Effects of N-glycans on the structure of human IgA2.

The transition of IgA antibodies into clinical development is crucial because they have the potential to create a new class of therapeutics with superior pathogen neutralization, cancer cell killing, and immunomodulation capacity compared to IgG. However, the biological role of IgA glycans in these processes needs to be better understood. This study provides a detailed biochemical, biophysical, and structural characterization of recombinant monomeric human IgA2, which varies in the amount/locations of attached glycans. Monomeric IgA2 antibodies were produced by removing the N-linked glycans in the CH1 and CH2 domains. The impact of glycans on oligomer formation, thermal stability, and receptor binding was evaluated. In addition, we performed a structural analysis of recombinant IgA2 in solution using Small Angle X-Ray Scattering (SAXS) to examine the effect of glycans on protein structure and flexibility. Our results indicate that the absence of glycans in the Fc tail region leads to higher-order aggregates. SAXS, combined with atomistic modeling, showed that the lack of glycans in the CH2 domain results in increased flexibility between the Fab and Fc domains and a different distribution of open and closed conformations in solution. When binding with the Fcα-receptor, the dissociation constant remains unaltered in the absence of glycans in the CH1 or CH2 domain, compared to the fully glycosylated protein. These results provide insights into N-glycans' function on IgA2, which could have important implications for developing more effective IgA-based therapeutics in the future.

Amino Acid Residues Controlling Domain Interaction and Interdomain Electron Transfer in Cellobiose Dehydrogenase.

The function of cellobiose dehydrogenase (CDH) in biosensors, biofuel cells, and as a physiological redox partner of lytic polysaccharide monooxygenase (LPMO) is based on its role as an electron donor. Before donating electrons to LPMO or electrodes, an interdomain electron transfer from the catalytic FAD-containing dehydrogenase domain to the electron shuttling cytochrome domain of CDH is required. This study investigates the role of two crucial amino acids located at the dehydrogenase domain on domain interaction and interdomain electron transfer by structure-based engineering. The electron transfer kinetics of wild-type Myriococcum thermophilum CDH and its variants M309A, R698S, and M309A/R698S were analyzed by stopped-flow spectrophotometry and structural effects were studied by small-angle X-ray scattering. The data show that R698 is essential to pull the cytochrome domain close to the dehydrogenase domain and orient the heme propionate group towards the FAD, while M309 is an integral part of the electron transfer pathway - its mutation reducing the interdomain electron transfer 10-fold. Structural models and molecular dynamics simulations pinpoint the action of these two residues on the domain interaction and interdomain electron transfer.

Resolving domain positions of cellobiose dehydrogenase by small angle X-ray scattering.

The interdomain electron transfer (IET) between the catalytic flavodehydrogenase domain and the electron-transferring cytochrome domain of cellobiose dehydrogenase (CDH) plays an essential role in biocatalysis, biosensors and biofuel cells, as well as in its natural function as an auxiliary enzyme of lytic polysaccharide monooxygenase. We investigated the mobility of the cytochrome and dehydrogenase domains of CDH, which is hypothesised to limit IET in solution by small angle X-ray scattering (SAXS). CDH from Myriococcum thermophilum (syn. Crassicarpon hotsonii, syn. Thermothelomyces myriococcoides) was probed by SAXS to study the CDH mobility at different pH and in the presence of divalent cations. By comparison of the experimental SAXS data, using pair-distance distribution functions and Kratky plots, we show an increase in CDH mobility at higher pH, indicating alterations of domain mobility. To further visualise CDH movement in solution, we performed SAXS-based multistate modelling. Glycan structures present on CDH partially masked the resulting SAXS shapes, we diminished these effects by deglycosylation and studied the effect of glycoforms by modelling. The modelling shows that with increasing pH, the cytochrome domain adopts a more flexible state with significant separation from the dehydrogenase domain. On the contrary, the presence of calcium ions decreases the mobility of the cytochrome domain. Experimental SAXS data, multistate modelling and previously reported kinetic data show how pH and divalent ions impact the closed state necessary for the IET governed by the movement of the CDH cytochrome domain.

Protein-protein interactions and reduced excluded volume increase dynamic binding capacity of dual salt systems in hydrophobic interaction chromatography.

