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Objective: The green fluorescent protein (GFP) and its derivatives are widely used in biomedical research. The manipulation of GFP-tagged proteins by GFP-specific binders, e.g. single-domain antibodies (nanobodies), is of increasing significance. It is therefore important to better understand the properties of antiGFP-GFP interaction in order to establish methodological applications. In this work the interaction of superfolder GFP (sfGFP) and its enhancer nanobody (aGFP enh ) was characterized further., Results: Previous calorimetric experiments demonstrated that the aGFP enh nanobody binds strongly to sfGFP with a nanomolar affinity. Here we show that this interaction results in a substantial structural stabilization of aGFP enh reflected in a significant increase of its melting temperature by almost 30 °C. The thermal stability of the sfGFP-aGFP enh complex is close to 85 °C in the pH range 7.0-8.5. For therapeutic applications thermoresistance is often an essential factor. Our results suggest that methodologies based on GFP-aGFP interaction can be applied under a wide range of physicochemical conditions. The aGFP enh nanobody seems to be suitable for manipulating sfGFP-labeled targets even in extreme thermophilic organisms., (© 2023. The Author(s).)

In this paper, we report on the utilization of micro-technology based tools to fight viral infections. Inspired by various hemoperfusion and immune-affinity capture systems, a blood virus depletion device has been developed that offers highly efficient capture and removal of the targeted virus from the circulation, thus decreasing virus load. Single-domain antibodies against the Wuhan (VHH-72) virus strain produced by recombinant DNA technology were immobilized on the surface of glass micro-beads, which were then utilized as stationary phase. For feasibility testing, the virus suspension was flown through the prototype immune-affinity device that captured the viruses and the filtered media left the column. The feasibility test of the proposed technology was performed in a Biosafety Level 4 classified laboratory using the Wuhan SARS-CoV-2 strain. The laboratory scale device actually captured 120,000 virus particles from the culture media circulation proving the feasibility of the suggested technology. This performance has an estimated capture ability of 15 million virus particles by using the therapeutic size column design, representing three times over-engineering with the assumption of 5 million genomic virus copies in an average viremic patient. Our results suggested that this new therapeutic virus capture device could significantly lower virus load thus preventing the development of more severe COVID-19 cases and consequently reducing mortality rate., (© 2023. The Author(s).)

The purpose of the recent work is to give a better explanation of how Dean vortices affect lateral focusing, and to understand how cell morphology can alter the focusing position compared to spherical particles. The position and extent of the focused region were investigated using polystyrene fluorescent beads with different bead diameters (Ø = 0.5, 1.1, 1.97, 2.9, 4.8, 5.4, 6.08, 10.2, 15.8, 16.5 µm) at different flow rates (0.5, 1, 2 µL/s). Size-dependent focusing generated a precise map of the equilibrium positions of the spherical beads at the end of the periodically altering channels, which gave a good benchmark for focusing multi-dimensional particles and cells. The biological samples used for experiments were rod-shaped Escherichia coli ( E. coli ), discoid biconcave-shaped red blood cells (RBC), round or ovoid-shaped yeast, Saccharomyces cerevisiae , and soft-irregular-shaped HeLa cancer-cell-line cells to understand how the shape of the cells affects the focusing position at the end of the channel.

High resolution analysis of N-glycans can be performed after their endoglycosidase mediated removal from proteins. N-glycosidase F peptide (PNGase F) is one the most frequently used enzyme for this purpose. Because of the significant demand for PNGase F both in basic and applied research, rapid and inexpensive methods are of great demand for its large-scale production, preferably in immobilizable form to solid supports or surfaces. In this paper, we report on the high-yield production of N-terminal 6His-PNGase F enzyme in a bacterial Escherichia coli SHuffle expression system. The activity profile of the generated enzyme was compared to commercially available PNGase F enzymes, featuring higher activity for the former. The method described here is thus suitable for the cost-effective production of PNGase F in an active, immobilizable form., (© 2022. The Author(s).)

