivatised oligosaccharides.

Surface-based tools, such as microarrays and optical biosensors, are being increasingly applied to the analysis of carbohydrate-protein interactions. A key to these developments is the presentation of the carbohydrate to the protein target. Dual polarisation interferometry (DPI) is a surface-based technique that permits the real-time measurement of the changes in thickness, refractive index, and mass of adsorbates 100-nm thick or less on the surface of a functionalised waveguide. DPI has been used to design and characterise a surface on which the orientation and density of the immobilised carbohydrates are suitable for studying their interactions with proteins and where non-specific binding is reduced to less than 5% of total binding. A thiol-functionalised surface was derivatised with a heterobifunctional cross-linker to yield a hydrazide surface. This was treated with oligosaccharides, derived from keratan sulphate, chondroitin sulphate, and heparin that possess a reducing end. To block the unreacted hydrazide groups, the surface was treated with an aldehyde-functionalised PEG, and the surfaces were then challenged with a variety of proteins.

Chemical and biological characterisation of a sensor surface for bioprocess monitoring.

This paper describes the step-wise fabrication and characterisation of a multi-layer dual polarization interferometry (DPI) based biosensor utilising Protein G (ProG) as the bio-recognition layer for the detection of a fragment antibody (Fab'). The biosensor is capable of monitoring the concentration of Fab' product within the extracellular medium of a fed-batch fermentation after leakage from Escherichia coli (E.coli). The activity, stability and functionality of each sensor layer were analysed in situ using DPI, whilst the chemical identity and homogeneity of the chemical layers were assessed ex situ using X-ray photoelectron spectroscopy (XPS) and secondary ion mass spectrometry (SIMS). Two different biotin linkers were found to produce hugely differing surfaces after the capture of NeutrAvidin™ (NA) and biotinylated Protein G (b-ProG). The hydrophilic (PEG)(4)-biotin linker resulted in a surface where the b-ProG layer was deposited and organised above the NA layer producing an active and stable surface, whilst the hydrophobic LC-biotin linker generated a surface where the b-ProG layer was buried within the NA layer leading to variable surfaces and poor binding of the Fab' target. The biosensor has a detection limit of 1.7 µg/ml with a dynamic range covering two orders of magnitude. The sensor can detect the onset of Fab' leakage as early as 2h following product induction, with high signal-to-noise ratios and little interference from extracellular components. Leakage of Fab' followed a biphasic profile, switching to a more rapid rate 20 h after induction, indicating accelerated product loss and the need for cultivation harvest.

The membrane insertion of helical antimicrobial peptides from the N-terminus of Helicobacter pylori ribosomal protein L1.

The interaction of two helical antimicrobial peptides, HPA3 and HPA3P with planar supported lipid membranes was quantitatively analysed using two complementary optical biosensors. The peptides are analogues of Hp(2-20) derived from the N-terminus of Helicobacter pylori ribosomal protein L1 (RpL1). The binding of these two peptide analogues to zwitterionic dimyristoyl-phosphatidylcholine (DMPC) and negatively charged membranes composed of DMPC/dimyristoylphosphatidylglycerol (DMPG) (4:1) was determined using surface plasmon resonance (SPR) and dual polarisation interferometry (DPI). Using SPR analysis, it was shown that the proline substitution in HPA3P resulted in much lower binding for both zwitterionic and anionic membranes than HPA3. Structural changes in the planar DMPC and DMPC/DMPG (4:1) bilayers induced by the binding of both Hp(2-20) analogues were then resolved in real-time with DPI. The overall process of peptide-induced changes in membrane structure was analysed by the real-time changes in bound peptide mass as a function of bilayer birefringence. The insertion of both HPA3 and HPA3P into the supported lipid bilayers resulted in a decrease in birefringence with increasing amounts of bound peptide which reflects a decrease in the order of the bilayer. The binding of HPA3 to each membrane was associated with a higher level of bound peptide and greater membrane lipid disordering and a faster and higher degree of insertion into the membrane than HPA3P. Furthermore, the binding of both HPA3 and HPA3P to negatively charged DMPC/DMPG bilayers also leads to a greater disruption of the lipid ordering. These results demonstrate the geometrical changes in the membrane upon peptide insertion and the extent of membrane structural changes can be obtained quantitatively. Moreover, monitoring the effect of peptides on a structurally characterised bilayer has provided further insight into the role of membrane structure changes in the molecular basis of peptide selectivity and activity and may assist in defining the mode of antimicrobial action.

Fabrication of carbohydrate surfaces by using nonderivatised oligosaccharides, and their application to measuring the assembly of sugar-protein complexes.

