Surface Binding Sites Research Articles

Functionalized gold nanoparticle-enhanced competitive assay for sensitive small-molecule metabolite detection using surface plasmon resonance.

The development of sensing systems for the measurement of small molecules is an active area of research. A sensor based approach for the measurement of metabolites can potentially provide the simplicity and portability required for widespread use. Rapid detection and quantitation of small-molecule metabolites can potentially emerge as an effective way to link the metabolite profile to the disease state. Surface plasmon resonance (SPR) combined with molecular recognition elements to deliver high specificity is a sensing platform that has been widely applied for a large range of biomolecules. However, direct detection of small molecules with SPR challenges the refractive index based detection mechanism. The work described here combines a periplasmic binding protein for recognition with target modified gold nanoparticles (AuNPs) in a competitive assay format for folic acid (FA) detection. Specifically, a SPR imaging substrate containing immobilized folate binding protein (FBP) is used to measure the adsorption of FA conjugated AuNPs. The immobilization of the FBP and the binding of the FA conjugated AuNPs are characterized and optimized. It is shown that free FA in solution can be quantitatively measured by competition for the surface binding sites with the functionalized AuNPs. We demonstrate that the dynamic range can be lowered from micromolar to nanomolar by simply decreasing the concentration of FA conjugated AuNPs, thus lowering the limit of detection to 2.9 nM. This type of competitive assay can be applied to a range of small molecules, which paves the way for future multiplexed analysis of metabolites using SPR.

The Importance of Surface-Binding Site towards Starch-Adsorptivity Level in α-Amylase: A Review on Structural Point of View.

Starch is a polymeric carbohydrate composed of glucose. As a source of energy, starch can be degraded by various amylolytic enzymes, including α-amylase. In a large-scale industry, starch processing cost is still expensive due to the requirement of high temperature during the gelatinization step. Therefore, α-amylase with raw starch digesting ability could decrease the energy cost by avoiding the high gelatinization temperature. It is known that the carbohydrate-binding module (CBM) and the surface-binding site (SBS) of α-amylase could facilitate the substrate binding to the enzyme's active site to enhance the starch digestion. These sites are a noncatalytic module, which could interact with a lengthy substrate such as insoluble starch. The major interaction between these sites and the substrate is the CH/pi-stacking interaction with the glucose ring. Several mutation studies on the Halothermothrix orenii, SusG Bacteroides thetaiotamicron, Barley, Aspergillus niger, and Saccharomycopsis fibuligera α-amylases have revealed that the stacking interaction through the aromatic residues at the SBS is essential to the starch adsorption. In this review, the SBS in various α-amylases is also presented. Therefore, based on the structural point of view, SBS is suggested as an essential site in α-amylase to increase its catalytic activity, especially towards the insoluble starch.

The influence of organic-binding metals on the biogas conversion of sewage sludge

The anaerobic conversion of sewage sludge to methane-rich biogas is an important bioenergy strategy that has been hindered by low conversion efficiency. The poorly understood mechanism of the influence of the key structural component in sludge is responsible for this. The influence of organic-binding metals (OBM), which account for a substantial proportion of metals in sludge, on biogas conversion of both sewage sludge and model sludge were explored in this study. It is observed that the net cumulative methane production of sludge decreased by 23% with the increase of OBM content, implying the crucial role of the OBM in anaerobic sludge digestion. Experimental results showed that the apparent activation energy of sludge organic solubilisation and the median particle size of sludge particulates increased with increasing OBM content, whereas the surface binding sites for enzymes decreased, indicating that the stability of the sludge floc was reinforced by the effect of OBM. Further analyses of the sludge structure revealed that a high OBM content (>2.5% total solids in the present study) compacted the sludge organic matter, restricted the molecular mobility and deteriorated the depolymerisation of the biopolymers by bridging and hydrogen-bonding interactions. This suggests that as a result of the effect of OBM, the hydrolysis and acidification of sludge particulate could be inhibited, resulting in poor biogas conversion. Moreover, it was further authenticated by the results from biochemical methane potential assay process. These findings can deepen the understanding of the role of OBM in sludge for biogas conversion and are important for the improvement of anaerobic sludge digestion.

