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Protein-ligand interaction is one of the highlights of molecular recognition. The most popular application of this type of interaction is drug development which requires a high throughput screening of a ligand that binds to the target protein. Our goal was to find a binding ligand with a simple detection, and once this type of ligand was found, other methods could then be used to measure the detailed kinetic or thermodynamic parameters. We started with the idea that the ligand NMR signal would disappear if it was bound to the non-tumbling mass. In order to create the non-tumbling mass, we tried the aggregates of a target protein, which was fused to the elastin-like polypeptide. We chose the maltose binding proteinas a test case, and we tried it with several sugars, which included maltose, glucose, sucrose, lactose, galactose, maltotriose, and β-cyclodextrin. The maltose signal in the H-1 NMR spectrum disappeared completely as hoped around the protein to ligand ratio of 1:3 at 298 K where the proteins aggregated. The protein signals also disappeared upon aggregation except for the fast-moving part, which resulted in a cleaner background than the monomeric form. Since we only needed to look for a disappearing signal amongst those from the mixture, it should be useful in high throughput screening. Other types of sugars except for the maltotriose and β-cyclodextrin, which are siblings of the maltose, did not seem to bind at all. We believe that our system would be especially more effective when dealing with a smaller target protein, so both the protein and the bound ligand would lose their signals only when the aggregates formed. We hope that our proposed method would contribute to accelerating the development of the potent drug candidates by simultaneously identifying several binders directly from a mixture. Supplementary Information The online version contains supplementary material available at 10.1007/s10858-021-00381-x.

The efficiency of enzymatic cyclodextrin production using cyclodextrin glycosyltransferases (CGTases) is limited by product inhibition. In this study, maltose binding site 2 (MBS2) of the β-CGTase from Bacillus circulans STB01 was modified to decrease product inhibition. First, two point mutants were prepared at position 599 (A599V and A599N). Then, two double mutants incorporating alanine at position 633 (A599N/Y633A and A599V/Y633A) were prepared. Finally, the entire MBS2 region was replaced by that of the α-CGTase from Paenibacillus macerans JFB05-01 to form multipoint mutant MBS2 β → α. All five mutants exhibited mixed-type product inhibition, although both the competitive and uncompetitive components of this inhibition were decreased. The total cyclization activities of A599N, A599V and A599V/Y633A were 15.6%, 76.8% and 70.9% lower than that of the wild-type, respectively, while that of A599N/Y633A was 22.4% higher. Among the mutants, only MBS2 β → α showed catalytic efficiency (kcat/Km) comparable with that of the wild-type. Moreover, A599N, A599N/Y633A and MBS2 β → α produced cyclodextrin yields 13.1%, 15.8% and 19.7% greater than that of the wild-type, respectively. These results suggest that A599N, A599N/Y633A and MBS2 β → α may be more suitable than the wild-type for cyclodextrin production.

The change in structure, function of proteinase K in the presence of maltose was studied using spectroscopic and computational techniques. UV–Vis spectroscopy results reveal with the addition of maltose, the peaks of proteinase K didn't show any shifts. The intrinsic fluorescence intensity was decreased regularly with rising the ligand concentration. These results suggest that the quenching mode is dynamic quenching. Far-UV circular dichroism (CD) studies showed a low decrease in the β-sheet, β-turn and the large increase in the α-helix. The activity of proteinase K was increased in the presence of maltose. Thus, maltose is an activator for proteinase K. Molecular docking results show a negative value for the Gibbs free energy. On the other hand, the thermal stability of proteinase K was investigated in the presence of maltose over the temperature range (298–353) K. Obtained data indicate the thermal stability of proteinase K will be reduced when the concentration of maltose increase. These results are confirmed by the molecular dynamic simulation technique.

Canonical ATP-binding cassette import systems rely on extracellular substrate binding proteins (SBP) for function. In gram-negative bacteria , SBPs are usually freely diffusible in the periplasm and, where studied, exist in excess over their cognate transporters. However, in vitro studies with the maltose transporter of Escherichia coli (MalFGK2) have demonstrated that mechanistically one copy of its SBP (MalE) per transport complex is sufficient for activity. To address whether such a condition is physiologically relevant, we have characterized a homolog of the E. coli system from the gram-negative bacterium Bdellovibrio bacteriovorus which has a single copy of a maltose binding domain fused to the MalF subunit. Both transporters share substrate specificity for maltose and linear maltodextrins. Specific ATPase and transport activities of the B. bacteriovorus transporter were comparable to those of the E. coli system assayed at a 1:1 M ratio of MalE to the transport complex. While MalEEc was able to additionally increase ATPase activity of MalFGK2Bb, the isolated MalE domain of B. bacteriovorus failed to stimulate the E. coli system. Strikingly, interactions of the MalE domain with the transmembrane subunits during the transport cycle as studied by site-specific cross-linking were found to differ from those observed for E. coli MalE-FGK2.

European honeybee, Apis mellifera, produces α-glucosidase (HBGase) that catalyzes the cleavage of an α-glycosidic bond of the non-reducing end of polysaccharides and has potential applications for malt hydrolysis in brewing industry. Characterized by their substrate specificities, HBGases have three isoforms including HBGase II, which prefers maltose to sucrose as a substrate. Previous study found that the catalytic efficiency of maltose hydrolysis of N226P mutant of HBGase II was higher than that of the wild type (WT), and the catalytic efficiency of maltose hydrolysis of WT was higher than those of H227Y and N226P-H227Y mutants. We hypothesized that N226P mutation probably caused maltose to bind with better affinity and position/orientation for hydrolysis than WT, while H227Y and N226P-H227Y mutations caused maltose to bind with worse affinity and position/orientation for hydrolysis than WT. Using this hypothesis, we performed molecular dynamics on the catalytically competent binding conformations of maltose/WT, maltose/N226P, maltose/H227Y, and maltose/N226P-H227Y complexes to elucidate effects of N226P and H227Y mutations on maltose binding in HBGase II active site. Our results reasonably support this hypothesis because the N226P mutant had better binding affinity, higher number of important binding residues, strong and medium hydrogen bonds as well as shorter distance between atoms necessary for hydrolysis than WT, while the H227Y and N226P-H227Y mutants had worse binding affinities, lower number of important binding residues and strong hydrogen bonds as well as longer distances between atoms necessary for hydrolysis than WT. Moreover, results of binding free energy and hydrogen bond interaction of residue 227 support the role of H227 as a maltose preference residue, as proposed by previous studies. Our study provides important and novel insight into how N226P and H227Y mutations affect maltose binding in HBGase II active site. This knowledge could potentially be used to engineer HBGase II to improve its efficiency.

