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A microcrystalline suspension ofBacillus lentussubtilisin (Savinase) produced during industrial large-scale production was analysed by X-ray powder diffraction (XRPD) and X-ray single-crystal diffraction (MX). XRPD established that the bulk microcrystal sample representative of the entire production suspension corresponded to space groupP212121, with unit-cell parametersa= 47.65,b= 62.43,c= 75.74 Å, equivalent to those for a known orthorhombic crystal form (PDB entry 1ndq). MX using synchrotron beamlines at the Diamond Light Source with beam dimensions of 20 × 20 µm was subsequently used to study the largest crystals present in the suspension, with diffraction data being collected from two single crystals (∼20 × 20 × 60 µm) to resolutions of 1.40 and 1.57 Å, respectively. Both structures also belonged to space groupP212121, but were quite distinct from the dominant form identified by XRPD, with unit-cell parametersa= 53.04,b = 57.55,c= 71.37 Å anda= 52.72,b= 57.13,c= 65.86 Å, respectively, and refined toR= 10.8% andRfree= 15.5% and toR= 14.1% andRfree= 18.0%, respectively. They are also different from any of the forms previously reported in the PDB. A controlled crystallization experiment with a highly purified Savinase sample allowed the growth of single crystals of the form identified by XRPD; their structure was solved and refined to a resolution of 1.17 Å with anRof 9.2% and anRfreeof 11.8%. Thus, there are at least three polymorphs present in the production suspension, albeit with the 1ndq-like microcrystals predominating. It is shown how the two techniques can provide invaluable and complementary information for such a production suspension and it is proposed that XRPD provides an excellent quality-control tool for such suspensions.

The enzymatic degradation of plant polysaccharides is emerging as one of the key environmental goals of the early 21st century, impacting on many processes in the textile and detergent industries as well as biomass conversion to biofuels. One of the well known problems with the use of nonstarch (nonfood)-based substrates such as the plant cell wall is that the cellulose fibers are embedded in a network of diverse polysaccharides, including xyloglucan, that renders access difficult. There is therefore increasing interest in the “accessory enzymes,” including xyloglucanases, that may aid biomass degradation through removal of “hemicellulose” polysaccharides. Here, we report the biochemical characterization of the endo-β-1,4-(xylo)glucan hydrolase from Paenibacillus polymyxa with polymeric, oligomeric, and defined chromogenic aryl-oligosaccharide substrates. The enzyme displays an unusual specificity on defined xyloglucan oligosaccharides, cleaving the XXXG-XXXG repeat into XXX and GXXXG. Kinetic analysis on defined oligosaccharides and on aryl-glycosides suggests that both the −4 and +1 subsites show discrimination against xylose-appended glucosides. The three-dimensional structures of PpXG44 have been solved both in apo-form and as a series of ligand complexes that map the −3 to −1 and +1 to +5 subsites of the extended ligand binding cleft. Complex structures are consistent with partial intolerance of xylosides in the −4′ subsites. The atypical specificity of PpXG44 may thus find use in industrial processes involving xyloglucan degradation, such as biomass conversion, or in the emerging exciting applications of defined xyloglucans in food, pharmaceuticals, and cellulose fiber modification.

Subtilisins represent a large class of microbial serine proteases. To date, there are three-dimensional structures of proteinaceous inhibitors from three families in complex with subtilisins in the Protein Data Bank. All interact with subtilisin via an exposed loop covering six interacting residues. Here we present the crystal structure of the complex between the Bacillus lentus subtilisin Savinase and the barley alpha-amylase/subtilisin inhibitor (BASI). This is the first reported structure of a cereal Kunitz-P family inhibitor in complex with a subtilisin. Structural analysis revealed that BASI inhibits Savinase in a novel way, as the interacting loop is shorter than loops previously reported. Mutational analysis showed that Thr88 is crucial for the inhibition, as it stabilises the interacting loop through intramolecular interactions with the BASI backbone.