Improved antibody-based ricin neutralization by affinity maturation is correlated with slower off-rate values.

While potent monoclonal antibodies against ricin were introduced over the years, the question whether increasing antibody affinity enables better toxin neutralization was not fully addressed yet. The aim of this study was to characterize the contribution of antibody affinity to the ricin neutralization potential of the antibody. cHD23 monoclonal antibody that targets the toxin B-subunit and interferes with its binding to membranal receptors, was isolated. In order to create antibody clones with improved affinity toward ricin, a scFv-phage display library containing mutated versions of the variable regions of cHD23 was constructed and clones with improved binding of ricin were isolated. Structural modeling of these mutants suggests that the inserted mutations may increase the antibody conformational flexibility thus improving its ability to bind ricin. While it was found that the selected clones exhibited improved neutralization of ricin, the correlation between the KD values and potency was only minor (r = 0.55). However, a positive correlation (r = 0.84) exist between the off-rate values (koff) of the affinity matured clones and their ability to neutralize ricin. As cell membranes display inordinately large amounts of potential surface binding sites for ricin, it is suggested that antibodies with improved off-rate values block the ability of the toxin to bind to target receptors, in a highly efficient manner. Currently, antibody-based therapy is the most effective treatment for ricin intoxication and it is anticipated that the findings of this study will provide useful information and a possible strategy to design an improved antibody-based therapy for the toxin.

Characterization of surface binding sites in glycoside hydrolases: A computational study.

Structural properties of carbohydrate surface binding sites (SBSs) were investigated with computational methods. Eighty-five SBSs of 44 enzymes in 119 Protein Data Bank (PDB) files were collected as a dataset. On the basis of SBSs shape, they were divided into 3 categories: flat surfaces, clefts, and cavities (types A, B, and C, respectively). Ligand varieties showed the correlation between shape of SBSs and ligands size. To reduce cut-off differences in each SBSs with different ligand size, molecular docking were performed. Molecular docking results were used to refine SBSs classification and binding sites cut-off. Docking results predicted putative ligands positions and displayed dependence of the ligands binding mode to the structural type of SBSs. Physicochemical properties of SBSs were calculated for all docking results with YASARA Structure. The results showed that all SBSs are hydrophilic, while their charges could vary and depended to ligand size and defined cut-off. Surface binding sites type B had highest average values of solvent accessible surface area. Analysis of interactions showed that hydrophobic interactions occur more than hydrogen bonds, which is related to the presence of aromatic residues and carbohydrates interactions.

Origin of Chain Length Specificites of Starch Branching Enzyme

Starch Branching enzyme (BE) is one of the three enzymes involved in starch biosynthesis. It is responsible for synthesizing the alpha-1,6-glucan branches, remodeling the linear alpha-1,4-glucan polymer to produce amylopectin. There are at least two isoforms of the enzyme in most plants, with each having distinct reactivities and product chain-length specificities. Though the chemistry of the active site of branching enzyme is relatively well studied, the origin of the chain length specificity is yet to be understood. Using protein crystallography and biochemical studies on rice branching enzyme, we aim to understand the factors controlling the chain-length specificity of branching enzymes. The mechanism of branching enzyme involves two steps: (1) An oligosaccharide (donor chain) binds to the enzyme and is cleaved by the action of a nucleophilic aspartate residue to form a covalently-linked enzyme-glucan intermediate. (2) A second oligosaccharide (acceptor chain) then reacts, by nucleophilic attack of one of its alpha-1,6-hydroxyl groups, to form a new alpha-1,6-branch. We have focused on discovering the surface glucan binding sites in branching enzyme because they are likely essential to understanding the specificity of the enzyme. To this end we have obtained a crystal structure of an oligosaccharide (M12)-bound rice branching enzyme, which reveals oligosaccharide binding from the outer surface of the enzyme almost to the active site. Mutations of the residues interacting with the M12 can substantially compromise the enzyme's activity, though none have affected the branch chain specificity. On the other hand, comparison of an isoamylase maltoheptaose (M7)-bound structure with rice branching enzyme suggested another potential glucan surface binding site. Interestingly, mutations in this new site did effect the chain-length specificity of the enzyme. These results suggest that the bound M12 is part of the acceptor chain and the newly identified binding site hosts the donor chain. Together, the data allow us to, for the first time propose a detailed mechanism for the enzyme, explaining how disparate surface glucan binding sites far from the active site create the enzyme's activity and specificity against its polymeric substrate.