An intensively investigated intermediate state of protein folding is the molten globule (MG) state, which contains secondary but hardly any tertiary structure. In previous work, we have determined the distances between interacting spins within maltose binding protein (MBP) in its native state using continuous wave and double electron-electron resonance (DEER) electron paramagnetic resonance (EPR) spectroscopy. Seven double mutants had been employed to investigate the structure within the two domains of MBP. DEER data nicely corroborated the previously available X-ray data. Even in its MG state, MBP is known to still bind its ligand maltose. We therefore hypothesized that there must be a defined structure around the binding pocket of MBP already in the absence of tertiary structure. Here we have investigated the functional and structural difference between native and MG state in the open and closed form with a new set of MBP mutants. In these, the spin-label positions were placed near the active site. Binding of its ligands leads to a conformational change from open to closed state, where the two domains are more closely together. The complete set of MBP mutants was analyzed at pH 3.2 (MG) and pH 7.4 (native state) using double-quantum coherence EPR. The values were compared with theoretical predictions of distances between the labels in biradicals constructed by molecular modeling from the crystal structures of MBP in open and closed form and were found to be in excellent agreement. Measurements show a defined structure around the binding pocket of MBP in MG, which explains maltose binding. A new and important finding is that in both states ligand-free MBP can be found in open and closed form, while ligand-bound MBP appears only in closed form because of maltose binding.

Endophytic fungi Chaetomium sp isolated from Phyllanthus niruri Linn. Mycelium powder was extracted by using ethyl acetate. Extract was fractionated using n-hexane, dichloromethane and ethanol 96%. The antimicrobial test was carried out using disc diffusion and microdilution methods. The antioxidant activity of the fraction was determined using hydrogen peroxide free radical scavenging and reducing power capacity activities. The cytotoxicity assay of the fraction against T47D breast cancer cell was carried out using dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide method (MTT). The in silico prediction of chemical substances which are reported exist in Chaetomium sp. performed using AutoDockVina embedded in PyRx version 8.0. Dichloromethane fraction was found as the most active sample against Escherichia coli (IC 50 20.76 mg/mL), Staphylococcus aureus (IC 50 70.15 mg/mL), Salmonella typhi (49.13 mg/mL) and was found as the most high phenolic content with value 47.44 mg GAE/g fraction, whereas the best antioxidant activity was performed by ethanol 96% fraction (85%). Cytotoxicity assay against T47D cell line showed dichloromethane fraction have highest activity with IC 50 10.76 mg/mL. The docking studies showed that compounds bearing xanthone structure were potential for maltose binding periplasmic and human aromatase associating with their potencies as antibacteria and anticancer. Endophytic fungi Chaetomium sp. was isolated from Phyllanthus niruri using n-hexane, dichloromethane and ethanol fractions was studied its various biological activities as antimicrobial, antioxidant and cytotoxic agent against breast cancer cell.

Lipid peroxidation is involved in many disorders and diseases such as cardiovascular disease, cancers, neurodegenerative diseases, and even aging. Lipid peroxidation products existing in blood or bodily fluids are very important biomarkers for the diagnosis of such diseases. In particular, 13(R,S)-hydroxy-9(E),11(E)-octadecadienoic acid (13-(E,E)-HODE) is an oxidiation product of linoleic acid, which is an important biomarker for many diseases such as diabetes and Alzheimer's disease. In this study, we successfully displayed the antigen-binding fragment of an antibody produced by hybridoma 1213-1 on the M13 phage and performed analysis of the antibody variable region genes. The blast results suggested that it is a novel antibody. We also developed a phage-antibody-based competitive ELISA and a novel Open Sandwich ELISA (OS ELISA) for the detection of 13-(E,E)-HODE. The OS ELISA showed a limit of detection (LOD) of 15.6 nM of 13-(E,E)-HODE and low cross-reactivity with other HODE such as 9-(E,E)-HODE. Another format of the open sandwich ELISA with purified maltose binding protein-fused VL and VH-phage showed a lower LOD of 2.2 nM of 13-(E,E)-HODE, which may be sensitive enough to detect the concentration of 13-(E,E)-HODE in patients’ blood samples. This is the first OS ELISA for the detection of lipids, and we believe it also represents the first molecular cloning of anti-HODE antibody genes.

The type-1 water soluble chlorophyll binding proteins (WSCP1) have been generally known as chlorophyll extractors and transporters from the thylakoid membrane to the chloroplast envelope, where the membrane bound chlorophyllase catabolizes the chlorophyll. Despite the type-2 WSCP, WSCP1 has been known to be located in the chloroplasts of the green plants. In the present study, the non-chloroplastic protein superfamily containing domain of unknown function 538 (DUF538) as functional homologue of type-1 WSCP has been identified in plants. The structural similarities/differences and the cellular locations of Celosia cristata DUF538 and Chenopodium album WSCP1 were predicted by using computational tools and the chlorophyll binding abilities of their purified maltose binding protein-fused forms were estimated by maltose binding affinity method. It was predicted that despite CaWSCP1, CcDUF538 is a non-chloroplastic protein. The chlorophyll binding abilities of the recombinant fusion forms of test WSCP1 and DUF538 were estimated to be about 58 and 56%, respectively. Considering DUF538 as stress-induced protein, it was speculated that they may form complex with chlorophyll molecules or their hydrolyzed products out of chloroplasts to proceed the chlorophyll breakdown and nitrogen/carbon recycling in stress-challenged plants.

We present polysaccharide-based nanoparticles able to associate and increase the catalytic activity of the maltose-binding MBP317-347 switch enzyme. Fluorescence quenching and molecular docking studies along with the partial resistance to increasing pH and ionic strength indicate that the increase in enzymatic activity is due to a specific interaction between the maltose binding pocket on MBP317-347 and alginate exposed on the surface of the nanoparticles. Finally, we show that the hybrid self co-assembled particles increase the half-life of MBP317-347 over six-fold at 37°C, thus reflecting their potential use as a macromolecular drug delivery system.

Bacteriophage base tailplate proteins were recently discovered to have hydrolytic activity towards disaccharides. The putative assignment of sugar binding sites was based on known lectin structures and identified residues a.a. 40–120 as the potential binding region for disaccharides [1]. To help verify the prediction, an in silico analysis was performed on the structure of a base tailplate protein gp31 from Klebsiella pneumoniae bacteriophage KP32 (PDB: 5MU4 ) which shows activity towards maltose but not trehalose [1]. Based on the information, a full surface docking was performed for both sugars which identified 2 regions different than originally predicted. The first region clearly favored maltose during the docking phase while the second one allowed for the energetically-equivalent binding of trehalose. To verify the assignment, a molecular dynamics simulation was performed to assess the stability of the docked substrates. MD simulations suggested that the first site included residues D131, D133, and E134, and was also superior for maltose binding while clearly disfavoring trehalose. Analysis of the putative catalytic mechanism suggested residues D131, D133 and E134 as critical for substrate binding. The residue D133 did participate in a stable substrate binding and was positioned near the scissile bond, potentially making it a catalytic residue. Catalytic residues were most likely D131 and D133, one of the two options proposed by Pyra et al. [1]. A comparison with known hydrolase mechanisms suggested that the enzyme most likely retains configuration during hydrolysis of maltose. The findings are discussed for other bacteriophage proteins regarding their potential specificities and catalytic mechanisms.