Background: A clinically relevant allergic reaction requires recognition of at least two different epitopes on the surface of the allergen by IgE. These epitopes may be specific or cross-reactive. Moreover, patterns of IgE reactivity may be patient-specific. The aim of our study was to compare specific and cross-reactive IgE epitopes and epitope patterns between individual patients. We used Bet v 1-related food allergy as a model. Methods: Five patients were investigated by cross-competitive ELISA for specific and cross-reacting IgE to Bet v 1, and its homologues Gly m 4 (soybean), Ara h 8 (peanut), and Pru av 1 (cherry). Allergen-specific as well as cross-reactive IgE epitopes were assessed by competitive immunoscreening of a phage-displayed random 7-mer peptide library using polyclonal purified IgE from individual sera. The resulting peptide mimics were mapped on the surface of the 3D-structure of the allergens using a computer-based algorithm. Results: Competitive immunoscreening and epitope mapping identified patient-specific IgE epitope patterns. However, one IgE-binding surface area that was recognized by all patients and two recognized by three patients were identified on all four proteins. These results are consistent with the determination of IgE cross-reactivity of the individual patients’ sera against the four recombinant allergens by cross-competitive ELISA. Conclusions: Selection of phage-displayed peptide mimics with serum IgE from allergic patients in combination with computer-based mapping of the peptide mimics onto the surface of the three-dimensional allergen structure is a promising novel tool to investigate IgE epitope specificity in individual patients. Such basic information on epitope structure may be used for prediction of cross-reactivity and potential allergenicity of novel foods.

Presently X-ray crystallography of protein-antibody complexes is still the most direct way of identifying B-cell epitopes. The objective of this study was to assess the potential of a computer-based epitope mapping tool (EMT) using antigenic amino acid motifs as a fast alternative in a number of applications not requiring detailed information, e.g. development of pharmaceutical proteins, vaccines and industrial enzymes. Using Gal d 4 as a model protein, the EMT was capable of identifying, in the context of the folded protein, amino acid positions known to be involved in antibody binding. The high sensitivity and positive predictive value of the EMT as well as the relevance of the structural associations suggested by the EMT indicated the existence of amino acid motifs that are likely to be involved in antigenic determinants. In addition, differential mapping revealed that sensitivity and positive predictive value were dependent on the minimum relative surface accessibility (RSA) of the amino acids included in the mapping, demonstrating that the EMTs accommodated for the fact that epitopes are three-dimensional entities with various degrees of accessibility. The comparison with existing prediction scales demonstrated the superiority of the EMT with respect to physico-chemical scales. The mapping tool also performed better than the available structural scales, but the significance of the differences remains to be established. Thus, the EMT has the potential of becoming a fast and simple alternative to X-ray crystallography for predicting structural antigenic determinants, if detailed epitope information is not required.

A novel method has been developed to fabricate nanoscale pits on Au(111) in aqueous environments by in situ scanning tunnelling microscopy (STM), based on critical interactions between tip and substrate. The most striking advantages of the present method are that the dimension and position of the pits can be controlled well in aqueous environments, and the operations are simple. Parameters affecting the pit formation and size have been systematically characterized to show that pit formation is dominated by bias voltage. A mechanism is proposed based on local surface reconstruction induced by electronic contact between tip and substrate.

Microscopic structures for molecular monolayers of L-cysteine and L-cystine assembled on Au(111) have been disclosed by employing electrochemistry and in situ scanning tunneling microscopy (STM). High-resolution STM images show that the adlayers of both cyteine and cystine exhibit highly-ordered networklike clusters with (3√3 × 6)R30° structure. By combining the surface coverage estimated from voltammetric data, each cluster is demonstrated to include six individual cysteine molecules or three cystine molecules. As a comparison, no cluster structure is observed for the 1-butanethiol adlayer prepared and examined under the same conditions as those for cysteine and cystine. This suggests that intermolecular and intramolecular hydrogen bonds among adsorbed cysteine or cystine molecules could be responsible for the origin of the cluster−network structures for the adlayers. Several models are proposed and used to explain the in situ STM observations in detail.