Deploying two salts in hydrophobic interaction chromatography can significantly increase dynamic binding capacities. Nevertheless, the mechanistic understanding of this phenomenon is lacking. Here, we investigate whether surface tension or ionic strength govern dynamic binding capacities of the chromatographic resin Toyopearl Butyl-650 M in dual salt systems. Small-angle X-ray scattering was employed to analyze the model proteins and the protein-resin adduct in the respective dual salt systems. The dual salt systems incorporate sodium citrate and a secondary sodium salt (acetate, sulfate, or phosphate). As model proteins, we used lysozyme, GFP, and a monoclonal antibody (adalimumab). Moreover, for the protein-resin adduct, we determined the model parameters of a self-avoiding random walk model fitted into the pair density distribution function of the SAXS data. Ionic strength is more predictive for dynamic binding capacities in HIC dual salt systems than surface tension. However, dynamic binding capacities still differ by up to 30 % between the investigated dual salt systems. The proteins exhibit extensive protein-protein interactions in the studied dual salt HIC buffers. We found a correlation of protein-protein interactions with the well-known Hofmeister series. For systems with elevated protein-protein interactions, adsorption isotherms deviate from Langmuirian behavior. This highlights the importance of lateral protein-protein interactions in protein adsorption, where monomolecular protein layers are usually assumed. SAXS analysis of the protein-resin adduct indicates an inverse correlation of the binding capacity and the excluded volume parameter. This is indicative of the deposition of proteins in the cavities of the stationary phase. We hypothesize that increasing protein-protein interactions allow the formation of attractive clusters and multilayers in the cavities, respectively.

Monomeric and homotrimeric solution structures of truncated human peroxidasin 1 variants.

Human peroxidasin 1 is a multidomain peroxidase situated in the basement membrane. The iron enzyme with covalently bound heme oxidizes bromide to hypobromous acid which facilitates the formation of distinct sulfilimine cross-links in the collagen IV network and therefore contributes to its mechanical stability. Additional to the catalytically active peroxidase domain peroxidasin comprises a leucine rich repeat domain, four Ig domains and a C-terminal von Willebrand factor type C module (VWC). Peroxidasin has been shown to form homotrimers involving two redox-sensitive cysteine residues and to undergo posttranslational C-terminal proteolytic cleavage. The present study on several recombinantly produced truncated peroxidasin variants showed that the VWC is not required for trimer formation whereas the alpha-helical linker region located between the peroxidase domain and the VWC is crucial for trimerization. Our data furthermore implies that peroxidasin oligomerization occurs intracellularly before C-terminal cleavage. For the first time we present overall solution structures of monomeric and trimeric truncated peroxidasin variants which were determined by rotary shadowing combined with transmission electron microscopy and by small-angle X-ray scattering (SAXS). A triangular arrangement of the peroxidase domains to each other within the homotrimer was revealed and this structure was confirmed by a model of trimeric peroxidase domains. Our SAXS data showed that the Ig domains are highly flexible and interact with the peroxidase domain and that within the homotrimer each alpha-helical linker region interacts with the respective adjacent peroxidase domain. The implications of our findings on the structure-function relationship of peroxidasin are discussed.

Antibody Binding Heterogeneity of Protein A Resins.

Protein A affinity chromatography is a core unit operation in antibody manufacturing. Nevertheless, there is not enough understanding of in-column antibody adsorption in the Protein A capture step. This work aims to investigate in situ the establishment of an antibody (trastuzumab) layer during Protein A chromatography both in terms of energetic contributions and uptake kinetics. Flow microcalorimetry is employed as a technique with an in situ operating detector, which provides an understanding of the thermodynamics of the adsorption process. In addition, the antibody uptake rate is also investigated in order to establish a correlation between its diffusion on the stationary phase and the associated thermodynamics. Two resins with different particle size, intraparticle porosity, and a Protein A ligand structure are studied: the synthetically engineered B-domain tetrameric MabSelect SuRe and the synthetically engineered C-domain hexameric TOYOPEARL AF-rProtein A HC. The uptake rate follows a pore diffusion model at low equilibrium time, showing a slower diffusivity after a certain time because of the heterogeneous binding nature of these two resins. In addition, the microcalorimetric studies show that adsorption enthalpy is highly favourable at low isotherm concentrations and evolves toward an equilibrium with increasing surface concentration. These data suggest that the relationship between adsorption enthalpy and the establishment of the antibody layer in the Protein A chain is consistent with heterogeneous adsorption.