Inhibition of cyclodextrin glycosyltransferases (CGTases) by their product cyclodextrins limits the efficiency of cyclodextrin production. In an effort to produce variants with good activity but reduced product inhibition, six mutants were constructed at position 603 of the CGTase from Bacillus circulans STB01, which exhibits mixed-type product inhibition. In a kinetic analysis, N603I showed reduced noncompetitive inhibition while N603K, N603H and N603R showed increased noncompetitive inhibition. Unexpectedly, N603E and N603D exhibited reductions in both competitive and noncompetitive product inhibition. Noncompetitive product inhibition is closely related to the interaction between the cyclodextrin and the enzyme in maltose binding site 2 (MBS2). Structural models led to the suggestion that there is increased interaction between maltose binding sites 1 and 2 in mutants N603E and N603D, which may have led to the unexpected results. N603D exhibited a 23.9% greater cyclodextrin yield per gram of enzyme than the wild-type, suggesting it has potential for industrial use. Further reductions in product inhibition may be gained through studies of maltose binding site interactions.

Microcystin with leucine and arginine (MCLR) is a hepatotoxic toxin produced by cyanobacteria present in watercourses that causes cell death and tissue necrosis at high exposure levels. In this study, a sensitive non-competitive, Open sandwich Enzyme-linked Immunosorbent Assay (OS-ELISA) with wide detectable range was developed for the quantification of MCLR. OS-ELISA is based on the principle of antigen-driving interaction enhancement between variable regions of an antibody. The genes for the variable regions of antibody 3A8 against MCLR were used to construct the phage display vector pDong1/Fab(3A8) to prepare assay elements. The OS-ELISA performed with phage-displayed antibody VH fragment and secreted light chain showed a limit of detection (LOD) of 0.14 nM and a wide working range from 0.14 to 10,000 nM MCLR. Another format of OS-ELISA that used purified Maltose Binding Protein-fused VL and VH-phage showed a lower LOD of 85 pM MCLR. Using the developed assay, the recovery rates for samples of lake or river water spiked with MCLR were found to range from 95.5% to 116.4%, suggesting that the assay has good potential to be used as a monitoring tool for MCLR in nature.

Cyclodextrin glucanotransferase (CGT) is an industrial enzyme used for the production of cyclodextrins (CDs). CGT folds into five domains A, B, C, D and E. Domain E has the ability to bind starch and cyclodextrins (CDs) and is considered the starch-binding domain of CGT. Two maltose binding sites (MBS1 and MBS2) have been identified in domain E. Domain E participates in the formation of a deep groove which is located on the protein surface of CGT and extends from the active site to MBS2. To investigate the function of domain E in the catalysis of CGT, CGT mutants were constructed using a CGT from Bacillus macerans. The truncated CGT (CGTAE), was constructed by deleting domain E. In the chimeric CGT (CGT-SBD), domain E was replaced with the starch binding domain (SBD) of glucoamylase I (GAI), which has a similar structure and function to domain E. The modified CGT (CGT+6) was constructed by inserting six amino acids between domains D and E. The six amino acids were inserted as a pseudo linker in order to perturb the alignment of the groove by increasing the distance between domains D and E. CGTAE exhibited no detectable activity, whereas CGT-SBD had very low cyclization, starch-degrading, and coupling activities. CGT+6 exhibited about 5060% of the wild type CGT (WT-CGT) activity, and its thennostability was lower than that of WT-CGT. The K^, value for the cyclization activity of CGT+6 was about 2.6 fold greater than that of WT-CGT. The efficiency of the cyclization reaction (kgat /K„) was also significantly reduced.

Receptor activator of nuclear factor-kappa B ligand (RANKL) is a critical factor in osteoclastogenesis. It makes osteoclasts differentiate and multinucleate in bone remodeling. In the present study, RANKL was expressed as a soluble maltose binding protein (MBP)-fusion protein using the Escherichia coli maltose binding domain tag system (pMAL) expression vector system. The host cell E. coli DH5α was cultured and induced by isopropyl β-D-1- thiogalactopyranoside for rRANKL expression. Cells were disrupted by sonication to collect soluble MBP-fused rRANKL. The MBP-fusion rRANKL was purified with MBP Trap affinity chromatography and treated with Tobacco Etch Virus nuclear inclusion endopeptidase (TEV protease) to remove the MBP fusion protein. Dialysis was then carried out to remove binding maltose from the cleaved rRANKL solution. The cleaved rRANKL was purified with a second MBP Trap affinity chromatography to separate unsevered MBP-fusion rRANKL and cleaved MBP fusion protein. The purified rRANKL was shown to have biological activity by performing in vitro cell tests. In conclusion, biologically active rRANKL was successfully purified by a simple two-step chromatography purification process with one column.

This study was designed to explore the effect of active immunization against maltose binding protein-gonadotropin releasing hormone I hexamer (MBP-GnRH-I6) on the reproductive function in cats. Each immunized cat was administered twice intramuscularly in the neck at 16 and 20 weeks old. The concentrations of the testosterone and estradiol and the level of anti-GnRH-I antibody in the serum were measured by radioimmunoassay and ELISA, respectively. The results showed that the weight and size of testicles and ovaries, and the concentrations of serum testosterone and estradiol in the immunized animals were lower than those of the control cats (P

CryoEM structure of MxB tubes at 4.6 Å resolution reveals novel interfaces responsible for assembly and anti–HIV-1 activity., Human dynamin–like, interferon-induced myxovirus resistance 2 (Mx2 or MxB) is a potent HIV-1 inhibitor. Antiviral activity requires both the amino-terminal region of MxB and protein oligomerization, each of which has eluded structural determination due to difficulties in protein preparation. We report that maltose binding protein–fused, full-length wild-type MxB purifies as oligomers and further self-assembles into helical arrays in physiological salt. Guanosine triphosphate (GTP), but not guanosine diphosphate, binding results in array disassembly, whereas subsequent GTP hydrolysis allows its reformation. Using cryo-electron microscopy (cryoEM), we determined the MxB assembly structure at 4.6 Å resolution, representing the first near-atomic resolution structure in the mammalian dynamin superfamily. The structure revealed previously described and novel MxB assembly interfaces. Mutational analyses demonstrated a critical role for one of the novel interfaces in HIV-1 restriction.