We provide a comprehensive approach to the formation and characterization of molecular monolayers of the blue copper protein Pseudomonas aeruginosa azurin on Au(111) in aqueous ammonium acetate solution. Main issues are adsorption patterns, reductive desorption, properties of the double layer, and long-range electrochemical electron transfer between the electrode and the copper center. Voltammetry, electrochemical impedance spectroscopy (EIS), in situ scanning tunneling microscopy (STM), and X-ray photoelectron spectroscopy (XPS) have been employed to disclose features of these issues. Zn-substituted azurin, cystine, and 1-butanethiol are investigated for comparison. Cyclic voltammetric and capacitance measurements show qualitatively that azurin is adsorbed at submicromolar concentrations over a broad potential range. The characteristics of reductive desorption suggest that azurin is adsorbed via its disulfide group to form a monolayer. The adsorption of this protein on Au(111) via a gold−sulfur binding m...

We report the self-assembly and electrochemical behaviour of the blue copper protein Pseudomonas aeruginosa azurin on Au(111) electrodes in aqueous acetate buffer (pH=4.6). The formation of monolayers of this protein is substantiated by electrochemical measurements. Capacitance results indicate qualitatively that the protein is strongly adsorbed at sub-μM concentrations in a broad potential range (about 700 mV). This is further supported by the attenuation of a characteristic cyclic voltammetric peak of Au(111) in acetate solution with increasing azurin concentration. Reductive desorption is clearly disclosed in NaOH solution (pH=13), strongly suggesting that azurin is adsorbed via its disulphide group. An anodic peak and a cathodic peak associated with the copper centre of azurin are finally observed in the differential pulse voltammograms. These peaks are, interestingly, indicative of long-range electrochemical electron transfer such as paralleled by intramolecular electron transfer between the disulphide anion radical and the copper atom in homogeneous solution, and anticipated by theoretical frames. Together with reported in situ scanning tunnelling microscopy (STM) results they constitute the first case for electrochemistry of self-assembled monolayers of azurin, even redox proteins. This integrated investigation provides a new approach to both structure and function of adsorbed redox metalloproteins at the molecular level. Keywords: Azurin, Self-assembled monolayers, Gold single-crystal electrodes, Long-range electrochemical electron transfer, Electrochemistry

In situ scanning tunneling microscopy (STM) of redox molecules, in aqueous solution, shows interesting analogies and differences compared with interfacial electrochemical electron transfer (ET) and ET in homogeneous solution. This is because the redox level represents a deep indentation in the tunnel barrier, with possible temporary electronic population. Particular perspectives are that both the bias voltage and the overvoltage relative to a reference electrode can be controlled, reflected in spectroscopic features when the potential variation brings the redox level to cross the Fermi levels of the substrate and tip. The blue copper protein azurin adsorbs on gold(111) via a surface disulfide group. Well resolved in situ STM images show arrays of molecules on the triangular gold(111) terraces. This points to the feasibility of in situ STM of redox metalloproteins directly in their natural aqueous medium. Each structure also shows a central brighter contrast in the constant current mode, indicative of 2- to 4-fold current enhancement compared with the peripheral parts. This supports the notion of tunneling via the redox level of the copper atom and of in situ STM as a new approach to long-range electron tunneling in metalloproteins.

We provide a theoretical frame for in situ scanning tunneling microscopy (STM) of adsorbate molecules with low-lying redox levels strongly coupled to the environmental nuclear motion. The STM process is viewed as a coherent two-step electron transfer (ET) involving electron exchange between the local redox level and the manifolds of electronic levels in the substrate and tip. The notion coherence is here taken to imply that the intermediate electron or hole state after the first ET step does not fully relax vibrationally before the second ET step. These views and the theoretical formalism are appropriate to in situ STM of large transition metal complexes and redox metalloproteins. The formalism offers two kinds of spectroscopic features. One is the relation between the tunnel current and the bias voltage at fixed overvoltage of either the tip or the substrate relative to a reference electrode. The other one is the tunnel current dependence on the overvoltage, at fixed bias voltage. Recent data on tunnelin...

