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Cellulose nanofibers (CNFs) are environmentally friendly materials with a high specific strength. To enhance the impact properties of the carbon fiber reinforced plastics (CFRPs), a method for inse...

Cellulose nanofibers (CNFs) are environmental-friendly nanofiller with high strength and lightweight. To improve the impact properties of carbon fiber reinforced plastics (CFRPs), the application o...

Therapeutic antibodies have many biopharmaceutical applications; however, characterization of their higher-order structures is a major concern in quality control. We have developed AF.2A1, an artificial protein, that specifically recognizes non-native, structured IgGs. We performed binding assays using various types of IgGs and fragments to investigate the mechanisms by which AF.2A1 interacts with the non-native IgG. AF.2A1 recognized the acid-stressed IgGs from human, mouse, and rat, but not rabbit. Binding assays using the human IgG1 fragments revealed that an interface emerged by deleting five C-terminal residues. We conclude that AF.2A1 recognizes an exposed hydrophobic core centered on the Trp417. Our results concur with those of the previous studies showing that C-terminal structural changes occur early during antibody denaturation and aggregation. Our findings explain the molecular rationale for using AF.2A1 in quality control of biopharmaceutical IgGs.

An efficient method to increase specific strength of carbon fiber reinforced plastics (CFRP) by using cellulose nanofibers (CNFs) was developed. Eliminating a complex hydrophobic treatment of CNF b...

Alternative autophagy is an Atg5/Atg7-independent type of autophagy that contributes to various physiological events. We here identify Wipi3 as a molecule essential for alternative autophagy, but which plays minor roles in canonical autophagy. Wipi3 binds to Golgi membranes and is required for the generation of isolation membranes. We establish neuron-specific Wipi3-deficient mice, which show behavioral defects, mainly as a result of cerebellar neuronal loss. The accumulation of iron and ceruloplasmin is also found in the neuronal cells. These abnormalities are suppressed by the expression of Dram1, which is another crucial molecule for alternative autophagy. Although Atg7-deficient mice show similar phenotypes to Wipi3-deficient mice, electron microscopic analysis shows that they have completely different subcellular morphologies, including the morphology of organelles. Furthermore, most Atg7/Wipi3 double-deficient mice are embryonic lethal, indicating that Wipi3 functions to maintain neuronal cells via mechanisms different from those of canonical autophagy., Unlike canonical macroautophagy, alternative autophagy does not require the factors Atg5 and Atg7. Here, the authors show that Wipi3 is essential for alternative autophagy, but not for canonical autophagy, and that Wipi3 functions to maintain neuronal cells via mechanisms different from those of canonical autophagy.

Backbone circularization is an effective technique for protein stabilization. Here, we investigated the effect of a connector, an engineered segment that connects two protein termini, on the conformational stability of previously designed circularized variants of granulocyte colony-stimulating factor (G-CSF). Heat tolerance and chemical denaturation analyses revealed that aggregation resistance and thermodynamic stability of the circularized variants were superior to those of linear G-CSF. Crystal structure and molecular dynamics (MD) simulation of the most thermodynamically stable variant (C166) revealed a high number of intramolecular hydrogen bonds in both the connector region and Helix D adjacent to the connector region in the folded structure. MD simulations and theoretical calculations involving different force fields indicated a reduction in the main chain entropy of C166 in the unfolded state and increase in the intramolecular hydrogen bond energy of C166 in the folded structure. Although backbone circularization is usually considered to alter chain entropy of the unfolded state, the data indicated that it could also improve the conformational enthalpy of the folded state. Further structural examination of the connector region confirmed that protein design based on a statistical analysis of local structures is an effective approach for predicting an optimum connector length to improve the conformational stability of backbone-circularized proteins. Protein design using backbone circularization with an optimum connector length will be useful for the development of effective and safe protein therapeutics. DATABASE: Structural data are available in Protein Data Bank under the accession number 5ZO6.

Protein-protein interactions that can be controlled by environmental triggers have immense potential in various biological and industrial applications. In the current study, we aimed to engineer a pH-dependent protein-protein interaction that employs intramolecular electrostatic repulsion through a structure-guided histidine substitution approach. We implemented this strategy on

Cellulose nanofibers (CNFs) have high strength. Therefore, many studies have been conducted to improve the mechanical properties of carbon fiber reinforced plastics (CFRP) by dispersing CNF in the matrix. However, since CNFs are hydrophilic, a hydrophobic treatment is required. Previously, authors showed the bending strength of CFRP could be enhanced by CNFs without a hydrophobic treatment. Applying the electro-activated deposition resin molding (ERM) method to fabricate CFRP, CNFs can be applied on the surface since the electro-activated deposition solution is essentially water-based and contains a polymer with epoxy groups. In this study, the CNF sheet was prepared by drying CNF dispersion in advance. Shortly after the ERM, the CNF sheet was applied to the surface of the CFRP. The mechanical properties of the CFRP were enhanced, the maximum elastic modulus and tensile strength were 2.3 times and 1.7 times of the CFRP specimen without the CNF sheet, respectively.