Hybrid magnetic Fe3O4@SiO2-poly(ethylene oxide)-maltose (Fe3O4@SiO2-PEO-mal) nanoparticles synthesized by our group can be used as affinity adsorption carriers for direct separation of maltose binding protein-fused Hep I (MBP-Hep I) from a crude enzyme solution in a magnetic field. In this work, different PEO molecular weights for Fe3O4@SiO2-PEO-mal nanoparticles were used for characterizing of MBP-Hep I immobilization. The results showed that all four kinds of Fe3O4@SiO2-PEO-mal magnetic nanoparticles (6k, 20k, 35k and 100k for PEO) exhibited excellent adsorption capacities and the adsorption ratio increased as the PEO molecular weight increased from 6k to 100k. All four kinds of immobilized MBP-Hep I exhibited significantly improved stability at 30 °C compared with free MBP-Hep I and their half-lives were 20–50 times that of the free MBP-Hep I. Fe3O4@SiO2-PEO-mal nanoparticles with a PEO molecular weight of 100k were best able to immobilize MBP-Hep I (Fe3O4@SiO2-PEO100k-mal-MBP-Hep I). The molecular weight distribution profiles and anticoagulant activities, obtained from heparin depolymerization by free Hep I, free MBP-Hep I and Fe3O4@SiO2-PEO100k-mal-MBP-Hep I were the same. Furthermore, Fe3O4@SiO2-PEO100k-mal-MBP-Hep I exhibited reasonable reusability during enzymatic production of low molecular weight heparins (LMWHs).

Many proteins rely on disulfide bonds formed between pairs of cysteines for the stability of their folded state and to keep regulatory control over their functions. The hepatitis B virus-encoded HBx oncoprotein is known to perform an overwhelming array of functions in the cell and has been implicated in the development of hepatocellular carcinoma. However, its structure has not been elucidated. HBx carries nine conserved cysteine residues that have proven to be crucial for its various functions. However, the status of disulfide bonds between the cysteine residues reported in previous studies remains discrepant because of the use of refolded recombinant HBx that may contain non-native disulfides. Now we have determined the disulfide linkages in soluble and biologically active recombinant maltose binding protein-HBx fusion protein using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. We report four disulfide linkages in HBx protein, viz., between Cys(7) and Cys(69), Cys(61) and Cys(115), Cys(78) and Cys(137), and Cys(17) and Cys(143), based on the differential mobility of corresponding disulfide-linked peptide ions under reducing and nonreducing conditions. Cys(148) was observed to be free. Site-directed mutagenesis of Cys(143) and Cys(148) with serine and functional analyses of these mutants affirmed the importance of these residues in the ability of HBx to potentiate Cdk2/cyclin E kinase activity and transcriptionally activate promoter reporter gene activity. Thus, this study identifies native disulfide linkages in the structure of a biologically active viral oncoprotein.

ATP-binding cassette (ABC) transporters couple the translocation of solutes across membranes to ATP hydrolysis. Crystal structures of the Escherichia coli maltose importer (MalFGK 2 ) in complex with its substrate binding protein (MalE) provided unprecedented insights in the mechanism of substrate translocation, leaving the MalE–transporter interactions still poorly understood. Using pulsed EPR and cross-linking methods we investigated the effects of maltose and MalE on complex formation and correlated motions of the MalK 2 nucleotide-binding domains (NBDs). We found that both substrate-free (open) and liganded (closed) MalE interact with the transporter with similar affinity in all nucleotide states. In the apo-state, binding of open MalE occurs via the N -lobe, leaving the C-lobe disordered, but upon maltose binding, closed MalE associates tighter to the transporter. In both cases the NBDs remain open. In the presence of ATP, the transporter binds both substrate-free and liganded MalE, both inducing the outward-facing conformation trapped in the crystal with open MalE at the periplasmic side and NBDs tightly closed. In contrast to ATP, ADP–Mg 2+ alone is sufficient to induce a semiopen conformation in the NBDs. In this nucleotide-driven state, the transporter binds both open and closed MalE with slightly different periplasmic configurations. We also found that dissociation of MalE is not a required step for substrate translocation since a supercomplex with MalE cross-linked to MalG retains the ability to hydrolyze ATP and to transport maltose. These features of MalE–MalFGK 2 interactions highlight the conformational plasticity of the maltose importer, providing insights into the ATPase stimulation by unliganded MalE.

A genetically encoded maltose biosensor was constructed, comprising maltose binding protein (MBP) flanked by a green fluorescent protein (GFP(2)) at the N-terminus and a Renilla luciferase variant (RLuc2) at the C-terminus. This Bioluminescence resonance energy transfer(2) (BRET(2)) system showed a 30% increase in the BRET ratio upon maltose binding, compared with a 10% increase with an equivalent fluorescence resonance energy transfer (FRET) biosensor. BRET(2) provides a better matched Forster distance to the known separation of the N and C termini of MBP than FRET. The sensor responded to maltose and maltotriose and the response was completely abolished by introduction of a single point mutation in the BRET(2) tagged MBP protein. The half maximal effective concentration (EC(50)) was 0.37 μM for maltose and the response was linear over almost three log units ranging from 10nM to 3.16 μM maltose for the BRET(2) system compared to an EC(50) of 2.3 μM and a linear response ranging from 0.3 μM to 21.1 μM for the equivalent FRET-based biosensor. The biosensor's estimate of maltose in beer matched that of a commercial enzyme-linked assay but was quicker and more precise, demonstrating its applicability to real-world samples. A similar BRET(2)-based transduction scheme approach would likely be applicable to other binding proteins that have a "venus-fly-trap" mechanism.

A new subfamily of glycosyl hydrolase family GH13 was recently proposed for α-amylases from Anoxybacillus species (ASKA and ADTA), Geobacillus thermoleovorans (GTA, Pizzo and GtamyII), Bacillus aquimaris (BaqA) and 95 other putative protein homologues. To understand this new GH13 subfamily, we report crystal structures of truncated ASKA (TASKA). ASKA is a thermostable enzyme capable of producing high levels of maltose. Unlike GTA, biochemical analysis showed that Ca2+ ion supplementation enhances the catalytic activities of ASKA and TASKA. The crystal structures reveal the presence of four Ca2+ ion binding sites, with three of these binding sites are highly conserved among Anoxybacillus α-amylases. This work provides structural insights into this new GH13 subfamily both in the apo form and in complex with maltose. Furthermore, structural comparison of TASKA and GTA provides an overview of the conformational changes accompanying maltose binding at each subsite.