Photoinduced Charge and Energy Transfer in Complexes of C-Type Cytochromes with Water-Soluble Porphyrins

Due to their rich and tunable spectral and redox properties as well as relatively simple synthesis protocols, water-soluble porphyrins are promising candidates as photosensitizers for artificial photosynthesis. Quenching of porphyrin static fluorescence by monoheme horse heart cytochrome (cyt) c is a well-known phenomenon. It is attributed to the fast charge separation (CS), though the CS state has not been observed. Similarly, we found quenching of static fluorescence of porphyrins in the presence of PpcA, a 3-heme c-type cytochrome from the cyt c7 family from Geobacter sulfurreducens. In this report using ultrafast visible transient absorbance spectroscopy we demonstrate that the excited state of protein-bound zinc tetraphenyl sulfonated porphyrin (ZnTPPS) decays with an apparent time constant of about 200 ps in the presence of cyt c and approximately 20 ps in the presence of PpcA. However, the spectral changes are dominated by the features of porphyrin singlet and triplet states. Only a small fraction of photosensitizer excited state energy is retained in the CS state suggesting a competing energy transfer mechanism. Unbound fraction of ZnTPPS shows triplet quenching as a second order process with similar low CS state yields for both cyt c and PpcA. Heteronuclear NMR and all-atom molecular dynamics simulations reveal the likely surface binding sites for ZnTPPS. However, small angle X-ray scattering measurements demonstrate propensity of ZnTPPS to induce formation of protein multimers under experimental conditions typical for our experiments. There results demonstrate an unexpected complexity of ZnTPPS binding interactions and photochemistry with c-type cytochromes and suggest the presence of a competing dominant energy transfer process.

Computational Model of the Effect of a Surface-Binding Site on the Saccharomycopsis fibuligera R64 α-Amylase to the Substrate Adsorption.

α-Amylase is one of the important enzymes in the starch-processing industry. However, starch processing requires high temperature, thus resulting in high cost. The high adsorptivity of α-amylase to the substrate allows this enzyme to digest the starch at a lower temperature. α-Amylase from Saccharomycopsis fibuligera R64 (Sfamy R64), a locally sourced enzyme from Indonesia, has a high amylolytic activity but low starch adsorptivity. The objective of this study was to design a computational model of Sfamy R64 with increased starch adsorptivity using bioinformatics method. The model structure of Sfamy R64 was compared with the positive control, ie, Aspergillus niger α-amylase. The structural comparison showed that Sfamy R64 lacks the surface-binding site (SBS). An SBS was introduced to the structure of Sfamy R64 by S383Y/S386W mutations. The dynamics and binding affinity of the SBS of mutant to the substrate were also improved and comparable with that of the positive control.

Affinity Electrophoresis for Analysis of Catalytic Module-Carbohydrate Interactions.

Affinity electrophoresis has long been used to study the interaction between proteins and large soluble ligands. The technique has been found to have great utility for the examination of polysaccharide binding by proteins, particularly carbohydrate binding modules (CBMs). In recent years, carbohydrate surface binding sites of proteins mostly enzymes have also been investigated by this method. Here, we describe a protocol for identifying binding interactions between enzyme catalytic modules and a variety of carbohydrate ligands.