Full characterization of the attached carbohydrate moieties of glycoproteins is of high importance for both the rapidly growing biopharmaceutical industry and the biomedical field. In this paper we report the design and production of three important 6HIS-tagged exoglycosidases (neuraminidase, β-galactosidase and hexosaminidase) to support rapid solid phase N-glycan sequencing with high robustness using immobilized enzymes. The exoglycosidases were generated in bacterial expression systems with high yield. Oriented immobilization via the 6HIS-tag portion of the molecules supported easy accessibility to the active sites and consequently high digestion performance. The three exoglycosidases were premixed in an appropriate matrix format and processed in a low-salt buffer to support long term storage. The digestion efficiencies of the immobilized enzymes were demonstrated by using solid phase sequencing in conjunction with capillary electrophoresis analysis of the products on a commercial glycoprotein therapeutic (palivizumab) and human serum derived fluorophore labeled glycans., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Prostate cancer represents the second highest malignancy rate in men in all cancer diagnoses worldwide. The development and progression of prostate cancer is not completely understood yet at molecular level, but it has been reported that changes in the N-glycosylation of prostate-specific antigen (PSA) occur during tumor genesis. In this paper we report on the development and implementation of a high-throughput capillary electrophoresis based glycan analysis workflow for urinary PSA analysis. The technology utilizes selective, high yield single domain antibody based PSA capture, followed by preconcentration and capillary electrophoresis coupled with laser-induced fluorescence detection, resulting in high resolution N-glycan profiles. Urinary PSA glycan profiles were compared to a commercially available PSA standard revealing differences in their α2,3- and α2,6-sialylated isomers, proving the excellent selectivity of the suggested workflow. This is important as sialylation classification plays an important role in the differentiation between indolent, significant and aggressive forms of prostate cancer., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Regular monitoring of arsenic concentrations in water sources is essential due to the severe health effects. Our goal was to develop a rapidly responding, sensitive and stable sensing layer for the detection of arsenic. We have designed flagellin-based arsenic binding proteins capable of forming stable filament structures with high surface binding site densities. The D3 domain of Salmonella typhimurium flagellin was replaced with an arsenic-binding peptide motif of different bacterial ArsR transcriptional repressor factors. We have shown that the fusion proteins developed retain their polymerization ability and have thermal stability similar to that of wild-type filament. The strong arsenic binding capacity of the monomeric proteins was confirmed by isothermal titration calorimetry (ITC), and dissociation constants (K d ) of a few hundred nM were obtained for all three variants. As-binding fibers were immobilized on the surface of a gold electrode and used as a working electrode in cyclic voltammetry (CV) experiments to detect inorganic arsenic near the maximum allowable concentration (MAC) level. Based on these results, it can be concluded that the stable arsenic-binding flagellin variant can be used as a rapidly responding, sensitive, but simple sensing layer in a field device for the MAC-level detection of arsenic in natural waters.

Reliable measurement of the binding kinetics of low molecular weight analytes to their targets is still a challenging task. Often, the introduction of labels is simply impossible in such measurements, and the application of label-free methods is the only reliable choice. By measuring the binding kinetics of Ni(II) ions to genetically modified flagellin layers, we demonstrate that: (1) Grating-Coupled Interferometry (GCI) is well suited to resolve the binding of ions, even at very low protein immobilization levels; (2) it supplies high quality kinetic data from which the number and strength of available binding sites can be determined, and (3) the rate constants of the binding events can also be obtained with high accuracy. Experiments were performed using a flagellin variant incorporating the C-terminal domain of the nickel-responsive transcription factor NikR. GCI results were compared to affinity data from titration calorimetry. We found that besides the low-affinity binding sites characterized by a micromolar dissociation constant (K d ), tetrameric FliC-NikR C molecules possess high-affinity binding sites with K d values in the nanomolar range. GCI enabled us to obtain real-time kinetic data for the specific binding of an analyte with molar mass as low as 59 Da, even at signals lower than 1 pg/mm 2 .

The environmental monitoring of Ni is targeted at a threshold limit value of 0.34 μM, as set by the World Health Organization. This sensitivity target can usually only be met by time-consuming and expensive laboratory measurements. There is a need for inexpensive, field-applicable methods, even if they are only used for signaling the necessity of a more accurate laboratory investigation. In this work, bioengineered, protein-based sensing layers were developed for Ni detection in water. Two bacterial Ni-binding flagellin variants were fabricated using genetic engineering, and their applicability as Ni-sensitive biochip coatings was tested. Nanotubes of mutant flagellins were built by in vitro polymerization. A large surface density of the nanotubes on the sensor surface was achieved by covalent immobilization chemistry based on a dithiobis(succimidyl propionate) cross-linking method. The formation and density of the sensing layer was monitored and verified by spectroscopic ellipsometry and atomic force microscopy. Cyclic voltammetry (CV) measurements revealed a Ni sensitivity below 1 μM. It was also shown that, even after two months of storage, the used sensors can be regenerated and reused by rinsing in a 10 mM solution of ethylenediaminetetraacetic acid at room temperature.