Nonspecific interactions between biomacromolecules have long been recognized as playing important roles in the thermodynamics and kinetics of biochemical reactions in cellular environments. Our previous study found that nonspecific interactions with bovine serum albumin (BSA) reduced the affinity of maltose binding protein (MBP) for its ligand maltose [1]. To probe the nonspecific interactions further, here we turned to second virial coefficient (B22) measurements. B22 contains information about the nature and strength of intermolecular interactions. Previous measurements have largely focused on self-interactions. We recently developed a method based on static light scattering that probes both self- and mutual interactions in a binary mixturesm[2]. We are applying this method to MBP-BSA mixtures under a wide range of solvent conditions (e.g., pH and salt concentration). We are also using maltose binding and mutations to induce conformational changes and modify the surface of MBP. At a neutral pH, our B22 results for the self- and mutual-interactions of MBP and BSA are all close to those predicted by pure steric repulsion, indicating that the net effects of soft interactions (i.e., electrostatic interaction and nonpolar attraction) for MBP-BMP, BSA-BSA, and MBP-BSA pairs are all nearly null. At pH 5, which is close to the isoelectric points of both proteins and hence charge-charge repulsion should be weakened, our results show moderately attractive soft interactions for the MBP-MBP and BSA-BSA pairs but strongly attractive soft interactions for the MBP-BSA pair. These measurements will provide valuable data for our computational efforts in parameterizing protein-protein interaction energy functions. Together, our experimental and computational studies will lead to quantitative understanding of protein-protein nonspecific interactions and how they influence biochemical reactions in cellular environments.[1] Wu D,et al.,J Phys Chem B, 2015, 119, 1891-8.[2] Miklos AC, et al., PLoS ONE 8(10): e74969.

International audience; Fully biodegradable protein−polymer conjugates, namely, MBP-16 PMeEP (maltose binding protein−poly methyl-ethylene phosphonate), have been 17 investigated in order to understand the role of polymer solvation on protein 18 flexibility. Using elastic and quasi-elastic incoherent neutron scattering, in 19 combination with partially deuterated conjugate systems, we are able to disentangle 20 the polymer dynamics from the protein dynamics and meaningfully address the 21 coupling between both components. We highlight that, in the dry state, the protein− 22 polymer conjugates lack any dynamical transition in accordance with the generally 23 observed behavior for dry proteins. In addition, we observe a larger flexibility of the 24 conjugated protein, compared to the native protein, as well as a lack of polymer− 25 glass transition. Only upon water hydration does the conjugate recover its dynamical 26 transition, leading to the conclusion that exclusive polymer solvation is insufficient to 27 unfreeze fluctuations on the picosecond−nanosecond time scale in biomolecules. 28 Our results also confirm the established coupling between polymer and protein dynamics in the conjugate.

Genetic fusion of two proteins frequently induces beneficial effects to the proteins, such as increased solubility, besides the combination of two protein functions. Here, we study the effects of the bacterial surface layer protein SgsE from Geobacillus stearothermophilus NRS 2004/3a on the folding of a C-terminally fused enhanced green fluorescent protein (EGFP) moiety. Although GFPs are generally unable to adopt a functional confirmation in the bacterial periplasm of Escherichia coli cells, we observed periplasmic fluorescence from a chimera of a 150-amino-acid N-terminal truncation of SgsE and EGFP. Based on this finding, unfolding and refolding kinetics of different S-layer-EGFP chimeras, a maltose binding protein-EGFP chimera, and sole EGFP were monitored using green fluorescence as indicator for the folded protein state. Calculated apparent rate constants for unfolding and refolding indicated different folding pathways for EGFP depending on the fusion partner used, and a clearly stabilizing effect was observed for the SgsE_C fusion moiety. Thermal stability, as determined by differential scanning calorimetry, and unfolding equilibria were found to be independent of the fused partner. We conclude that the stabilizing effect SgsE_C exerts on EGFP is due to a reduction of degrees of freedom for folding of EGFP in the fused state.

The review is on molecular imaging reporter gene expression probes to generate the image contrast. These probes utilize the imaging technology specific to microscopic physical, physiological, metabolic events and changes to differentiate pathology from normal tissue. Reporter gene expression technique generates the specific information at micro- and nanoscopic scale with details of specific molecular or metabolic event. The present paper reviews a database of measurable imaging methods of transgene expression of maltose binding domain with resource of protein molecules in bioimaging. Molecular bioimaging is based on imaging the transgene expression products and offers multimodal real-time genomic kinetics. With this aim, gene expression probes combined with adjunct molecular imaging techniques are identified and their applications are defined for: 1. noninvasive in vivo imaging methods for specific molecular expression and interactions of protein-protein domains, 2. to visualize time-based molecular events in the tissues, 3. to visualize and follow-up trafficking and targeting of cells, 4. to optimize conditions of gene therapy and monitoring drug therapeutics, 5. to develop rapid drug chemosensitivity assay to image the drug effect at cellular and molecular level by multimodal radioimaging, optical and bioluminescence imaging. The feasibility of multimodal molecular imaging techniques on one platform is reported to visualize the cells in animal body after administration of binding domain ligand into animals through oral, intraperitoneal, intravenous, and intrathecal routes. The binding domain ligand acts as a secretory signal or plasma membrane trafficking signal domain. It can be visualized by positron emission tomography imaging. Today, the main challenge in developing multimodal multifunctional double labeled molecular imaging probes is to achieve goal of rapid, reproducible multimodal imaging (optical, physical, biosensor) or radiolabel mapping (PET, CT) in noninvasive and quantitative manner that can give information in time-dependent experimental, developmental, environmental and therapeutic influence on gene products and proteins in animals or patients. This patent review will be useful resource database of molecules as 'bioinformatics of potential reporters' and multimodal imaging methods on same platform resource in selecting molecules to develop imaging techniques of cells or molecular events during influence of drugs, metabolism, physiology, experimental, environmental and genetics in the body.

Nowadays, identification of the novel physio-biological and therapeutic functions of plant cysteine proteinase inhibitors “plant cystatins / phytocystatins” are the great of interests for molecular biologists. Whether for biochemical, structural or functional studies, their successful expression along with an easy purification method is required. To date, fusion tags are the best available tools that meet all those requirements. We report here the cloning and simple functional expression and purification of a barley putative cystatin in Escherichia coli cells. For the first time, a part of barley coding sequence containing a predicted active cystatin was amplified by polymerase chain reaction and expressed as maltose binding fusion protein in TB1 strain of E. coli cells using pMALc2X over-expression vector system without affecting the bacterial growth. The expressed product was purified by single step affinity chromatography from the soluble fraction of induced culture at a yield of about 37 mg/ liter of bacterial cell culture. The purified fused protein could efficiently inhibit papain activity in vitro without the cleavage of the fusion partner.

We describe a phage display methodology for engineering synthetic antigen binders (sABs) that recognize either the apo or the ligand-bound conformation of maltose-binding protein (MBP). sABs that preferentially recognize the maltose-bound form of MBP act as positive allosteric effectors by substantially increasing the affinity for maltose. A crystal structure of a sAB bound to the closed form of MBP reveals the basis for this allosteric effect. We show that sABs that recognize the bound form of MBP can rescue the function of a binding-deficient mutant by restoring its natural affinity for maltose. Furthermore, the sABs can enhance maltose binding in vivo, as they provide a growth advantage to bacteria under low-maltose conditions. The results demonstrate that structure-specific sABs can be engineered to dynamically control ligand-binding affinities by modulating the transition between different conformations.