The pearl necklace model in protein A chromatography: Molecular mechanisms at the resin interface.

Staphylococcal protein A chromatography is an established core technology for monoclonal antibody purification and capture in the downstream processing. MabSelect SuRe involves a tetrameric chain of a recombinant form of the B domain of staphylococcal protein A, called the Z-domain. Little is known about the stoichiometry, binding orientation, or preferred binding. We analyzed small-angle X-ray scattering data of the antibody-protein A complex immobilized in an industrial highly relevant chromatographic resin at different antibody concentrations. From scattering data, we computed the normalized radial density distributions. We designed three-dimensional (3D) models with protein data bank crystallographic structures of an IgG1 (the isoform of trastuzumab, used here; Protein Data Bank: 1HZH) and the staphylococcal protein A B domain (the native form of the recombinant structure contained in MabSelect SuRe resin; Protein Data Bank: 1BDD). We computed different binding conformations for different antibody to protein A stoichiometries (1:1, 2:1, and 3:1) and compared the normalized radial density distributions computed from 3D models with those obtained from the experimental data. In the linear range of the isotherm we favor a 1:1 ratio, with the antibody binding to the outer domains in the protein A chain at very low and high concentrations. In the saturation region, a 2:1 ratio is more likely to occur. A 3:1 stoichiometry is excluded because of steric effects.

Freestanding ultrathin films for separation of small molecules in an aqueous environment.

Alternative separation methods operating in an aqueous environment are of increasing importance for further progress of molecular separation in life sciences and other industrial sectors working towards a biobased economy. By spincoating, membranes with thicknesses under 100 nm and 20 cm2 surface area were prepared from an epoxy based resin. For the first time such ultrathin epoxy films were used for the selective separation of small molecules and metabolites within an aqueous environment. Initially, selectivity is demonstrated by the separation of two dyes of similar size (0.7 and 1.4 nm diameter). By variation of the precursor concentrations, both mechanical stability and selectivity for molecular transport are shown to be tunable. The observed transport properties of the different membranes correlated with their biaxial moduli and ultimate tensile strengths which were in the range of 0.3-3.5 GPa and 10-44 MPa, respectively. These observations agreed with the conclusion drawn from FTIR analysis that variations in the covalent crosslinking density determine the emergent properties. Finally, permeation rates for small molecules of industrial relevance were assessed to confirm a size based diffusion cutoff for compounds with hydrodynamic diameters below 2 nm.

Antibody adsorption in protein-A affinity chromatography - in situ measurement of nanoscale structure by small-angle X-ray scattering.

Protein-A chromatography is the most widely used chromatography step in downstream processing of antibodies. A deeper understanding of the influence of the surface topology on a molecular/nanoscale level on adsorption is essential for further improvement. It is not clear if the binding is homogenous throughout the entire bead network. We followed the protein absorption process and observed the formation of a protein layer on fibers of chromatography resin in a time-resolved manner in nanoscale. To characterize the changes in the antibody-protein-A ligand complex, small angle X-ray scattering was employed using a miniaturized X-ray-transparent chromatography column packed with a MabSelect SuRe resin. Antibody-free MabSelect SuRe resin fiber had an average radius of 12 nm and the protein layer thickness resulting from antibody adsorption was 5.5 and 10.4 nm for fiber and junctions, respectively under applied native conditions. We hypothesize that an average of 1.2 antibodies were adsorbed per protein-A ligand tetramer bound to the outermost units. In contrast to previous studies, it was therefore possible for the first time to directly correlate the nanostructure changes inside the column, which is otherwise a black box, with the adsorption and elution process.

Freely suspended perforated polymer nanomembranes for protein separations.