Background: Oral and enteral mucositis due to high-dose cytostatic treatment administered during autologous and allogeneic stem-cell transplantation increases mortality. Salivary secretory immunoglobulin A (sIgA) is a basic pillar of local immunity in the first line of defense. Altered salivary sialoglycoprotein carbohydrates are important in the pathologies in the oral cavity including inflammation, infection and neoplasia. Therefore, we assessed whether changes in the salivary and serum IgA glycosylation correlated with development and severity of oral mucositis., Methods: Using capillary electrophoresis, comparative analysis of serum and salivary IgA total N-glycans was conducted in 8 patients with autologous peripheral stem-cell transplantation (APSCT) at four different stages of transplantation (day -3/-7, 0, +7, +14) and in 10 healthy controls., Results: Fourteen out of the 31 structures identified in serum and 6 out of 38 in saliva showed significant changes upon transplantation compared with the control group. Only serum core fucosylated, sialylated bisecting biantennary glycan (FA2BG2S2) showed significant differences between any two stages of transplantation (day -3/-7 and day +14; p = 0.0279)., Conclusion: Our results suggest that changes in the serum IgA total N-glycan profile could serve as a disease-specific biomarker in patients undergoing APSCT, while analysis of salivary IgA N-glycan reflects the effect of APSCT on local immunity.

Aims: Application of capillary electrophoresis with laser induced fluorescence detection (CE-LIF) to identify the N-glycosylation structures of serum and saliva IgA from healthy controls and patients with malignant hematological diseases having cytostatic treatment induced mild oral mucosal lesions., Background: Altered N-glycosylation of body fluid glycoproteins can be an effective indicator of most inflammatory processes. Immunoglobulin A (IgA) is the second highest abundant immunoglobulin and has a major role in the immune-defense against potential pathogen attacks. While IgA is abundant in serum, secretory immunoglobulin A (sIgA) is one of the most prevalent proteins in mucosal surfaces, such as in saliva., Objective: Our aim was to investigate the changes of IgA glycosylation in serum and saliva as a response to an administered cytostatic treatment in patients with malignant hematological disorders., Methods: Capillary electrophoresis with laser induced fluorescent detection (CE-LIF) was used to analyze the N-glycosylation profiles of Z(IgA1) partitioned immunoglobulin A in pooled serum and saliva of 10 control subjects and 8 patients with malignant hematological diseases having cytostatic treatment induced mild oral mucosal lesions., Results: Eight of 31 and four of 38 N-glycans in serum and saliva, respectively, showed significant (p<0.05) differences upon comparison to the control group. Thirteen glycans were present in the saliva but not in the serum, on the other hand, six structures were found in the serum samples not present in the saliva., Conclusion: The developed Z(IgA1) partitioning and the high resolution CE-LIF based glyocoanalytical methods provided an efficient and sensitive workflow to detect and monitor IgA glycosylation alterations in serum and saliva with the scope for widespread molecular medicinal use., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The export signal recognized by the flagellum-specific export machinery is harbored within the highly conserved 26-47 segment of the disordered N-terminal part of Salmonella flagellin. In this work, we aimed to further localize the essential part of the export signal by deletion analysis and investigated how the length of the spacer segment preceding the signal affects export efficiency. Export signal variants were attached to a reporter protein, the CCP2 domain of human C1r protein, and export efficiency of the fusion constructs was studied. Our results suggest that almost any continuous oligopeptide of 8-10 residues within the 26-47 segment can efficiently direct flagellar export if preceded by a spacer segment of at least 15 amino acids without any specific sequential requirement., (© 2018 Federation of European Biochemical Societies.)

In this work we addressed the problem how to fabricate self-assembling tubular nanostructures displaying target recognition functionalities. Bacterial flagellar filaments, composed of thousands of flagellin subunits, were used as scaffolds to display single-domain antibodies (nanobodies) on their surface. As a representative example, an anti-GFP nanobody was successfully inserted into the middle part of flagellin replacing the hypervariable surface-exposed D3 domain. A novel procedure was developed to select appropriate linkers required for functional internal insertion. Linkers of various lengths and conformational properties were chosen from a linker database and they were randomly attached to both ends of an anti-GFP nanobody to facilitate insertion. Functional fusion constructs capable of forming filaments on the surface of flagellin-deficient host cells were selected by magnetic microparticles covered by target GFP molecules and appropriate linkers were identified. TEM studies revealed that short filaments of 2-900 nm were formed on the cell surface. ITC and fluorescent measurements demonstrated that the fusion protein exhibited high binding affinity towards GFP. Our approach allows the development of functionalized flagellar nanotubes against a variety of important target molecules offering potential applications in biosensorics and bio-nanotechnology.