Redox metalloproteins exhibit interesting features such as long-range electron transfer (ET), cooperative effects etc. of importance in relation to fundamental ET theory, and mapped in considerable detail. Adsorption and interfacial electrochemical ET of metalloproteins at metallic surfaces is also broadly important in a range of contexts, and has been addressed by spectroscopic, voltammetric, and thermodynamic methods. In situ scanning tunneling (STM) and atomic force microscopy (AFM) have opened new perspectives for addressing adsorbed metalloproteins in their natural functional aqueous medium at the molecular level. In addition to broadly recognized problems of in situ STM AFM imaging, sample preparation, mobility, and adsorbate stability are, however, particular problems. We illustrate here the perspectives by recent in situ STM imaging of covalently bound horse heart cytochrome c on polycrystalline platinum, and of chemisorbed Pseudomonas aeruginosa azurin on Au(111). Molecular resolution is achieved, but azurin gives by far the best images which show, moreover, an interesting submolecular feature. This is likely to be associated with the disulphide group as a natural unit for gentle linking, facile ET routes through the protein, and tunnel enhancement by the low-lying redox level of the copper atom. The particular electronic-vibrational three-level configuration in in situ STM of metalloproteins. finally, offers a new way of distinction between superexchange, coherent, and sequential ET modes in the long-range ET patterns of metalloproteins.

AFM instrument with no need for instrumental modification, but can easily be adapted to other commercial instruments. The cell is a closed system with insignificant sample evaporation. It is a chemically and mechanically robust two-component system which enables fast assembly and testing prior to insertion and minimizes leakage problems. The cell is also laterally flexible, facilitating scanning of large areas, holds inlets for rapid flushing and change of solution, and contains an optical device for adjusting the laser beam deflection in aqueous and gas ambient environments.

Scanning tunnel (STM) and atomic force microscopy (AFM) in the in situ mode under potentiostatic control have opened new perspectives for mapping the two-dimensional organization of surface adsorbates in aqueous solution. In situ STM and AFM, however, also raise recognized problems. In the context of biological macromolecules, sample immobilization and the mechanism of the imaging process are, for example, outstanding issues. We have shown that the blue single-copper redox protein azurin is well suited for gentle surface immobilization and mapping. Azurin has a surface disulphide group which adsorbs to gold and facile electron tunnel routes between this group and the copper atom. Azurin adsorbed on Au(111) can be imaged to molecular resolution by in situ STM and shows regular arrays of individual structures corresponding well to the known molecular size of azurin. The current falls off approximately exponentially with increasing distance with a decay constant of 0.4–0.5 A −1 . In comparison in situ AFM shows structures laterally convoluted with the tip while the vertical extension is in the same range as the structural size of azurin. The results are of interest in relation to electron tunnel mechanisms of redox metalloproteins and in technological contexts such as electrochemical biosensors, microbial corrosion and broadly for protein adsorption from biological liquids.

Laccases (E.C. 1.10.3.2) are multicopper oxidases catalytically active in the oxidation of diphenolics and related compounds by molecular dioxygen. The laccases contain a single-copper type I center and a trinuclear cluster of a single-copper type II and a dinuclear type III center. The oxidation of four equivalents of substrate near the type I copper and the sequential transfer of electrons to the trinuclear cluster are coupled with four-electron reduction of O2 to H2O at the latter site. Extensive efforts have been given to kinetic and structural characterization of numerous laccases to elucidate the catalytic mechanism, where laccase (sub)monolayer voltammetry has been a core approach. In this report, we address voltammetry and electrocatalysis of O2 reduction of (sub)monolayers of several laccases in new ways. These are based on the use of single-crystal, atomically planar bare Au(111)-electrode surfaces or surfaces modified by thiol-based self-assembled molecular monolayers. These well-defined surfaces enable introducing electrochemical scanning tunneling microscopy directly in aqueous biological media in which the enzymes are operative (in situ STM), to the level of resolution of the single enzyme molecule in electrocatalytic action. Enzyme-electrode electronic contact and intramolecular electron transfer triggered by the electrode potential or by O2-substrate binding to the enzyme, followed at the single-molecule level, are the most important observations of this study. © 2011 American Chemical Society.