Selective removal of nanometer-sized compounds such as proteins from fluids is an often challenging task in many scientific and industrial areas. Addressing such tasks with highly efficient and selective membranes is desirable since commonly used chromatographic approaches are expensive and difficult to scale up. Nanomembranes, molecularly thin separation layers, have been predicted and shown to possess outstanding properties but in spite ultra-fast diffusion times and high-resolution separation, to date they generally lack either of two crucial characteristics: compatibility with biological fluids and low-cost production. Here we report the fast and easy fabrication of highly crosslinked polymer membranes based on a thermoset resin (poly[(o-cresyl glycidyl ether)-co-formaldehyde (PCGF) cured with branched polyethyleneimine (PEI)) with nanoscale perforations of 25 nm diameter. During spin casting, microphase separation of a polylactide-co-glycolide induces the formation of nanometer sized domains that serve as templates for perforations which penetrate the 80 nm thick membranes. Ultrathin perforated nanomembranes can be freely suspended on the cm scale, exhibit high mechanical strength, low surface energies and a sharp permeability cutoff at a hydrodynamic diameter of 10 nm suitable for protein separations.

Small cause, large effect: Structural characterization of cutinases from Thermobifida cellulosilytica.

We have investigated the structures of two native cutinases from Thermobifida cellulosilytica, namely Thc_Cut1 and Thc_Cut2 as well as of two variants, Thc_Cut2_DM (Thc_Cut2_ Arg29Asn_Ala30Val) and Thc_Cut2_TM (Thc_Cut2_Arg19Ser_Arg29Asn_Ala30Val). The four enzymes showed different activities towards the aliphatic polyester poly(lactic acid) (PLLA). The crystal structures of the four enzymes were successfully solved and in combination with Small Angle X-Ray Scattering (SAXS) the structural features responsible for the selectivity difference were elucidated. Analysis of the crystal structures did not indicate significant conformational differences among the different cutinases. However, the distinctive SAXS scattering data collected from the enzymes in solution indicated a remarkable surface charge difference. The difference in the electrostatic and hydrophobic surface properties could explain potential alternative binding modes of the four cutinases on PLLA explaining their distinct activities. Biotechnol. Bioeng. 2017;114: 2481-2488. © 2017 Wiley Periodicals, Inc.

Structure of human promyeloperoxidase (proMPO) and the role of the propeptide in processing and maturation.

Myeloperoxidase (MPO) is synthesized by neutrophil and monocyte precursor cells and contributes to host defense by mediating microbial killing. Although several steps in MPO biosynthesis and processing have been elucidated, many questions remained, such as the structure-function relationship of monomeric unprocessed proMPO versus the mature dimeric MPO and the functional role of the propeptide. Here we have presented the first and high resolution (at 1.25 Å) crystal structure of proMPO and its solution structure obtained by small-angle X-ray scattering. Promyeloperoxidase hosts five occupied glycosylation sites and six intrachain cystine bridges with Cys-158 of the very flexible N-terminal propeptide being covalently linked to Cys-319 and thereby hindering homodimerization. Furthermore, the structure revealed (i) the binding site of proMPO-processing proconvertase, (ii) the structural motif for subsequent cleavage to the heavy and light chains of mature MPO protomers, and (iii) three covalent bonds between heme and the protein. Studies of the mutants C158A, C319A, and C158A/C319A demonstrated significant differences from the wild-type protein, including diminished enzymatic activity and prevention of export to the Golgi due to prolonged association with the chaperone calnexin. These structural and functional findings provide novel insights into MPO biosynthesis and processing.

Globular Head-Displayed Conserved Influenza H1 Hemagglutinin Stalk Epitopes Confer Protection against Heterologous H1N1 Virus.

Significant genetic variability in the head region of the influenza A hemagglutinin, the main target of current vaccines, makes it challenging to develop a long-lived seasonal influenza prophylaxis. Vaccines based on the conserved hemagglutinin stalk domain might provide broader cross-reactive immunity. However, this region of the hemagglutinin is immunosubdominant to the head region. Peptide-based vaccines have gained much interest as they allow the immune system to focus on relevant but less immunogenic epitopes. We developed a novel influenza A hemagglutinin-based display platform for H1 hemagglutinin stalk peptides that we identified in an epitope mapping assay using human immune sera and synthetic HA peptides. Flow cytometry and competition assays suggest that the identified stalk sequences do not recapitulate the epitopes of already described broadly neutralizing stalk antibodies. Vaccine constructs displaying 25-mer stalk sequences provided up to 75% protection from lethal heterologous virus challenge in BALB/c mice and induced antibody responses against the H1 hemagglutinin. The developed platform based on a vaccine antigen has the potential to be either used as stand-alone or as prime-vaccine in combination with conventional seasonal or pandemic vaccines for the amplification of stalk-based cross-reactive immunity in humans or as platform to evaluate the relevance of viral peptides/epitopes for protection against influenza virus infection.