Microbial α-amylase: A biomolecular overview

α-Amylase is an important amylolytic enzyme participating in hydrolysis of starch, the most common carbohydrate in nature. Compared to plant and animal origins, microbial α-amylase is the most popular source of industrial α-amylase. As such, high productive and favourable α-amylases for wider range of applications are highly sought after demands. The expression of α-amylase is regulated by its structural gene, amyR, DegU-P, PrsA lipoprotein, cutinase and other similar flanking genes, components of gene expression regulatory systems, molecular chaperones and enzymes. Moreover, the characteristics of α-amylase are closely related to the structures of constitutive domains and conserved regions, particularly the functional regions such as Ca2+-binding sites, non-catalytic carbohydrate-binding modules and surface-binding sites. Recent production of α-amylase based on genetic engineering and academic researches focused on mechanisms of catalysis greatly benefit from these biomolecular studies. Despite rapid developments, no reviews have systematically summarized these fundamental biomolecular studies. This review outlines microbial α-amylase at gene and structure levels by covering these significant aspects. The computer analytical tools are also reported, especially frequently used databases. A deeper understanding of the biomolecular basis of microbial α-amylase will significantly pave greater opportunities for industrial α-amylase and open our minds towards its related or even other enzymes.

Evaluation of the Significance of Starch Surface Binding Sites on Human Pancreatic α-Amylase.

Starch provides the major source of caloric intake in many diets. Cleavage of starch into malto-oligosaccharides in the gut is catalyzed by pancreatic α-amylase. These oligosaccharides are then further cleaved by gut wall α-glucosidases to release glucose, which is absorbed into the bloodstream. Potential surface binding sites for starch on the pancreatic amylase, distinct from the active site of the amylase, have been identified through X-ray crystallographic analyses. The role of these sites in the degradation of both starch granules and soluble starch was probed by the generation of a series of surface variants modified at each site to disrupt binding. Kinetic analysis of the binding and/or cleavage of substrates ranging from simple maltotriosides to soluble starch and insoluble starch granules has allowed evaluation of the potential role of each such surface site. In this way, two key surface binding sites, on the same face as the active site, are identified. One site, containing a pair of aromatic residues, is responsible for attachment to starch granules, while a second site featuring a tryptophan residue around which a malto-oligosaccharide wraps is shown to heavily influence soluble starch binding and hydrolysis. These studies provide insights into the mechanisms by which enzymes tackle the degradation of largely insoluble polymers and also present some new approaches to the interrogation of the binding sites involved.

Hydrogen Recombination Rates on Silica from Atomic-Scale Calculations

A combined molecular dynamics (MD) and Monte Carlo approach was used to bridge time scales, enabling calculations of surface recombination rates for hydrogen on silica. MD was used for temperatures between 10 and 600 K at a high pressure of 10 atm, yielding recombination coefficients between 0.1 and 1. For the lower pressures more common in applications, low recombination rates make the corresponding calculations intractably expensive. A Monte Carlo technique, informed by the MD simulations, was designed to bridge the essential time scales. Distinct weak and strong surface binding sites for atomic hydrogen with densities of approximately 10 nm–2 were found using grand canonical Monte Carlo (GCMC) simulations, which, in turn, were used to obtain Eley–Rideal rate constants based on semiequilibrium theory. Monte Carlo variational transition state theory (MCVTST) was used to calculate Langmuir–Hinshelwood and thermal desorption rate constants for hydrogen atoms in strong and weak adsorption sites. Calculated reaction rates were used in a Langmuir kinetics model to estimate the recombination coefficient γ for T = 10–2000 K at gas-phase radical densities between 1012 and 1016 cm–3, yielding values of γ = 10–4–0.9.

Monomolecular Siloxane Film as a Model of Single Site Catalysts.