Many researches on strengthening the matrix of Carbon Fiber Reinforced Plastic (CFRP) using cellulose nanofibers (CNFs) have been reported. However, hydrophobic treatment to modify the surface of C...

Aggregates of therapeutic proteins that can contaminate drug products during manufacture is a growing concern for the pharmaceutical industry because the aggregates are potentially immunogenic. Electron microscopy is a typical, indispensable method for imaging nanometer- to micrometer-sized structures. Nevertheless, it is not ideal because it must be performed with ex situ monitoring under high-vacuum conditions, where the samples could be altered by staining and drying. Here, we introduce a scanning electron-assisted dielectric microscopy (SE-ADM) technique for in-solution imaging of monoclonal immunoglobulin G (IgG) aggregates without staining and drying. Remarkably, SE-ADM allowed assessment of the size and morphology of the IgG aggregates in solution by completely excluding drying-induced artifacts. SE-ADM was also beneficial to study IgG aggregation caused by temporary acid exposure followed by neutralization, pH-shift stress. A box-counting analysis of the SE-ADM images provided fractal dimensions of the larger aggregates, which complemented the fractal dimensions of the smaller aggregates measured by light scattering. The scale-free or self-similarity nature of the fractal aggregates indicated that a common mechanism for antibody aggregation existed between the smaller and larger aggregates. Consequently, SE-ADM is a useful method for characterizing protein aggregates to bridge the gaps that occur among conventional analytical methods, such as those related to in situ/ ex situ techniques or size/morphology assessments.

For the efficient manufacturing of carbon fiber reinforced plastics (CFRP), the electro-activated deposition resin molding (ERM) method is developed. The carbon fiber fabric is immersed in an electro-activated deposition solution containing polymer with epoxy groups. By energization , an epoxy resin is precipitated on the surface of the carbon fiber, and impregnation occurred in the solution. In this study, applying the ERM method, CFRP specimen with the hole was manufactured. Furthermore, the reinforcement by arranging the fiber circumferentially around the hole was installed. The carbon fiber fabric was sewn using the tailored fiber placement (TFP) machine. As a results, the effect of the reinforcement was confirmed. The tensile strength of CFRP with the hole and the reinforcement was the same as in the case without the hole. From the finite element analysis , it was confirmed that the reinforcement around the hole reduced the stress concentration.

Cellulose nanofiber (CNF) is high strength and lightweight, additionally, it is produced by sustainable natural resources such as wood. Therefore, it has been reported the mechanical properties of CFRP can be improved by dispersing CNF in the matrix. However, as CNF is hydrophilic, a hydrophobic treatment was applied in many previous studies. For efficient CFRP manufacturing, the authors have developed an electrochemical resin molding method by using aqueous electro-activated deposition which contains the polymer having an epoxy group. In this method, the carbon fiber fabric is immersed in the electro-activated deposition solution and energized, epoxy resin is precipitated around the carbon fiber and impregnated. In this study, shortly after electro-activated deposition, CNF without a hydrophobic treatment was applied to the surface of the resin-impregnated carbon fiber fabric. The mechanical properties of CFRP could be enhanced, and optimum weight fraction of CNF was revealed.

Aggregation of protein therapeutics can lead to immunogenicity and loss of function in vivo. Its effective prevention requires an understanding of the conformational and colloidal stability of protein and the improvement of both. Granulocyte colony-stimulating factor (G-CSF), which is one of the most widely used protein therapeutics, was previously shown to be conformationally stabilized by connecting its N- and C-termini with amide bonds (backbone circularization). In this study, we investigated whether circularization affects the colloidal stability of proteins. Colloidal stability was indirectly assessed by analyzing the aggregation behavior of G-CSF variants using analytical ultracentrifugation (AUC) and small-angle X-ray scattering (SAXS). Consequently, we found that the unfolded structure of circularized G-CSF was more compact than non-circularized G-CSF, and that backbone circularization improved its aggregation resistance against chemical denaturation by guanidine hydrochloride (GdnHCl). The improved aggregation resistance suggests that the expansion tolerance of circularized G-CSF in the unfolded state increased its colloidal stability. Thus, backbone circularization is an excellent method for enhancing the colloidal and the conformational stability of protein with minimal sequence changes. It is therefore expected to be effective in extending the storage stability of protein therapeutics, enhancing their biological stability.

Alternative autophagy is an autophagy-related protein 5 (Atg5)-independent type of macroautophagy. Unc51-like kinase 1 (Ulk1) is an essential initiator not only for Atg5-dependent canonical autophagy but also for alternative autophagy. However, the mechanism as to how Ulk1 differentially regulates both types of autophagy has remained unclear. In this study, we identify a phosphorylation site of Ulk1 at Ser746, which is phosphorylated during genotoxic stress-induced alternative autophagy. Phospho-Ulk1746 localizes exclusively on the Golgi and is required for alternative autophagy, but not canonical autophagy. We also identify receptor-interacting protein kinase 3 (RIPK3) as the kinase responsible for genotoxic stress-induced Ulk1746 phosphorylation, because RIPK3 interacts with and phosphorylates Ulk1 at Ser746, and loss of RIPK3 abolishes Ulk1746 phosphorylation. These findings indicate that RIPK3-dependent Ulk1746 phosphorylation on the Golgi plays a pivotal role in genotoxic stress-induced alternative autophagy., Unlike canonical macroautophagy, alternative autophagy does not require the factors Atg5 and Atg7, but does require Ulk1. Here the authors show that phosphorylation of Ulk1 at Ser746 by RIPK3 is required for alternative autophagy initiation at the Golgi in response to genotoxic stress.