Enhanced cutinase-catalyzed hydrolysis of polyethylene terephthalate by covalent fusion to hydrophobins.

Cutinases have shown potential for hydrolysis of the recalcitrant synthetic polymer polyethylene terephthalate (PET). We have shown previously that the rate of this hydrolysis can be enhanced by the addition of hydrophobins, small fungal proteins that can alter the physicochemical properties of surfaces. Here we have investigated whether the PET-hydrolyzing activity of a bacterial cutinase from Thermobifida cellulosilytica (Thc_Cut1) would be further enhanced by fusion to one of three Trichoderma hydrophobins, i.e., the class II hydrophobins HFB4 and HFB7 and the pseudo-class I hydrophobin HFB9b. The fusion enzymes exhibited decreased kcat values on soluble substrates (p-nitrophenyl acetate and p-nitrophenyl butyrate) and strongly decreased the hydrophilicity of glass but caused only small changes in the hydrophobicity of PET. When the enzyme was fused to HFB4 or HFB7, the hydrolysis of PET was enhanced >16-fold over the level with the free enzyme, while a mixture of the enzyme and the hydrophobins led only to a 4-fold increase at most. Fusion with the non-class II hydrophobin HFB9b did not increase the rate of hydrolysis over that of the enzyme-hydrophobin mixture, but HFB9b performed best when PET was preincubated with the hydrophobins before enzyme treatment. The pattern of hydrolysis by the fusion enzymes differed from that of Thc_Cut1 as the concentration of the product mono(2-hydroxyethyl) terephthalate relative to that of the main product, terephthalic acid, increased. Small-angle X-ray scattering (SAXS) analysis revealed an increased scattering contrast of the fusion proteins over that of the free proteins, suggesting a change in conformation or enhanced protein aggregation. Our data show that the level of hydrolysis of PET by cutinase can be significantly increased by fusion to hydrophobins. The data further suggest that this likely involves binding of the hydrophobins to the cutinase and changes in the conformation of its active center.

Surfaces energies of monoliths by inverse liquid chromatography and contact angles.

Seven porous chromatographic columns, termed monoliths, and seven nonporous sheets were produced from polymethacrylates. Their surfaces were activated by different densities of butyl and phenyl ligands. We determined the retention times of highly dilute molecular probes in monoliths and accessed contact angles of pure molecular probes of sheets. We calculated surface energies for both systems. We applied theories of Young, Dupré, and van Oss and compared the results of both types of experiments with respect to Lifshitz-van der Waals and Lewis acid and Lewis base contributions and find agreement but an additive constant.

Self-assembly of "S-bilayers", a step toward expanding the dimensionality of S-layer assemblies.

Protein-based assemblies with ordered nanometer-scale features in three dimensions are of interest as functional nanomaterials but are difficult to generate. Here we report that a truncated S-layer protein assembles into stable bilayers, which we characterized using cryogenic-electron microscopy, tomography, and X-ray spectroscopy. We find that emergence of this supermolecular architecture is the outcome of hierarchical processes; the proteins condense in solution to form 2-D crystals, which then stack parallel to one another to create isotropic bilayered assemblies. Within this bilayered structure, registry between lattices in two layers was disclosed, whereas the intrinsic symmetry in each layer was altered. Comparison of these data to images of wild-type SbpA layers on intact cells gave insight into the interactions responsible for bilayer formation. These results establish a platform for engineering S-layer assemblies with 3-D architecture.

Liquid formulations for stabilizing IgMs during physical stress and long-term storage.

PURPOSE: To develop a liquid formulation for IgMs to survive physical stress and storage. METHODS: Stabilizing formulations for 8 monoclonal immunoglobulin (IgMs) were found using differential scanning calorimetry (DSC). In these formulations, the IgMs were subjected to stress and storage and analyzed by size exclusion chromatography and fluorescence activated cell sorting. Structure was analyzed using small-angle X-ray scattering (SAXS). RESULTS: The highest conformational stability was found near the isoelectric point and further enhanced by addition of sorbitol, sucrose and glycine. For 2 IgMs, the pH optimum for conformational and storage stability did not correspond. Lowering the pH led to the desired storage stability. Optimized formulations prevented aggregation and fragmentation from shear stress, freeze-thaw cycles, accelerated storage and real time storage at 4°C and -20°C for 12 months. Optimized formulations also preserved immunoreactivity for 12 months. SAXS indicated that IgM in stabilizing conditions was closer to the structural IgM model (2RCJ) and less susceptible for aggregation. CONCLUSIONS: A long-term stabilizing formulation for 8 IgMs was found comprising 20% sorbitol and 1 M glycine at pH 5.0-5.5 which may have broad utility for other IgMs. Formulation development using DSC and accelerated storage was evaluated in this study and may be used for other proteins.