With the aim of creating one-dimensional magnetic nanostructures, we genetically engineered flagellar filaments produced by Salmonella bacteria to display iron- or magnetite-binding sites, and used the mutant filaments as templates for both nucleation and attachment of the magnetic iron oxide magnetite. Although nucleation from solution and attachment of nanoparticles to a pre-existing surface are two different processes, non-classical crystal nucleation pathways have been increasingly recognized in biological systems, and in many cases nucleation and particle attachment cannot be clearly distinguished. In this study we tested the magnetite-nucleating ability of four types of mutant flagella previously shown to be efficient binders of magnetite nanoparticles, and we used two other mutant flagella that were engineered to periodically display known iron-binding oligopeptides on their surfaces. All mutant filaments were demonstrated to be efficient as templates for the synthesis of one-dimensional magnetic nanostructures under ambient conditions. Both approaches resulted in similar final products, with randomly oriented magnetite nanoparticles partially covering the filamentous biological templates. In an external magnetic field, the viscosity of a suspension of the produced magnetic filaments showed a twofold increase relative to the control sample. The results of magnetic susceptibility measurements were also consistent with the magnetic nanoparticles occurring in linear structures. Our study demonstrates that biological templating can be used to produce one-dimensional magnetic nanostructures under benign conditions, and that modified flagellar filaments can be used for creating model systems in which crystal nucleation from solution can be experimentally studied.

This work aimed at developing a novel method for fabricating 1 D magnetite nanostructures with the help of mutated flagellar filaments. We constructed four different flagellin mutants displaying magnetite-binding motifs: two contained fragments of magnetosome-associated proteins from magnetotactic bacteria (MamI and Mms6), and synthetic sequences were used for the other two. A magnetic selection method identified the MamI mutant as having the highest binding affinity to magnetite. Filaments built from MamI loop-containing flagellin subunits were used as templates to form chains of magnetite nanoparticles along the filament by capturing them from suspension. Our study represents a proof-of-concept that flagellar filaments can be engineered to facilitate formation of 1 D magnetite nanostructures under ambient conditions. In addition, it proves the interaction between MamI and magnetite, with implications for the role of this protein in magnetotactic bacteria., (© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Unlabelled: Biomimetic coatings with cell-adhesion-regulating functionalities are intensively researched today. For example, cell-based biosensing for drug development, biomedical implants, and tissue engineering require that the surface adhesion of living cells is well controlled. Recently, we have shown that the bacterial flagellar protein, flagellin, adsorbs through its terminal segments to hydrophobic surfaces, forming an oriented monolayer and exposing its variable D3 domain to the solution. Here, we hypothesized that this nanostructured layer is highly cell-repellent since it mimics the surface of the flagellar filaments. Moreover, we proposed flagellin as a carrier molecule to display the cell-adhesive RGD (Arg-Gly-Asp) peptide sequence and induce cell adhesion on the coated surface. The D3 domain of flagellin was replaced with one or more RGD motifs linked by various oligopeptides modulating flexibility and accessibility of the inserted segment. The obtained flagellin variants were applied to create surface coatings inducing cell adhesion and spreading to different levels, while wild-type flagellin was shown to form a surface layer with strong anti-adhesive properties. As reference surfaces synthetic polymers were applied which have anti-adhesive (PLL-g-PEG poly(l-lysine)-graft-poly(ethylene glycol)) or adhesion inducing properties (RGD-functionalized PLL-g-PEG). Quantitative adhesion data was obtained by employing optical biochips and microscopy. Cell-adhesion-regulating coatings can be simply formed on hydrophobic surfaces by using the developed flagellin-based constructs. The developed novel RGD-displaying flagellin variants can be easily obtained by bacterial production and can serve as alternatives to create cell-adhesion-regulating biomimetic coatings., Statement of Significance: In the present work, we show for the first time that., (Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Export of external flagellar proteins requires a signal located within their N-terminal disordered part, however, these regions do not share any significant sequence similarity suggesting that the secondary/tertiary structure might be important for recognition by the export gate. NMR experiments were performed to reveal the conformational properties of the flagellin signal sequence in vitro. It assumed a largely disordered fluctuating structure in aqueous environment, but acquired a folded structure containing an amphipathic helical portion in 50% MeOH or upon addition of SDS micelles which are known to promote hydrophobic interactions. Our observations raise the possibility that the signal sequence may partially undergo amphipathic helical ordering upon interaction with the recognition unit of the flagellar export machinery in a similar way as revealed for protein import into intracellular eukaryotic organelles mediated by targeting signals of high diversity., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Flagellin, the main component of flagellar filaments, is a protein possessing polymerization ability. In this work, a novel fusion construct of xylanase A from B. subtilis and Salmonella flagellin was created which is applicable to build xylan-degrading catalytic nanorods of high stability. The FliC-XynA chimera when overexpressed in a flagellin deficient Salmonella host strain was secreted into the culture medium by the flagellum-specific export machinery allowing easy purification. Filamentous assemblies displaying high surface density of catalytic sites were produced by ammonium sulfate-induced polymerization. FliC-XynA nanorods were resistant to proteolytic degradation and preserved their enzymatic activity for a long period of time. Furnishing enzymes with self-assembling ability to build catalytic nanorods offers a promising alternative approach to enzyme immobilization onto nanostructured synthetic scaffolds.