The grafting of active peptides onto structurally stable scaffold proteins is effective for the generation of functional proteins. In this study, we aimed to develop a grafting method using ubiquitin as a scaffold protein. Ubiquitin is a small protein consisting of 76 amino acid residues that is highly stable against heat and pH stress, which are desirable characteristics for a scaffold protein. Moreover, its structure is maintained even if it is split or additional residues are inserted. Therefore, we assumed that grafting of an active peptide into ubiquitin would result in a functional protein. As a proof of concept, we developed the ubiquitin-based binder (UbB), into which the p53 (17-28) peptide was inserted between Ile36 and Pro37. The p53 (17-28) peptide, utilized as a model active peptide in this work, is known to bind to mouse double minute 2 homolog (Mdm2). Size exclusion chromatography and circular dichroism indicated that UbB maintained a similar structure to that of ubiquitin. The affinity for Mdm2 measured by surface plasmon resonance was 292 times greater than that of the p53 (17-28) peptide. These observations indicate that ubiquitin is a robust scaffold for peptide grafting. We hope that this study will aid further development of ubiquitin-based protein engineering.

A cycle of dropping and shaking a vial containing antibody solution was reported to induce aggregation. In this study, antibody solutions in glass prefillable syringes with or without silicone oil lubrication were subjected to the combined stresses of dropping and shaking, using a friability testing apparatus. Larger numbers of subvisible particles were generated, regardless of silicone oil lubrication, upon combination stress exposure than that with shaking stress alone. Nucleation of antibody molecules upon perturbation by an impact of dropping and adsorption of antibody molecules to the syringe surface followed by film formation and antibody film desorption were considered key steps in the particle formation promoted by combination stress. A larger number of silicone oil droplets was released when silicone oil-lubricated glass syringes containing phosphate buffer saline were exposed to combination stress than that observed with shaking stress alone. Polysorbate 20, a non-ionic surfactant, effectively reduced the number of protein particles, but failed to prevent silicone oil release upon combination stress exposure. This study indicates that stress-stability assays using the friability testing apparatus are effective for assessing the stability of biopharmaceuticals under the combined stresses of dropping and shaking, which have not been tested in conventional stress-stability assays.

Therapeutic IgG becomes unstable under various stresses in the manufacturing process. The resulting non-native IgG molecules tend to associate with each other and form aggregates. Because such aggregates not only decrease the pharmacological effect but also become a potential risk factor for immunogenicity, rapid analysis of aggregation is required for quality control of therapeutic IgG. In this study, we developed a homogeneous assay using AlphaScreen and AF.2A1. AF.2A1 is a 25-residue artificial protein that binds specifically to non-native IgG generated under chemical and physical stresses. This assay is performed in a short period of time. Our results show that AF.2A1-AlphaScreen may be used to evaluate the various types of IgG, as AF.2A1 recognizes the non-native structure in the constant region (Fc region) of IgG. The assay was effective for detection of non-native IgG, with particle size up to ca. 500 nm, generated under acid, heat, and stirring conditions. In addition, this technique is suitable for analyzing non-native IgG in CHO cell culture supernatant and mixed with large amounts of native IgG. These results indicate the potential of AF.2A1-AlphaScreen to be used as a high-throughput evaluation method for process monitoring as well as quality testing in the manufacturing of therapeutic IgG.

Antibodies are therapeutic proteins that are becoming indispensable for the treatment of serious diseases. Their efficacies depend on their folded structures, the loss of which, through unfolding or aggregation, should be closely monitored during their manufacture, storage, and dosing for safe usage. In downstream processes, exposure of the crude antibody solution to acid, followed by neutralization, is an established standard protocol for antibody purification and inactivation of viruses in antibody preparations. Nevertheless, this treatment also triggers an unwanted side reaction, i.e., antibody aggregation. The aggregates continuously evolve at neutral pH. It remains unclear whether the aggregates progressively incorporate the native, folded, monomers into themselves, enabling aggregate growth as seen in amyloid fibrils. In the present study, the diffusion of fluorescently labeled monoclonal humanized immunoglobulin G1 monomers in aggregates produced by pH-shift stress was tracked by fluorescence correlation spectroscopy. This method was used in addition to monitoring aggregate formation by dynamic light scattering. The diffusing velocities of the monomers indicated that the folded monomers were not involved in aggregate formation, in contrast with unfolded monomers. On the basis of the results, we propose a bifurcated pathway for the refolding and aggregation of antibodies triggered by a pH shift from acidic to neutral. In the scheme, the refolded monomers survive until their unfolding. The insights in this study will contribute to the manufacture of aggregation-resistant therapeutic antibodies.