Cellulases, including cellobiohydrolases and endoglucanases, are important enzymes involved in the breakdown of the polysaccharide cellulose. These catalysts have found widescale industrial applications, particularly in the paper and textile industries, and are now finding use in `second-generation' conversion of biomass to biofuels. Despite this considerable biotechnological application, and undoubted future potential, uncertainty remains as to the exact reaction mechanism of the inverting cellulases found in the GH6 family of carbohydrate-active enzymes. In order to gain additional understanding as to how these societally beneficial biocatalysts function, the crystal structure of a GH6 cellobiohydrolase from Chaetomium thermophilum, CtCel6A, has been solved. This structure reveals a distorted α/β-barrel fold comprising a buried tunnel-like active site quite typical of Cel6A enzymes. Analysis of an enzyme-product complex (cellobiose in the -3 and -2 subsites and cellotetraose in subsites +1 to +4) supports the hypothesis that this group of enzymes act via an atypical single-displacement mechanism. Of particular note in this analysis is an active-centre metal ion, Li(+), the position of which matches the position of the positively charged anomeric carbon of the oxocarbenium-ion-like transition state.

Thermostable enzymes employ various structural features dictated at the amino-acid sequence level that allow them to maintain their integrity at higher temperatures. Many hypotheses as to the nature of thermal stability have been proposed, including optimized core hydrophobicity and an increase in charged surface residues to enhance polar solvent interactions for solubility. Here, the three-dimensional structure of the family GH11 xylanase from the moderate thermophileThermobifida fuscain its trapped covalent glycosyl-enzyme intermediate complex is presented. Interactions with the bound ligand show fewer direct hydrogen bonds from ligand to protein than observed in previous complexes from other species and imply that binding of the xylan substrate involves several water-mediated hydrogen bonds.

Cyclic voltammetry of Pseudomonas aeruginosa azurin on polycrystalline gold is reversible ( E 0 = 360 mV vs she; 50 mM ammonium acetate) but the voltammetric signals decay with time constants of about 3 × 10 −3 s −1 . No signal is observed for monocrystalline Au(111). Cys3Ser azurin is electrochemically inactive on either type of gold electrode but shows a reversible although decaying peak (362 mV, 50 mM ammonium acetate; decay time constant ≈2 × 10 −3 s −1 ) on edge-plane pyrolytic graphite. Ex situ and in situ atomic force microscopy (AFM) of the azurins on Au(111) show initially arrays of protein structures of lateral 100–200 A and vertical ≈50 A extension. These could be individual molecular images convoluted with the tip curvature. As scanning proceeds the structures in the ex situ mode collect into large two-dimensional assemblies while the adsorbed protein in the in situ mode is largely swept into the solution, recovering the free Au(111) surface. The cyclic voltammetry and AFM data are consistent with time dependent adsorption of the azurins on gold via the disulphide bridge (wild-type) or free thiol group (Cys3Ser mutant).

The resolution of scanning tunnelling microscopy (STM) and other scanning probe microscopies is unprecedented but the techniques are fraught with limitations as analytical tools. These limitations and their relationship to the physical mechanisms of image contrast are first discussed. Some new options based on in situ STM, which hold prospects for molecular- and mesoscopic-scale analytical chemistry, are then reviewed. They are illustrated by metallic electro-crystallisation and -dissolution, and in situ STM spectroscopy of large redox molecules. The biophysically oriented analytical options of in situ atomic force microscopy, and analytical chemical perspectives for the new microcantilever sensor techniques are also discussed.