Achieving structurally well-defined catalytic species requires a fundamental understanding of surface chemistry. Detailed structural characterization of the catalyst binding sites in situ, such as single site catalysts on silica supports, is technically challenging or even unattainable. Octadecyltrioxysilane (OTOS) monolayers formed from octadecyltrimethoxysilane (OTMS) at the air-liquid interface after hydrolysis and condensation at low pH were chosen as a model system of surface binding sites in silica-supported Zn(2+) catalysts. We characterize the system by grazing incidence X-ray diffraction, X-ray reflectivity (XR), and X-ray fluorescence spectroscopy (XFS). Previous X-ray and infrared surface studies of OTMS/OTOS films at the air-liquid interface proposed the formation of polymer OTOS structures. According to our analysis, polymer formation is inconsistent with the X-ray observations and structural properties of siloxanes; it is energetically unfavorable and thus highly unlikely. We suggest an alternative mechanism of hydrolysis/condensation in OTMS leading to the formation of structurally allowed cyclic trimers with the six-membered siloxane rings, which explain well both the X-ray and infrared results. XR and XFS consistently demonstrate that tetrahedral [Zn(NH3)4](2+) ions bind to hydroxyl groups of the film at a stoichiometric ratio of OTOS:Zn ∼ 2:1. The high binding affinity of zinc ions to OTOS trimers suggests that the six-membered siloxane rings are binding locations for single site Zn/SiO2 catalysts. Our results show that OTOS monolayers may serve as a platform for studying silica surface chemistry or hydroxyl-mediated reactions.

Using Carbohydrate Interaction Assays to Reveal Novel Binding Sites in Carbohydrate Active Enzymes.

Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical for the activity of their cognate enzyme, though they are not readily detected in the sequence of a protein, but normally require a crystal structure of a complex for their identification. A variety of methods, including affinity electrophoresis (AE), insoluble polysaccharide pulldown (IPP) and surface plasmon resonance (SPR) have been used to study auxiliary binding sites. These techniques are complementary as AE allows monitoring of binding to soluble polysaccharides, IPP to insoluble polysaccharides and SPR to oligosaccharides. Here we show that these methods are useful not only for analyzing known binding sites, but also for identifying new ones, even without structural data available. We further verify the chosen assays discriminate between known SBS/CBM containing enzymes and negative controls. Altogether 35 enzymes are screened for the presence of SBSs or CBMs and several novel binding sites are identified, including the first SBS ever reported in a cellulase. This work demonstrates that combinations of these methods can be used as a part of routine enzyme characterization to identify new binding sites and advance the study of SBSs and CBMs, allowing them to be detected in the absence of structural data.

Biochip-based instruments development for space exploration: influence of the antibody immobilization process on the biochip resistance to freeze-drying, temperature shifts and cosmic radiations

AbstractDue to the diversity of antibody (Ab)-based biochips chemistries available and the little knowledge about biochips resistance to space constraints, immobilization of Abs on the surface of the biochips dedicated to Solar System exploration is challenging. In the present paper, we have developed ten different biochip models including covalent or affinity immobilization with full-length Abs or Ab fragments. Ab immobilizations were carried out in oriented/non-oriented manner using commercial activated surfaces withN-hydroxysuccinic ester (NHS-surfaces) or homemade surfaces using three generations of dendrimers (dendrigraft of poly L-lysine (DGL) surfaces). The performances of the Ab -based surfaces were cross-compared on the following criteria: (i) analytical performances (expressed by both the surface density of immobilized Abs and the amount of antigens initially captured by the surface) and (ii) resistance of surfaces to preparation procedure (freeze-drying, storage) or spatial constraints (irradiation and temperature shifts) encountered during a space mission. The latter results have been expressed as percentage of surface binding capacity losses (or percentage of remaining active Abs). The highest amount of captured antigen was achieved with Ab surfaces having full-length Abs and DGL-surfaces that have much higher surface densities than commercial NHS-surface. After freeze-drying process, thermal shift and storage sample exposition, we found that more than 80% of surface binding sites remained active in this case. In addition, the resistance of Ab surfaces to irradiation with particles such as electron, carbon ions or protons depends not only on the chemistries (covalent/affinity linkages) and strategies (oriented/non-oriented) used to construct the biochip, but also on the type, energy and fluence of incident particles. Our results clearly indicate that full-length Ab immobilization on NHS-surfaces and DGL-surfaces should be preferred for potential use in instruments for planetary exploration.