ATG5 and ATG7 are considered to be essential molecules for the induction of autophagy. However, we found that cells lacking ATG5 or ATG7 can still form autophagosomes/autolysosomes and perform autophagic protein degradation when subjected to certain types of stress. Although the lipidation of LC3 is accepted as a good indicator of autophagy, this did not occur during ATG5/ATG7-independent alternative autophagy. Unlike conventional autophagy, autophagosomes appeared to be generated in a Rab9-dependent manner by the fusion of the phagophores with vesicles derived from the trans-Golgi and late endosomes. Therefore, mammalian autophagy can occur via at least two different pathways; the ATG5/ATG7-dependent conventional pathway and an ATG5/ATG7-independent alternative pathway.

This article presented the data related to the research article entitled “Calibration-free concentration analysis for an analyte prone to self-association” (H. Imamura, S. Honda, 2017) [1]. The data included surface plasmon resonance (SPR) responses of the variants of protein G with different masses under mass transport limitation. The friction factors of the proteins analyzed by an ultracentrifugation were recorded. Calculation of the SPR response of the proteins was also described.

The strength enhancement of the matrix of CFRP has been a major research subject. In particular, strength of the surface resin layer of CFRP is quite important since the bending stress is most effective on the surface but also exposed to foreign object debris (FOD). One of the methods to strengthen resin matrix of CFRP is to disperse cellulose nanofibers (CNF), therefore, many studies have been conducted. However, due to hydrophilicity of CNF, a hydrophobic treatment is indispensable. Although various hydrophobic methods have been proposed, special chemical substances are required and resulted in higher manufacturing costs. In previous study, authors showed that the mechanical properties of CFRP were enhanced by CNF without hydrophobic treatment using the electro-activated deposition resin molding (ERM) method. In the ERM method, a non-crimp carbon fiber fabric (NCF) is immersed in the electro-activated deposition solution which contains polymer with epoxy group. Then, by energization, the resin precipitates around the carbon fiber and is efficiently impregnated to the NCF in the solution. After the ERM, CNF can be applied on the surface of CFRP since the electro-activated deposition solution is water-based. After thermal curing, CNF is embedded on the surface resin layer of CFRP. In this study, the analysis to understand the effect of CNF on the mechanical properties was carried out. As a practical application, the I-shaped cross-sectional beam (I-beam) was selected for the analysis. It was experimentally revealed that the bending strength of I-beam was enhanced by embedding of CNF. With assumed the material constants based on the tensile tests of the CFRP, the bending properties with dispersing CNF on the surface were analyzed by using the finite element method. As a result, the relationship between load and deflection could be well simulated, and the effect of CNF on the bending properties was clarified.

Autophagy is an evolutionarily conserved process that degrades subcellular constituents. Mammalian cells undergo two types of autophagy; Atg5-dependent conventional autophagy and Atg5-independent alternative autophagy, and the molecules required for the latter type of autophagy are largely unknown. In this study, we analyzed the molecular mechanisms of genotoxic stress-induced alternative autophagy, and identified the essential role of p53 and damage-regulated autophagy modulator (Dram1). Dram1 was sufficient to induce alternative autophagy. In the mechanism of alternative autophagy, Dram1 functions in the closure of isolation membranes downstream of p53. These findings indicate that Dram1 plays a pivotal role in genotoxic stress-induced alternative autophagy.

To strengthen carbon fiber reinforced plastics (CFRPs), fiber placement along the principal stress directions have been studied as a promising approach. However, a method to sufficiently impregnate a resin between the curved carbon fibers has not established thus far, although arbitrary arrangement of carbon fibers is feasible using the automated fiber placement (AFP) machine. Previously, the authors had developed an electrodeposition resin molding method based on electrophoresis. In this method, the carbon fiber preforms sewn by the AFP machine is immersed in the electrodeposition solution containing polymers with epoxy groups, and optimal current is applied to impregnate resin between carbon fibers. In this study, we showed that a CFRP specimen could be fabricated with arranging the carbon fibers along the principal stress direction, resulting in approximately 10% higher tensile strength than that of the specimen with unidirectional carbon fibers.

Ideal quality control of therapeutic antibodies involves analytical techniques with high-sensitivity, high-resolution, and high-throughput performance. Few technologies that assess the physicochemical heterogeneity of antibodies, however, meet all the required demands. We developed a biosensing method for the quality control of therapeutic antibodies based on an artificial protein, AF.2A1, which discriminates between the native and the non-native three-dimensional structures of immunoglobulin G (IgG). AF.2A1 specifically recognized non-native IgG spiked into a solution of native IgG, thereby making it possible to detect contamination by a small amount of non-native IgG, which is difficult using conventional size-based separation or spectroscopic techniques. Using AF.2A1 as an analytical probe, we determined the concentration of non-native IgG formed under various pH conditions. The probe was also applicable to accelerated tests of the long-term stability of a therapeutic antibody, allowing monitoring of the formation of non-native IgG at elevated temperatures and extended periods of storage. AF.2A1, a proteinous probe, can be combined with established methods or devices to achieve high-throughput assays and provides the potential for probe-based biosensing for the quality control of therapeutic antibodies.