Substrate-binding proteins (SBP) are associated with a wide variety of protein complexes. The proteins are part of ATP-binding cassette transporters for substrate uptake, ion gradient driven transporters, DNA-binding proteins, as well as channels and receptors from both pro-and eukaryotes. A wealth of structural and functional data is available on SBPs, with over 120 unique entries in the Protein Data Bank (PDB). Over a decade ago these proteins were divided into three structural classes, but based on the currently available wealth of structural data, we propose a new classification into six clusters, based on features of their three-dimensional structure. (C) 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Immunization using recombinant maltose binding protein-gonadotropin releasing hormone (MBP-GnRH6) altered both testicular development and transcription of the pituitary GnRH receptor (GnRHR) gene in boars. Scrotal measurement and blood samples were taken at 4-week interval after immunization at 9 weeks of age. The concentrations of testosterone and anti-GnRH antibodies in serum were determined by radioimmunoassay and enzyme-linked immunosorbent assay, respectively. The results showed that active immunization with MBP-GnRH6 increased the serum concentration of anti-GnRH antibodies (P0.05) and reduced the serum concentration of testosterone (P0.05) as compared with MBP controls. At 25 weeks of age, boars were sacrificed and testes were evaluated histologically. Testicular development was suppressed in the MBP-GnRH6 immunized animals as compared with MBP immunized boars. MBP-GnRH6 immunized pigs exhibited mounting behavior 4 weeks later than MBP immunized boars. No mature spermatozoa were observed from MBP-GnRH6 immunized animals. By real-time quantitative PCR analysis, the amount of GnRHR mRNA in the pituitary tissue was found to be significantly lower in MBP-GnRH6 immunized animals than in controls (P0.05). These data demonstrate that recombinant MBP-GnRH6 was effective in immunological castration in boars.

Among the numerous bacterial Type II restriction enzymes, EcoRI endonuclease is the most extensively studied and is widely used in recombinant DNA technology. Its heterologous overexpression as recombinant protein has already been studied. However, very limited information concerning its fused product is available thus far. In the present study, the EcoRI restriction endonuclease gene was cloned and expressed as a part of maltose-binding fusion protein under the control of strong inducible tac promoter in TB1 strain of Escherichia coli cells. Transformed cells containing pMALc2X-EcoRI recombinant plasmid were unable to grow under experimental conditions. However, fused EcoRI protein was purified (with the yield of 0.01 mg/l of bacterial culture) by affinity chromatography from E. coli cells induced at the late exponential phase of growth. Restriction quality test revealed that the purified product could restrict a control plasmid DNA in vitro.

The principal objective of this study was to explore protein conformational changes using fluorescence resonance energy transfer (FRET) technology. Maltose binding protein (MBP) was adopted as a target model, due to its well-characterized structure and ligand specificity. To the best of our knowledge, this is the first report to provide information regarding the biological distance between the two lobes of MBP upon maltose binding. For the FRET pair, ECFP and EYFP were used as the donor and the acceptor, and were linked genetically to the C-terminal and N-terminal regions of MBP (ECFP:MBP:EYFP), respectively. After the FRET reaction, maltose-treated MBP was shown to exhibit a considerable energy transfer (FRET efficiency (E)= approximately 0.11, Distance (D)= approximately 6.93 nm) at the ensemble level, which was regarded as reflective of the increase in donor quenching and the upshift in acceptor emission intensity, thereby suggesting that the donor and the acceptor had been brought close together as the result of structural alterations in MBP. However, upon glucose treatment, no FRET phenomenon was detected, thereby implying the specificity of interaction between MBP and maltose. The in vitro FRET results were also confirmed via the acceptor photobleaching method. Therefore, our data showed that maltose-stimulated conformational changes of MBP could be measured by FRET, thereby providing biological information, including the FRET efficiency and the intramolecular distance.

Methylglyoxal is mainly catabolized by two major enzymatic pathways. The first is the ubiquitous detoxification pathway, the glyoxalase pathway. In addition to the glyoxalase pathway, aldose reductase pathway also plays a crucial role in lowering the levels of methylglyoxal. The gene encoding aldose reductase (ALR) has been cloned from Leishmania donovani, a protozoan parasite causing visceral leishmaniasis. DNA sequence analysis revealed an open reading frame (ORF) of approximately 855 bp encoding a putative protein of 284 amino acids with a calculated molecular mass of 31.7 kDa and a predicted isoelectric point of 5.85. The sequence identity between L. donovani ALR (LdALR) and mammals and plants is only 36-44%. The ORF is a single copy gene. A protein with a molecular mass that matched the estimated approximately 74 kDa according to the amino acid composition of LdALR with a maltose binding tag present at its N-terminal end was induced by heterologous expression of LdALR in Escherichia coli. In the presence of glutathione, recombinant LdALR reduced methylglyoxal with a K(m) of approximately 112 microM. Comparative structural analysis of the human ALR structure with LdALR model suggests that the active site anchoring the N-terminal end of the glutathione is highly conserved. However, the C-terminal end of the glutathione backbone is expected to be exposed in LdALR, as the residues anchoring the C-terminal end of the glutathione backbone come from the three loop regions in human, which are apparently shortened in the LdALR structure. Thus, the computational analysis provides clues about the expected mode of glutathione binding and its interactions with the protein. This is the first report of the role of an ALR in the metabolic disposal of methylglyoxal in L. donovani and of thiol binding to a kinetoplastid aldose reductase.

Pollination involves a series of complex cellular interactions and signal transduction events. Numerous reports have suggested a central role for protein kinases in pollen germination and pollen tube growth and a large number of receptor-like kinases have been detected exclusively in pollen in higher plants. However, few are well characterized, especially for the receptor-like cytoplasmic kinases. Here we report a receptor-like kinase gene, OsRLCK1, which belongs to the receptor-like cytoplasmic kinase VIII subfamily. Real-time quantitative polymerase chain reaction analysis and whole mount RNA in situ hybridization showed that OsRLCK1 is a pollen-specific gene and expressed only in the mature pollen. When expressed in the onion epidermal cells, the OsRLCK1-GFP fusion protein was diffused throughout the cell, indicating its cytoplasmic and nuclear localization. The Maltose Binding Protein-OsRLCK1 recombinant protein was found to be capable of autophosphorylation on threonine residue, showing that it encodes a functional kinase. These results suggest that OsRLCK1 is likely to play a role in a signaling pathway associated with pollen performance during pollination in rice.