Stereochemical Recognition of Helicenes on Metal Surfaces.

The chiral recognition among biomolecules is fundamentally important for many processes of life, including the stereochemistry of evolution. Of special interest is chiral recognition during crystallization of racemates, when either homochiral recognition leads to a conglomerate of homochiral crystals or heterochiral recognition dominates resulting in a racemic compound. The complex nature of molecular recognition at the level of nucleation and crystal growth renders it difficult to understand and calls for manageable model systems. Notably, the approach of studying aggregation of molecules at surfaces under well-defined conditions includes the benefit of the availability of a multitude of highly sensitive investigation methods, of which scanning tunneling microscopy (STM) with its submolecular resolution is tremendously valuable. Heterogeneous nucleation at surfaces is strongly favored over homogeneous nucleation in solution; hence, surfaces are significantly involved in stereochemical recognition during crystallization. Helicenes are a fascinating class of chiral compounds with outstanding optical activity. These π-conjugated, ortho-fused, aromatic hydrocarbons are promising candidates for organic electronic devices such as sensors, circular dichroic photonics, liquid crystal displays or spin filters. But in particular the defined footprint of their terminal benzo rings on a surface makes them interesting for studying stereochemical recognition with different single crystalline surfaces and the impact this has, in turn, on intermolecular recognition. In this Account, we describe the self-assembly of helicenes on metal surfaces with the focus on stereochemical recognition in two-dimensional structures. Using the isomeric all-carbon helicenes, heptahelicene and dibenzohelicene as examples, different aggregation phenomena on different surfaces of single crystalline copper, silver, and gold are investigated. By means of STM different modes of transmission of molecular handedness from single molecules into extended two-dimensional supramolecular structures are identified. For the problem of racemate versus conglomerate crystallization, the impact of surface and molecular structure and their interplay are analyzed. This leads to detailed conclusions about the importance of the match of molecular and surface binding sites for long-range self-assembly. The absence of polar groups puts emphasis on van der Waals interaction and their maximization by steric overlap of molecular parts in enantiomeric and diastereomeric interactions. With STM as a manipulation tool, dimers are manually separated in order to analyze their chiral composition. And finally, new nonlinear cooperative effects induced by small enantiospecific bias are discovered that lead to single enantiomorphism in two-dimensional racemate crystals as well as in racemic multilayered films. By means of these model studies many details that govern chiral recognition at surfaces are rationalized.

ChemInform Abstract: Analysis of Surface Binding Sites (SBS) Within GH62, GH13, and GH77

AbstractReview: 57 refs.

Unique carbohydrate binding platforms employed by the glucan phosphatases.

Glucan phosphatases are a family of enzymes that are functionally conserved at the enzymatic level in animals and plants. These enzymes bind and dephosphorylate glycogen in animals and starch in plants. While the enzymatic function is conserved, the glucan phosphatases employ distinct mechanisms to bind and dephosphorylate glycogen or starch. The founding member of the family is a bimodular human protein called laforin that is comprised of a carbohydrate binding module 20 (CBM20) followed by a dual specificity phosphatase domain. Plants contain two glucan phosphatases: Starch EXcess4 (SEX4) and Like Sex Four2 (LSF2). SEX4 contains a chloroplast targeting peptide, dual specificity phosphatase (DSP) domain, a CBM45, and a carboxy-terminal motif. LSF2 is comprised of simply a chloroplast targeting peptide, DSP domain, and carboxy-terminal motif. SEX4 employs an integrated DSP-CBM glucan-binding platform to engage and dephosphorylate starch. LSF2 lacks a CBM and instead utilizes two surface binding sites to bind and dephosphorylate starch. Laforin is a dimeric protein in solution and it utilizes a tetramodular architecture and cooperativity to bind and dephosphorylate glycogen. This chapter describes the biological role of glucan phosphatases in glycogen and starch metabolism and compares and contrasts their ability to bind and dephosphorylate glucans.