The purification process of an antibody in manufacturing involves temporal exposure of the molecules to low pH followed by neutralization-pH-shift stress-which causes aggregation. It remains unclear how aggregation triggered by pH-shift stress grows at neutral pH and how it depends on the temperature in an ambient range. We used static and dynamic light scattering to monitor the time-dependent evolution of the aggregate size of the pH-shift stressed antibody between 4.0 and 40.0 °C. A power-law relationship between the effective molecular weight and the effective hydrodynamic radius was found, indicating that the aggregates were fractal with a dimension of 1.98. We found that the aggregation kinetics in the lower-temperature range, 4.0-25.0 °C, were well described by the Smoluchowski aggregation equation. The temperature dependence of the effective aggregation rate constant gave 13 ± 1 kcal/mol of endothermic activation energy. Temporal acid exposure creates an enriched population of unfolded protein molecules that are competent of aggregating. Therefore, the energetically unfavorable unfolding step is not required and the aggregation proceeds faster. These findings provide a basis for predicting the growth of aggregates during storage under practical, ambient conditions.

For the thermal management of electric devices, the application of phase change materials (PCMs) such as paraffin has been studied. However, as long as the PCM changes to liquid phase, a sealed structure is required, which is a constraint for weight reduction. Therefore, the authors have focused on trans-1,4-polybutadiene (TPBD), a semi-crystalline polymer that accumulates heat by crystal structure transition at 50~80 °C in the solid phase. However, the strength of TPBD is insufficient and thermal conductivity is not enough to dissipate the heat. In this study, a method to homogeneously apply TPBD to the surface of carbon fiber reinforced plastics (CFRP) was developed using cellulose nanofiber (CNF) and aqueous electrodeposition solution containing a polymer with epoxy group. The heat-storable CFRP was obtained, the maximum exoergic and endoergic heats of 30 J/g were confirmed, and its tensile strength was approximately 61% of CFRP without TPBD/CNF.

In chemical biology, the elucidation of chemical target is crucial for successful drug development. Because MHC class I molecules present peptides from intracellular damaged proteins, it might be possible to identify targets of a chemical by analyzing peptide sequences on MHC class I. Therefore, we treated cells with the autophagy-inducing chemical TMD-457 and identified the peptides presented on MHC class I. Many of the peptides were derived from molecules involved in ER trafficking and ER stress, which were confirmed by morphological and biochemical analyses. Therefore, our results demonstrate that analyzing MHC class I peptides is useful for the detection of chemical targets.

The quality of preparations of therapeutic IgG molecules, widely used for the treatment of various diseases, should be maintained during storage and administration. Nevertheless, recent studies demonstrate that IgG aggregation is one of the most critical immunogenicity risk factors that compromises safety and efficacy of therapeutic IgG molecules in the clinical setting. During the IgG manufacturing process, 0.22-μm membrane filters are commonly used to remove aggregates. However, particles with a diameter below 0.22 μm (small aggregates) are not removed from the final product. The residual species may grow into large aggregates during the storage period. In the current study, we devised a strategy to suppress IgG aggregate growth by removing aggregation precursors using the artificial protein AF.2A1. This protein efficiently binds the Fc region of non-native IgG conformers generated under chemical and physical stresses. Magnetic beads conjugated with AF.2A1 were used to remove non-native monomers and aggregates from solutions of native IgG and from native IgG solutions spiked with stressed IgG. The time-dependent growth of aggregates after the removal treatment was monitored. The removal of aggregation precursors, i.e., non-native monomers and nanometer aggregates (

This work presents a method to fabricate the heat-storable CFRP, a multifunctional composite that combines strength, lightweight and heat storability. For mobile electric devices, thermal management system using phase change material (PCM) has been developed. However, as long as the PCM changes to liquid phase, it is difficult to reduce the weight since the sealed container is indispensable. Hence, the authors have focused trans-1,4-polybutadiene (TPBD), which is crystalline polymer that accumulates heat by crystal structure transition in solid phase. In this study, TPBD was incorporated into CFRP. To avoid exposing TPBD to high-temperature and high-pressure processes, the electrodeposition resin molding (ERM) method was applied. That is, a non-crimp carbon fiber fabric (NCF) was immersed in the electrodeposition solution containing epoxy polymers in which the pulverized TPBD was dispersed. Applying a current, resin was deposited and impregnated between carbon fibers. Simultaneously, TPBD was incorporated to obtain heat-storable CFRP.

Backbone circularization is a powerful approach for enhancing the structural stability of polypeptides. Herein, we present the crystal structure of the circularized variant of the granulocyte colony-stimulating factor (G-CSF) in which the terminal helical region was circularized using a short, two-amino acid connector. The structure revealed that the N- and C-termini were indeed connected by a peptide bond. The local structure of the C-terminal region transited from an α helix to 310 helix with a bend close to the N-terminal region, indicating that the structural change offset the insufficient length of the connector. This is the first-ever report of a crystal structure of the backbone of a circularized protein. It will facilitate the development of backbone circularization methodology.