Flagella, the locomotion organelles of bacteria, extend from the cytoplasm to the cell exterior. External flagellar proteins are synthesized in the cytoplasm and exported by the flagellar type III secretion system. Soluble components of the flagellar export apparatus, FliI, FliH, and FliJ, have been implicated to carry late export substrates in complex with their cognate chaperones from the cytoplasm to the export gate. The importance of the soluble components in the delivery of the three minor late substrates FlgK, FlgL (hook-filament junction) and FliD (filament-cap) has been convincingly demonstrated, but their role in the transport of the major filament component flagellin (FliC) is still unclear. We have used continuous ATPase activity measurements and quartz crystal microbalance (QCM) studies to characterize interactions between the soluble export components and flagellin or the FliC:FliS substrate-chaperone complex. As controls, interactions between soluble export component pairs were characterized providing Kd values. FliC or FliC:FliS did not influence the ATPase activity of FliI alone or in complex with FliH and/or FliJ suggesting lack of interaction in solution. Immobilized FliI, FliH, or FliJ did not interact with FliC or FliC:FliS detected by QCM. The lack of interaction in the fluid phase between FliC or FliC:FliS and the soluble export components, in particular with the ATPase FliI, suggests that cells use different mechanisms for the export of late minor substrates, and the major substrate, FliC. It seems that the abundantly produced flagellin does not require the assistance of the soluble export components to efficiently reach the export gate., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The surface adsorption of the protein flagellin was followed in situ using optical waveguide lightmode spectroscopy (OWLS). Flagellin did not show significant adsorption on a hydrophilic waveguide, but very rapidly formed a dense monolayer on a hydrophobic (silanized) surface. The homogeneous and isotropic optical layer model, which has hitherto been generally applied in OWLS data interpretation for adsorbed protein films, failed to characterize the flagellin layer, but it could be successfully modeled as an uniaxial thin film. This anisotropic modeling revealed a significant positive birefringence in the layer, suggesting oriented protein adsorption. The adsorbed flagellin orientation was further evidenced by monitoring the surface adsorption of truncated flagellin variants, in which the terminal protein regions or the central (D3) domain were removed. Without the terminal regions the protein adsorption was much slower and the resulting films were significantly less birefringent, implying that intact flagellin adsorbs on the hydrophobic surface via its terminal regions.

The exact role of the metal ion, usually Mg(2+), in the catalysis of human 3-phosphoglycerate kinase, a well-studied two-domain enzyme, has not been clarified. Here we have prepared single and double alanine mutants of the potential metal-binding residues, D374 and D218. While all mutations weaken the catalytic interactions with Mg(2+), they surprisingly strengthen binding of both MgADP and MgATP, and the effects are even more pronounced for ADP and ATP. Thermodynamic parameters of binding indicate an increase in the binding entropy as a reason for the strengthening. In agreement with the experimental results, computer-simulated annealing calculations for the complexes of these mutants have supported the mobility of the nucleotide phosphates and, as a consequence, formation of their new interaction(s) within the active site. A similar type of mobility is suggested to be a characteristic feature of the nucleotide site of the wild-type enzyme, too, both in its inactive open conformation and in the active closed conformation. This mobility of the nucleotide phosphates that is regulated by the aspartate side chains of D218 and D374 through the complexing Mg(2+) is suggested to be essential in enzyme function.