Identification of the gC1qR sites for the HIV-1 viral envelope protein gp41 and the HCV core protein: Implications in viral-specific pathogenesis and therapy.

A substantial body of evidence accumulated over the past 20 years supports the concept that gC1qR is a major pathogen-associated pattern recognition receptor (PRR). This conclusion is based on the fact that, a wide range of bacterial and viral ligands are able to exploit gC1qR to either suppress the host's immune response and thus enhance their survival, or to gain access into cells to initiate disease. Of the extensive array of viral ligands that have affinity for gC1qR, the HIV-1 envelope glycoprotein gp41, and the core protein of hepatitis C virus (HCV) are of major interest as they are known to contribute to the high morbidity and mortality caused by these pathogens. While the HCV core protein binds gC1qR and suppresses T cell proliferation resulting in a significantly diminished immune response, the gp41 employs gC1qR to induce the surface expression of the NK cell ligand, NKp44L, on uninfected CD4(+) T cells, thereby rendering them susceptible to autologous destruction by NKp44 receptor expressing NK cells. Because of the potential for the design of peptide-based or antibody-based therapeutic options, the present studies were undertaken to define the gC1qR interaction sites for these pathogen-associated molecular ligands. Employing a solid phase microplate-binding assay, we examined the binding of each viral ligand to wild type gC1qR and 11 gC1qR deletion mutants. The results obtained from these studies have identified two major HCV core protein sites on a domain of gC1qR comprising of residues 144-148 and 196-202. Domain 196-202 in turn, is located in the last half of the larger gC1qR segment encoded by exons IV-VI (residues 159-282), which was proposed previously to contain the site for HCV core protein. The major gC1qR site for gp41 on the other hand, was found to be in a highly conserved region encoded by exon IV and comprises of residues 174-180. Interestingly, gC1qR residues 174-180 also constitute the cell surface-binding site for soluble gC1qR (sgC1qR), which can bind to the cell surface in an autocrine/paracrine manner via surface expressed fibrinogen or other membrane molecules. The identification of the sites for these viral ligands should therefore provide additional targets for the design of peptide-based or antigen-based therapeutic strategies.

Surface Selective 1H and 27Al MAS NMR Observations of Strontium Oxide Doped γ-Alumina

High-surface area γ-alumina is industrially used as a catalyst support. Catalytically active elements are doped onto the support, where they can bind to AlO4, AlO5 or AlO6 sites on the surface. Pretreating the surface with alkaline earth oxides can alter the availability of these surface sites, hence affecting the catalytic activity. The surface binding sites of strontium oxide (SrO) on γ-alumina were previously unknown. Direct 27Al magic angle spinning nuclear magnetic resonance (MAS NMR) could not detect AlO5 sites at 9.4 T, so 1H–27Al cross-polarisation (CP) MAS NMR was used to preferentially select the surface environment signals. We directly observed the three surface environments on dehydrated γ-alumina as a function of SrO impregnation up to 4 wt% SrO. We found that Sr2+ preferentially binds to AlO5 and AlO6 surface sites. 1H MAS NMR revealed SrO impregnation causes a reduction in the terminal (−0.3 ppm) and bridging (2.2 ppm) hydroxyl environments, as well as the promotion of a new peak in between these sites, at 0.5 ppm. By using 1H–27Al CP/MAS NMR the relative proportions of surface sites on γ-alumina can be determined, allowing an optimal level of SrO doping that can saturate all the AlO5 sites. Importantly, this provides a method of subsequently depositing catalytically active elements on just the AlO4 or AlO6 sites, which can provide a different catalytic activity or stability compared to the AlO5 binding site.