SummaryProtein engineering that exploits known functional peptides holds great promise for generating novel functional proteins. Here we propose a combinatorial approach, termed adaptive assembly, which provides a tailor-made protein scaffold for a given functional peptide. A combinatorial library was designed to create a tailor-made scaffold, which was generated from β hairpins derived from a 10-residue minimal protein “chignolin” and randomized amino acid sequences. We applied adaptive assembly to a peptide with low affinity for the Fc region of human immunoglobulin G, generating a 54-residue protein AF.p17 with a 40,600-fold enhanced affinity. The crystal structure of AF.p17 complexed with the Fc region revealed that the scaffold fixed the active conformation with a unique structure composed of a short α helix, β hairpins, and a loop-like structure. Adaptive assembly can take full advantage of known peptides as assets for generating novel functional proteins.

Antibody therapeutics are now in widespread use and provide a new approach for treating serious diseases such as rheumatic diseases and cancer. Monoclonal antibodies used as therapeutic agents must be of high quality, and their safety must be guaranteed. Aggregated antibody is a degradation product that may be generated during the manufacturing process. To maintain the high quality and safety of antibody therapeutics, it is necessary to understand the mechanism of aggregation and to develop technologies to strictly control aggregate formation. Here, we extensively investigated the conformational and colloidal characteristics of isolated antibody constant domains, and provided insights into the molecular mechanism of antibody aggregation. Isolated domains (CH2, CH3, CL, and CH1-CL dimer) of human immunoglobulin G were synthesized, solubilized using 49 sets of solution conditions (pH 2-8 and 0-300 mM NaCl), and characterized using circular dichroism, intrinsic tryptophan fluorescence, and dynamic light scattering. Salt-induced conformational changes and oligomer formation were kinetically analyzed by NaCl-jump measurements (from 0 to 300 mM at pH 3). Phase diagrams revealed that the domains have different conformational and colloidal stabilities. The unfolded fractions of CH3 and CH2 at pH 3 were larger than that of CL and CH1-CL dimer. The secondary and tertiary structures and particle sizes of CH3 and CH2 showed that, in non-native states, these domains were sensitive to salt concentration. Kinetic analyses suggest that oligomer formation by CH3 and CH2 proceeds through partially refolded conformations. The colloidal stability of CH3 in non-native states is the lowest of the four domains under the conditions tested. We propose that the impact of IgG constant domains on aggregation follows the order CH3 > CH2 > CH1-CL dimer > CL; furthermore, we suggest that CH3 plays the most critical role in driving intact antibody aggregation under acidic conditions.

Monoclonal immunoglobulin G (IgG) is a multidomain protein. It has been reported that the conformational and colloidal stabilities of each domain are different, and it is predicted that limited domains participate in IgG aggregation. In contrast, the influence of interdomain interactions on IgG aggregation remains unclear. The fragment crystallizable (Fc) region is also a multidomain protein consisting of two sets of CH2 and CH3 domains. Here, we have analyzed the conformational change and aggregate size of an aglycosylated Fc region induced by both acid and salt stresses and have elucidated the influence of interdomain interactions between CH2 and CH3 domains on the conformational and colloidal stabilities of the aglycosylated Fc region. Singular value decomposition analyses demonstrated that the CH2 and CH3 domains unfolded almost independently from each other in the aglycosylated Fc region. Meanwhile, the colloidal stabilities of the CH2 and CH3 domains affect the aggregation process of the unfolded ag...

Calibration-free concentration analysis (CFCA) based on surface plasmon resonance uses the diffusion coefficient of an analyte to determine the concentration of that analyte in a bulk solution. In general, CFCA is avoided when investigating analytes prone to self-association, as the heterogeneous diffusion coefficient results in a loss of precision. The derivation for self-association of the analyte was presented here. By using the diffusion coefficient for the monomeric state, CFCA provides the lowest possible concentration even though the analyte is self-associated.

Protein-protein interaction in response to environmental conditions enables sophisticated biological and biotechnological processes. Aiming toward the rational design of a pH-sensitive protein-protein interaction, we engineered pH-sensitive mutants of streptococcal protein G B1, a binder to the IgG constant region. We systematically introduced histidine residues into the binding interface to cause electrostatic repulsion on the basis of a rigid body model. Exquisite pH sensitivity of this interaction was confirmed by surface plasmon resonance and affinity chromatography employing a clinically used human IgG. The pH-sensitive mechanism of the interaction was analyzed and evaluated from kinetic, thermodynamic, and structural viewpoints. Histidine-mediated electrostatic repulsion resulted in significant loss of exothermic heat of the binding that decreased the affinity only at acidic conditions, thereby improving the pH sensitivity. The reduced binding energy was partly recovered by "enthalpy-entropy compensation." Crystal structures of the designed mutants confirmed the validity of the rigid body model on which the effective electrostatic repulsion was based. Moreover, our data suggested that the entropy gain involved exclusion of water molecules solvated in a space formed by the introduced histidine and adjacent tryptophan residue. Our findings concerning the mechanism of histidine-introduced interactions will provide a guideline for the rational design of pH-sensitive protein-protein recognition.