The flagellum-specific export system is a specialized type III export machinery, which exports external flagellar proteins through the central channel of the flagellar filament. A number of evidence indicates that short segments within the disordered N-terminal region of flagellar axial proteins are recognized by the flagellum-specific export apparatus. Recently, we have demonstrated that the 26-47 segment of Salmonella typhimurium flagellin is capable of mediating flagellar export. N-terminal flagellin segments containing the export signal combined with a hexahistidine tag can be attached to heterologous proteins (preferentially in the size range of 9-40 kDa) facilitating their secreted expression and easy purification from the medium. Certain over-expressed proteins that are easily degraded within the cells are found intact in the medium implying a potential application of this expression system for proteins of high proteolytic susceptibility.

The aim of our work is to furnish enzymes with polymerization ability by creating fusion constructs with the polymerizable protein, flagellin, the main component of bacterial flagellar filaments. The D3 domain of flagellin, exposed on the surface of flagellar filaments, is formed by the hypervariable central portion of the polypeptide chain. D3 is not essential for filament formation. The concept in this project is to replace the D3 domain with suitable monomeric enzymes without adversely affecting polymerization ability, and to assemble these chimeric flagellins into tubular nanostructures. To test the feasibility of this approach, xylanase A (XynA) from B. subtilis was chosen as a model enzyme for insertion into the central part of flagellin. With the help of genetic engineering, a fusion construct was created in which the D3 domain was replaced by XynA. The flagellin-XynA chimera exhibited catalytic activity as well as polymerization ability. These results demonstrate that polymerization ability can be introduced into various proteins, and building blocks for rationally designed assembly of filamentous nanostructures can be created.

The hypervariable D3 domain of Salmonella flagellin, composed of residues 190-283, is situated at the outer surface of flagellar filaments. A flagellin mutant deprived of the complete D3 domain (ΔD3&#95;FliC) exhibited a significantly decreased thermal stability (T(m) 41.9 °C) as compared to intact flagellin (T(m) 47.3 °C). However, the stability of filaments formed from ΔD3&#95;FliC subunits was virtually identical with that of native flagellar filaments. While D3 comprises the most stable part of monomeric flagellin playing an important role in the stabilization of the other two (D1 and D2) domains, the situation is reversed in the polymeric state. Upon filament formation, ordering of the disordered terminal regions of flagellin in the core part of the filament results in the stabilization of the radially arranged D1 and D2 domains, and there is a substantial increase of stability even in the distant outermost D3 domain, which is connected to D2 via a pair of short antiparallel β-strands. Our experiments revealed that crosslinking the ends of the isolated D3 domain through a disulfide bridge gives rise to a stabilization effect reminiscent of that observed upon polymerization. It appears that the short interdomain linker between domains D2 and D3 serves as a stabilization center that facilitates propagation of the conformational signal from the filament core to the outer part of filament. Because D3 is a largely independent part of flagellin, its replacement by heterologous proteins or domains might offer a promising approach for creation of various fusion proteins possessing polymerization ability., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Recently, we have demonstrated that the 26-47 segment of Salmonella enterica serovar Typhimurium flagellin is capable of mediating flagellar export. In order to reveal whether other parts of the N-terminal region have any significant influence on secretion, a series of plasmids were constructed containing the lac promoter followed by the 26-47, 2-65, or 2-192 portion of Salmonella flagellin, to which various heterologous proteins of different size were fused (18 constructs overall). Essentially, all three segments could drive protein export; however, the nature of the attached polypeptide also had a significant effect on secretion efficiency. When low export efficiency was observed, it was mainly caused by inclusion body formation. Our data provide strong support for the idea that a short segment within the disordered N-terminal region of axial proteins is recognized by the flagellar type III export machinery. The 26-47 segment of flagellin contains all of the necessary information to direct translocation of attached polypeptide chains. This short (positions 26 to 47) flagellin segment attached to recombinant proteins can be used for secreted protein expression. Certain fusion proteins that are easily degraded within the cells were found to be intact in the medium, implying a potential application of this expression system for proteins with high proteolytic susceptibility.

The energetic changes accompanying domain closure of 3-phosphoglycerate kinase, a typical hinge-bending enzyme, were assessed. Calorimetric titrations of the enzyme with each substrate, both in the absence and presence of the other one, provide information not only about the energetics of substrate binding, but of the associated conformational changes, including domain closure. Our results suggest that conformational rearrangements in the hinge generated by binding of both substrates provide the main driving force for domain closure overcoming the slightly unfavourable contact interactions between the domains.