This way up. Dual polarisation interferometry was used to design and characterise a surface on which the orientation and density of immobilised carbohydrates was suitable for studying their interactions with proteins. Lactoferrin was shown to adopt two orientations: "end-on" or "side-on", while for FGF-2 a single monolayer of protein was observed. The new surface can be used to elucidate the binding of proteins to carbohydrates and the geometry of the complexes, a frequently controversial area. Surface-based tools, such as microarrays and optical biosensors, are being increasingly applied to the analysis of carbohydrate-protein interactions. A key to these developments is the presentation of the carbohydrate to the protein target. Dual polarisation interferometry (DPI) is a surface-based technique that permits the real-time measurement of the changes in thickness, refractive index and mass of adsorbates 100 nm thick or less on the surface of a functionalised waveguide. DPI has been used to design and characterise a surface on which the orientation and density of the immobilised carbohydrates is suitable for studying their interactions with proteins and where nonspecific binding is reduced to less than 5 % of total binding. A thiol-functionalised surface was derivatised with a heterobifunctional crosslinker to yield a hydrazide surface. This was treated with oligosaccharides, derived from keratan sulfate (KS) chondroitin sulfate (CS) and heparin, that possess a reducing end. To block the unreacted hydrazide groups, the surface was treated with an aldehyde-functionalised PEG. The heparin DP-10 surfaces were then used to determine the performance of the immobilised DP-10 with respect to binding of two well-characterised proteins, lactoferrin (Lf) and fibroblast growth factor-2. The results show that Lf could adopt two different orientations, at high protein loadings the protein layer thickness corresponded to an "end-on" orientation of Lf, whilst rinsing with buffer saw the Lf molecules adopt a "side-on" configuration. In the case of FGF-2, a single monolayer of protein bound to DP-10 was observed. These results demonstrate that the new surface can be used to resolve key questions relating to the binding of proteins to carbohydrates, including, when used in DPI, the resolution of the geometry of complexes, an area that is frequently controversial.

Interaction of an artificial antimicrobial peptide with lipid membranes.

Antimicrobial peptides constitute an important part of the innate immune defense and are promising new candidates for antibiotics. Naturally occurring antimicrobial peptides often possess hemolytic activity and are not suitable as drugs. Therefore, a range of new synthetic antimicrobial peptides have been developed in recent years with promising properties. But their mechanism of action is in most cases not fully understood. One of these peptides, called V4, is a cyclized 19 amino acid peptide whose amino acid sequence has been modeled upon the hydrophobic/cationic binding pattern found in Factor C of the horseshoe crab (Carcinoscorpius rotundicauda). In this work we used a combination of biophysical techniques to elucidate the mechanism of action of V4. Langmuir-Blodgett trough, atomic force microscopy, Fluorescence Correlation Spectroscopy, Dual Polarization Interference, and confocal microscopy experiments show how the hydrophobic and cationic properties of V4 lead to a) selective binding of the peptide to anionic lipids (POPG) versus zwitterionic lipids (POPC), b) aggregation of vesicles, and above a certain concentration threshold to c) integration of the peptide into the bilayer and finally d) to the disruption of the bilayer structure. The understanding of the mechanism of action of this peptide in relation to the properties of its constituent amino acids is a first step in designing better peptides in the future.

Optical anisotropy of supported lipid structures probed by waveguide spectroscopy and its application to study of supported lipid bilayer formation kinetics.

Supramolecular conformation and molecular orientation was monitored during supported lipid bilayer (SLB) formation using dual polarization interferometry (DPI). DPI was shown to enable real time sensitive determination of birefringence of the lipid bilayer together with thickness or refractive index (with the other a fixed value). This approach removes differences in mass loading due to anisotropy, so the mass becomes solely a function of the lipid d n/d c value. DPI measurements show highly reproducible qualitative and quantitative results for adsorption of liposomes of different lipid compositions and in buffers with or without CaCl 2. The packing of solvent-free self-assembled SLBs is shown to differ from other preparation methods. Birefringence analysis accompanied by mass and thickness measurements shows characteristic features of vesicle adsorption and SLB formation kinetics previously not demonstrated by evanescent optical techniques, including indications of percolation-type rupture of clusters of liposomes on the surface and correlated adsorption kinetics induced by liposome charge repulsion. Our study demonstrates that understanding of mechanistic details for an adsorption process for which conformational changes and ordering occur can be elucidated using DPI and greatly enhanced by modeling of optical birefringence. The data is in some respects more detailed than what can be obtained with conventional biosensing techniques like surface plasmon resonance and complementary to methods such as the quartz crystal microbalance.

Characterisation of membrane mimetics on a dual polarisation interferometer.

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 degrees from surface normal, and possessed a mass of 1.05+/-0.10 ng mm(-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 ng mm(-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 ng mm(-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.

Apolipoprotein E isoprotein-specific interactions with tissue plasminogen activator.

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 detectable 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.

A new quantitative optical biosensor for protein characterisation.

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".