On the ideology of minimalism, we have designed a micro-protein consisting of only 10 amino acids. The molecule, CLN025, exhibits a β-hairpin structure in both crystalline state and aqueous solution. Thermal unfolding is shown to be reversible and can be fitted as a two-state transition. Molecular dynamics simulation starting from an extended conformation leads to a funnel-shaped surface and scale-free network, being the structures located at the bottom and the hub similar to the experimental ones. Considering these consequences, we proposed the concept of “ideal protein” as a way to clarify the differences between “protein” and “peptide”.

To investigate fundamental processes conserved in all eukaryotic cells, the budding yeast Saccharomyces cerevisiae has being widely used as a model organism. In particular, the yeast two-hybrid system is a powerful technique for analyzing protein–protein interactions and protein function in living cells. Here, we describe several approaches for investigating and regulating the activity of target proteins using the yeast guanine nucleotide-binding protein (G-protein) signaling machinery as the readout. These approaches are rapid and easy-to-use tools that support the design of regulatory factors against receptors, enzymes, and other proteins that have been identified as potential drug target molecules.

Endothelin-1, a 21 amino acid polypeptide produced by vascular endothelial cells, has potent vasoactive properties. The purpose of this study was to estimate the effects of exogenous big endothelin-1 on fetal lamb circulation.Regional blood flow was measured by the colored microsphere technique during continuous infusion (60 minutes) of big endothelin-1, or saline (control), in 12 chronically instrumented sheep fetuses.After 60 minutes of big endothelin-1 administration, the fetal plasma endothelin-1 concentration increased significantly from 24.0 +/- 6.7 to 49.7 +/- 31.4 pg/mL (P = .018) without significant changes in fetal arterial blood gases. Continuous infusion of big endothelin-1 decreased blood flow in most organs except the brain and the heart. After the big endothelin-1 infusion, the blood flow to the brain significantly increased from 158 +/- 51 to 174 +/- 71 mL/min/100 g (P = .002); the blood flow to the heart also increased significantly from 171 +/- 95 to 200 +/- 112 mL/min/100 g (P = .001), respectively.Continuous infusion of endothelin-1 decreases blood flow in most of organs except the brain and the heart. It is likely that endothelin-1 plays an important role in fetal redistribution of blood flow.

Various mutants of the protein fragment, barnase module-1 (1-24) were investigated in order to reveal the structural principle of amyloid-like fibrils. By means of circular dichroism spectroscopy, X-ray diffraction, electron microscopy, and thioflavin T binding assay, we found that the molecules containing two beta-strands and an intervening turn structure are assembled to form a cross-beta structure. Stabilization by both the hydrophobic interactions and hydrogen bonding between the respective paired side-chains on the coupled beta-strands was essential for fibril formation. These two types of interaction can also arrange the corresponding residues in lines on both sheet surfaces of protofilaments with a cross-beta structure. This leads to the most probable fibril structure constructed with the line-matching interactions between protofilaments. Consideration of the geometrical symmetry resulted in our finding that a limited number of essential models for molecular packing in fibril structure are stable, which would rationally explain the occurrence of two or three morphologies from an identical molecular species. The ribbon-like fibrils exhibited striped texture along the axis, which was assigned to a stacked two-sheet repeat as a structural unit. The comprehensively proposed structural model, that is, the sheet-sheet interaction between left-handed cross-beta structures, results in a slightly right-handed twist of beta-sheet stacking, which reasonably elucidates the intrinsic sizes of the fibril width and its helical period along the fibril axis, as the bias in the orientation of the hydrogen-bonded beta-strand pair at the lateral edge is larger than that at the central protofilament.

A short C-terminal fragment of immunoglobulin-binding domain of streptococcal protein G is known to form nativelike beta-hairpin at physiological conditions. To understand the cooperative folding of the short peptide, eight Ala-substituted mutants of the fragment were investigated with respect to their structural stabilities by analyzing temperature dependence of NMR signals. On comparison of the obtained thermodynamic parameters, we found that the nonpolar residues Tyr45 and Phe52 and the polar residues Asp46 and Thr49 are crucial for the beta-hairpin folding. The results suggest a strong interaction between the nonpolar side chains that participates in a putative hydrophobic cluster and that the polar side chains form a fairly rigid conformation around the loop (46-51). We also investigated the complex formation of the mutants with N-terminal fragment at the variety of temperature to get their thermal unfolding profiles and found that the mutations on the residues Asp46 and Thr49 largely destabilized the complexes, while substitution of Asp47 slightly stabilized the complex. From these results, we deduced that both the hydrophobic cluster formation and the rigidity of the loop (46-51) cooperatively stabilize the beta-hairpin structure of the fragment. These interactions which form a stable beta-hairpin may be the initial structural scaffold which is important in the early folding events of the whole domain.