Surface energies of hydrophobic interaction chromatography media by inverse liquid chromatography.

Hydrophobicity of hydrophobic interaction chromatography media is currently ranked according to retention of reference proteins. A new method, suitable for porous media, is presented here to determine the surface energy and its Lifshitz-van-der-Waals, Lewis acid and Lewis base contributions. The theory of van Oss has been adapted for data obtained by inverse liquid chromatography. Furthermore, this method is characterized by the independence of the determination of the phase ratio. The retention of probes with different molecular properties was used to calculate the surface energy and the Lifshitz-van-der-Waals as well as Lewis acid and Lewis base contributions to the surface energy. The media with polymethacrylate backbone had a higher surface energy (γ ≈ 200 mJ/m(2)) and Lifshitz-van-der-Waals contribution (γ(LW) ≈ 140 mJ/m(2)) than the agarose-based media (γ ≈ 90-180 mJ/m(2) and γ(LW) ≈ 50-160 mJ/m(2)).

Nanobiotechnology with S-layer proteins as building blocks.

One of the key challenges in nanobiotechnology is the utilization of self- assembly systems, wherein molecules spontaneously associate into reproducible aggregates and supramolecular structures. In this contribution, we describe the basic principles of crystalline bacterial surface layers (S-layers) and their use as patterning elements. The broad application potential of S-layers in nanobiotechnology is based on the specific intrinsic features of the monomolecular arrays composed of identical protein or glycoprotein subunits. Most important, physicochemical properties and functional groups on the protein lattice are arranged in well-defined positions and orientations. Many applications of S-layers depend on the capability of isolated subunits to recrystallize into monomolecular arrays in suspension or on suitable surfaces (e.g., polymers, metals, silicon wafers) or interfaces (e.g., lipid films, liposomes, emulsomes). S-layers also represent a unique structural basis and patterning element for generating more complex supramolecular structures involving all major classes of biological molecules (e.g., proteins, lipids, glycans, nucleic acids, or combinations of these). Thus, S-layers fulfill key requirements as building blocks for the production of new supramolecular materials and nanoscale devices as required in molecular nanotechnology, nanobiotechnology, biomimetics, and synthetic biology.

Single-molecule force spectroscopy reveals the individual mechanical unfolding pathways of a surface layer protein.

Surface layers (S-layers) represent an almost universal feature of archaeal cell envelopes and are probably the most abundant bacterial cell proteins. S-layers are monomolecular crystalline structures of single protein or glycoprotein monomers that completely cover the cell surface during all stages of the cell growth cycle, thereby performing their intrinsic function under a constant intra- and intermolecular mechanical stress. In gram-positive bacteria, the individual S-layer proteins are anchored by a specific binding mechanism to polysaccharides (secondary cell wall polymers) that are linked to the underlying peptidoglycan layer. In this work, atomic force microscopy-based single-molecule force spectroscopy and a polyprotein approach are used to study the individual mechanical unfolding pathways of an S-layer protein. We uncover complex unfolding pathways involving the consecutive unfolding of structural intermediates, where a mechanical stability of 87 pN is revealed. Different initial extensibilities allow the hypothesis that S-layer proteins adapt highly stable, mechanically resilient conformations that are not extensible under the presence of a pulling force. Interestingly, a change of the unfolding pathway is observed when individual S-layer proteins interact with secondary cell wall polymers, which is a direct signature of a conformational change induced by the ligand. Moreover, the mechanical stability increases up to 110 pN. This work demonstrates that single-molecule force spectroscopy offers a powerful tool to detect subtle changes in the structure of an individual protein upon binding of a ligand and constitutes the first conformational study of surface layer proteins at the single-molecule level.