Two DNA fragments encoding PDZ domain(21–110 residues) and BAR domain(150–360 residues) from PICK1 (1–416 residues) were amplified by PCR and then introduced into vectors, pET-32M and pMAL-c2X respectively to generate recombinant plasmids, pE-pdz and pM-bar. Having been separately transferred into the hosts E. coli BL21 and E. coli JM109, these two strains can express fusion proteins: His-tagged PDZ(PDZ domain) and maltose binding protein-BAR(MBP-BAR domain) respectively, as confirmed by both SDS-PAGE and Western blotting. The interaction between these two domains is dose-dependence, as identified by a pull-down test. Moreover, it has been shown from the ELISA analysis that the actual amount of PDZ bound to MBP-BAR-amylose beads reaches (16 ± 0.5)%, as calculated by the molar ratio of PDZ to MBP-BAR. In addition, the interaction between BAR(bait) and PDZ(prey) in vivo was also examined with a yeast two-hybrid system.

Summary Bordetella pertussis is the causative agent of whooping cough, a major childhood pathogen; acellular vaccines consisting of purified B. pertussis antigens such as filamentous haemagglutinin (FHA) are commonly used to prevent pertussis. Despite the importance of FHA in B. pertussis pathogenesis and its inclusion in most acellular vaccines, the functional importance of individual domains in the induction of protective immunity is largely unknown. In this study, we have purified a recombinant FHA protein from Escherichia coli consisting of a 42 kDa maltose binding domain of E. coli and the 43 kDa type I immunodominant domain of FHA. The fusion protein (Mal85) was purified from E. coli cell lysates via affinity chromatography with an amylose column. Mal85 was then delivered to BALB/c mice intranasally encapsulated in liposomes, formulated with ProtollinTM or in conjuction with an immunostimulatory CpG oligonucleotide. Mice were also vaccinated intraperitoneally with alum-adsorbed Mal85. Sera from all treatment groups showed strong IgG responses to Mal85 and recognized native FHA. Specific salivary IgA was induced in mice vaccinated with Mal85 in liposomes, ProtollinTM and delivered with CpG. Vaccination with Mal85 encapsulated in liposomes or formulated with ProtollinTM provided protection against aerosol challenge with B. pertussis in BALB/c mice. These data indicate that the type I domain of FHA is a protective antigen in mice and may serve as a candidate for inclusion in new acellular pertussis vaccines.

Two environmentally sensitive, long-wavelength fluorescent phenoxazine derivatives, INR and IANR, were synthesized with linkers for conjugation to the thiol group of cysteine in binding proteins. The linkers were designed based on the attachment sites at two different positions on the phenoxazine, which were chosen in order to study the orientation of the dye with respect to the binding protein. Conjugation of the dyes to the S337C maltose binding protein (MBP) mutant provided conjugates of these dyes that are capable of detecting maltose with different sensitivities. The dye INR gave a 3-fold (+200%) change in fluorescence intensity upon maltose binding when conjugated to S337C MBP with a binding constant (K(d)) of 435 microM. The fluorescence change for IANR was only 20% and the K(d) was 1.4 mM. Conformational analysis of the dyes by molecular modeling suggested that the linker in IANR imparted greater conformational freedom to the dye, resulting in little change in environment between the open and the closed-form conformations. The linker in INR, on the other hand, showed restricted motion, which placed the dye in different environments in the open and closed forms of the protein. Thus, design and placement of the linker play a critical role in the performance of these dyes as environmentally sensitive probes.

The aim of this study was to investigate the efficacy of active immunisation against recombinant maltose binding protein-gonadotropin releasing hormone I (MBP-GnRH-I6) in preventing boar taint. The concentrations of testosterone and anti-GnRH-I antibodies in serum were determined by radioimmunoassay and enzyme linked immunosorbent assay, respectively. The mRNA levels of 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase (17β-HSD) in testes were analysed by real time quantitative PCR. The concentrations of androstenone and 3-methylindole in backfat were assayed by HPLC. Active immunisation against MBP-GnRH-I6 increased the serum concentration of anti-GnRH-I antibodies (Pβ-HSD and 17β-HSD mRNA in testis. However, it reduced the serum concentrations of testosterone (P>0.05) and androstenone. The concentration of 3-methylindole in backfat (P>0.05) was not changed. Thus, we conclude that vaccination against MBP-GnRH-I6 is a practical and effective method to suppress the synthesis of androstenone.

Analysis of the genome sequence of Caulobacter crescentus predicts 67 TonB-dependent outer membrane proteins. To demonstrate that among them are proteins that transport nutrients other than chelated Fe 3+ and vitamin B 12 —the substrates hitherto known to be transported by TonB-dependent transporters—the outer membrane protein profile of cells grown on different substrates was determined by two-dimensional electrophoresis. Maltose induced the synthesis of a hitherto unknown 99.5-kDa protein, designated here as MalA, encoded by the cc2287 genomic locus. MalA mediated growth on maltodextrins and transported [ 14 C]maltodextrins from [ 14 C]maltose to [ 14 C]maltopentaose. [ 14 C]maltose transport showed biphasic kinetics, with a fast initial rate and a slower second rate. The initial transport had a K d of 0.2 μM, while the second transport had a K d of 5 μM. It is proposed that the fast rate reflects binding to MalA and the second rate reflects transport into the cells. Energy depletion of cells by 100 μM carbonyl cyanide 3-chlorophenylhydrazone abolished maltose binding and transport. Deletion of the malA gene diminished maltose transport to 1% of the wild-type malA strain and impaired transport of the larger maltodextrins. The malA mutant was unable to grow on maltodextrins larger than maltotetraose. Deletion of two C. crescentus genes homologous to the exbB exbD genes of Escherichia coli abolished [ 14 C]maltodextrin binding and transport and growth on maltodextrins larger than maltotetraose. These mutants also showed impaired growth on Fe 3+ -rhodotorulate as the sole iron source, which provided evidence of energy-coupled transport. Unexpectedly, a deletion mutant of a tonB homolog transported maltose at the wild-type rate and grew on all maltodextrins tested. Since Fe 3+ -rhodotorulate served as an iron source for the tonB mutant, an additional gene encoding a protein with a TonB function is postulated. Permeation of maltose and maltotriose through the outer membrane of the C. crescentus malA mutant was slower than permeation through the outer membrane of an E. coli lamB mutant, which suggests a low porin activity in C. crescentus . The pores of the C. crescentus porins are slightly larger than those of E. coli K-12, since maltotetraose supported growth of the C. crescentus malA mutant but failed to support growth of the E. coli lamB mutant. The data are consistent with the proposal that binding of maltodextrins to MalA requires energy and MalA actively transports maltodextrins with K d values 1,000-fold smaller than those for the LamB porin and 100-fold larger than those for the vitamin B 12 and ferric siderophore outer membrane transporters. MalA is the first example of an outer membrane protein for which an ExbB/ExbD-dependent transport of a nutrient other than iron and vitamin B 12 has been demonstrated.