Thermodynamic investigations of flagellin from Salmonella typhimurium and its proteolytic fragments were conducted by differential scanning calorimetry (DSC) and circular dichroism (CD) melting measurements. A new method of analysis for a multi-state transition based on our original theoretical treatment of thermodynamic equations has been developed to analyze those data. The analysis of DSC curves confirmed the three thermodynamic domains of flagellin. The thermodynamic parameters of each domain were revised from those previously reported and the new values of the parameters have a good correlation to the apparent molecular masses of the morphological domains. CD melting measurements at far and near-UV wavelengths showed sequential unfolding of the domains. Therefore, we could reasonably assign the thermodynamically identified domains to the morphological domains. Further analysis of both DSC and CD data provided insights into the folding energetics of the multidomain structure of flagellin. An inner domain (Df1) of flagellin in the filament unfolds through a relatively broad transition, while the two outer domains unfold cooperatively and show sharp transitions. This indicates that the interdomain interactions between Df1 and D2 has different characteristics from the apparently more intimate interactions between D2 and D3. These characteristics suggest that flagellin is organized with relatively flexible domains and rigid domains, which appears to be responsible for the well-regulated assembly mechanism of the bacterial flagellar filament.

To elucidate early stages in protein folding, we have adopted a fragment reconstitution method for small proteins. This approach is expected to provide nuclei for protein folding and to allow us to investigate folding mechanisms. In previous work [Kobayashi, N., et al. (1995) FEBS Lett. 366, 99-103.] we demonstrated the association of two complementary fragments, derived from the immunoglobulin G-binding domain B1 of streptococcal Protein G, and showed the structural similarity between the reconstituted domain and the uncleaved wild-type domain. In this work we have further characterized the reconstituted domain as well as the uncleaved domain thermodynamically by means of differential scanning calorimetry (DSC) and circular dichroism (CD) measurements. Although composed of short peptide fragments not linked by covalent bonds, the reconstituted domain showed a typical folding/unfolding curve in both DSC and CD melting measurements and behaved like a globular protein. The domain was not very stable, and the small value of the Gibbs free energy corresponded to the class of the weakest protein-protein binding systems. The denaturation temperature of 0. 78 mM solution was 313 K at pH 5.9 as measured by DSC, which was more than 40 degrees lower than the uncleaved domain. This apparent instability was primarily caused by entropic disadvantage attributed to a bimolecular reaction. The temperature dependence of the enthalpy change from the folded to the unfolded state was almost identical for the reconstituted domain and the uncleaved one. This indicates that most of the noncovalent intramolecular interactions stabilizing the native structure, such as hydrogen bonding and hydrophobic interactions, are regenerated in the reconstituted domain. By comparing the equilibrium constants of the reconstituted and uncleaved domains, we determined the effective concentration to be approximately 6 M at 298 K. Structure-based estimation of the thermodynamic properties from the values of accessible surface areas showed that approximately 35% of the total heat capacity change and approximately 25% of the total enthalpy change can be attributed to the interchain interaction at 298 K. Furthermore, the folding/unfolding equilibrium of beta-hairpin structure of the fragment 41-56 alone was also characterized. These analyses allow us to envision the microdomain folding mechanism of the Protein G B1 domain, in which segment 41-56 first forms a stable beta-hairpin structure and then collides with segment 1-40, followed by spontaneous folding of the whole molecule.

To obtain a selection marker gene functional in a thermophilic bacterium, Thermus thermophilus, an in vivo-directed evolutionary strategy was conducted on a hygromycin B phosphotransferase gene (hyg) from Streptomyces hygroscopicus. The expression of wild-type hyg in T. thermophilus provided hygromycin B (HygB) resistance up to 60 °C. Through selection of mutants showing HygB resistance at higher temperatures, eight amino acid substitutions and the duplication of three amino acids were identified. A variant containing seven substitutions and the duplication (HYG10) showed HygB resistance at a highest temperature of 74 °C. Biochemical and biophysical analyses of recombinant HYG and HYG10 revealed that HYG10 was in fact thermostabilized. Modeling of the three-dimensional structure of HYG10 suggests the possible roles of the various substitutions and the duplication on thermostabilization, of which three substitutions and the duplication located at the enzyme surface suggested that these mutations made the enzyme more hydrophilic and provided increased stability in aqueous solution.

Strains of Thermus thermophilus produce unique carotenoids called thermozeaxanthins and their colonies are light-yellow pigmented. Here, we developed a new cloning system allowing for the rapid and convenient detection of recombinants by color screening based on carotenoid production in T. thermophilus. We constructed two cloning vectors that overexpress the crtB gene encoding a phytoene synthase under the strong promoter of the slpA gene. Phytoene synthase is one of essential enzymes for the production of carotenoids. We also isolated a carotenoid-overproducing mutant that formed orange colonies. Because disruption of crtB in the carotenoid-overproducing mutant resulted in white colonies, we used the disruptant as a host strain. Whereas transformants carrying a new cloning vector, pTRK1-PRslpA-crtBcas, grew into unusual red-pigmented colonies probably because of the extreme accumulation of thermozeaxanthins, those carrying the vector with a foreign DNA inserts formed white colonies. Thus, recombinants can be detected easily by color screening (red/white screening) in T. thermophilus. This cloning system requires no additional chromogenic substrate in the medium. We also constructed a promoter-probe vector, pTRK1-crtBmcs-PP, employing the open reading frame of crtB with multiple cloning sites. Using this vector, a series of colony-color phenotypes is observed probably depending on promoter activities of foreign DNA inserts, which enables the rapid probing of promoters. These vectors are useful to simplify cloning procedures and to identify the promoters of different strengths in T. thermophilus.