Mga is a transcriptional regulator in the pathogenStreptococcus pyogenesthat positively activates several important virulence genes involved in colonization and immune evasion in the human host. A naturally occurring mutant of Mga that is defective in its ability to activate transcription has been identified in the serotype M50 strain B514-Sm. Sequence alignment of the defective M50 Mga with the fully functional Mga from serotypes M4 and M49 revealed only three amino acid changes that might result in a defective protein. Electrophoretic mobility shift assays using purified M50 and M4 maltose binding protein-Mga found that both exhibited DNA-binding activity towards regulated promoters. Thus, the significance of each residue for the functionality of M50 Mga was explored through introduction of “gain-of-function” mutations based on M4 Mga. Transcriptional studies of the mutant alleles under both constitutive (PrpsL) and autoactivated (Pmga4) promoters illustrated that an arginine-to-methionine change at position 461 of M50 Mga protein fully restored activation of downstream genes. Western blot analyses of steady-state Mga levels suggest that the M461 residue may play a role in overall conformation and protein stability of Mga. However, despite the conservation of the M461 protein among all other Mga proteins, it does not appear to be necessary for activity in a divergent M6 Mga. These studies highlight the potential differences that exist between divergent Mga proteins in this important human pathogen.

Within the framework of reported information on the Sec pathway mechanism, a mathematical model for the periplasmic translocation of fusion proteins in bacteria was developed. The mathematical model includes all stages of the targeting stage and assume that the ATP-driven translocation stage is completed in a single step. The equations for the targeting stage involved cytoplasmic folding rate and SecB binding kinetics. Rate equations for the translocation stage were derived using King-Altman and network reduction techniques. Experimental data for maltose binding protein–glucose isomerase fusion protein (MBP-GI) translocation and reported data for MBP translocation were used to estimate the parameters. The simulation results show that the model =ts well to the experimental data of cytoplasmic and periplasmic MBP-GI distributions. When the values of the targeting stage parameters, k1 and k3 or the concentration of SecB are changed, the model correctly predicts the expected changes in the MBP-GI distribution to the cytoplasmic and periplasmic spaces. The SecB complex and the preprotein concentrations predicted attain steady state immediately, within seconds, and their amounts are very low when compared to MBP-GI in either compartment. The model can be made applicable to any protein that uses the Sec pathway, and with ATP and Sec pathway protein limitations. ? 2003 Elsevier Ltd. All rights reserved.

A preliminary structural analysis of Vibrio cholerae zonula occludens toxin (ZOT) was made by equilibrium denaturation and circular dichroism. ZOT is a structurally unstable protein in aqueous solution (ΔG(H2O) 3.82 kcal/mol), the putative intra- and extracellular domains unfold co-operatively, with complete denaturation via observed conformational intermediates. Refolding of denatured ZOT is not dependent on disulphide bridge formation. Partial refolding of a maltose binding protein–ZOT fusion did not prevent its specific binding to the ZOT receptor on Caco-2 cells. Immuno-gold labelling showed that the ZOT receptor localises to the intercellular contacts between cells in a confluent monolayer.

The Streptomyces coelicolor bldG gene encodes a protein showing similarity to the SpoIIAA and RsbV anti-anti-sigma factors of Bacillus subtilis. Purified maltose binding protein-BldG could be phosphorylated in vitro by wild-type S. coelicolor crude extract, and both the phosphorylated and unphosphorylated forms of BldG could be detected in vivo using isoelectric focusing. ATP was shown to serve as the phosphoryl group donor, and phosphorylation of BldG was abolished when the putative phosphorylation site was changed from a serine to an alanine residue. A bldG mutant strain expressing the non-phosphorylatable BldG protein was unable to undergo morphological differentiation or produce antibiotics even after prolonged incubation, suggesting that phosphorylation of BldG is necessary for proper development in S. coelicolor.

LamB (maltoporin) is essential for the uptake of maltose and malto-oligosaccharides across the outer membrane of Escherichia coli. Purified LamB was reconstituted in artificial lipid bilayer membranes forming channels in the permanently open configuration at neutral pH. Almost complete channel closure was observed when the pH on both sides of the membrane was lowered to pH 4. When LamB was added to only one side of the membrane, the cis-side, and the pH was lowered at either side of the membrane, the cis- or the trans-side, the response to pH was asymmetric, suggesting preferential orientation of maltoporin channels and pH- dependent closure of only one side of the channel. In experiments with LamB mutants in which major external loops L4, L6, and/or L9 were deleted, we identified the surface-exposed loops L4 and L6 as the cause of pH-mediated closure. The pH dependence of the LamB channel is consistent with the assumption that it inserts in a preferential orientation into the lipid bilayer. About 70-80% of the reconstituted channels are oriented with the extracellular entrance toward the side to which the protein was added (the cis-side) and with the periplasmic opening on the opposite side (the trans-side). The possibility of closing the channels, which are oriented in the reverse direction by low pH at the trans-side, allowed the deduction of channel asymmetry with respect to carbohydrate binding kinetics. Whereas maltose binding was found to be almost symmetric with respect to the channel orientation, the sucrose and trehalose binding to LamB was asymmetric. The results are discussed in respect to possible physiological function of the pH-dependent closure of maltoporin.

The affinity of the maltose binding protein−nitro reductase fusion (MBP−NR) to electropolymerized films of N-(3-pyrrol-1-ylpropyl)-4,4‘-bipyridinium (PPB) has been studied in aqueous medium by the quartz crystal microbalance (QCM) technique. It was found that the MBP domain of MBP−NR exhibits specific binding toward PPB films through the maltose binding site and that the immobilized MBP−NR retains its enzymatic activity toward trinitrotoluene (TNT) reduction. Although some MBP−NR was nonspecifically adsorbed onto a maltose-covered PPB film as well as to a bare Au electrode, in both cases the adsorbed enzyme exhibited no catalytic activity. Nitro reductase (NR) also appeared to adsorb onto the PPB films but did not exhibit any enzymatic activity. The electropolymerization of N-(3-pyrrol-1-ylpropyl)-4,4‘-bipyridinium (PPB) onto a Au electrode in acetonitrile solution was studied by electrochemical quartz crystal microbalance (EQCM). It was found that the extent of polymerization upon pyrrole-centered oxidat...

Maltosyltransferase (MTase) from the hyperthermophile Thermotoga maritima represents a novel maltodextrin glycosyltransferase acting on starch and malto-oligosaccharides. It catalyzes the transfer of maltosyl units from α-1,4-linked glucans or malto-oligosaccharides to other α-1,4-linked glucans, malto-oligosaccharides or glucose. It belongs to the glycoside hydrolase family 13, which represents a large group of (β/α) 8 barrel proteins sharing a similar active site structure. The crystal structures of MTase and its complex with maltose have been determined at 2.4 A and 2.1 A resolution, respectively. MTase is a homodimer, each subunit of which consists of four domains, two of which are structurally homologous to those of other family 13 enzymes. The catalytic core domain has the (β/α) 8 barrel fold with the active-site cleft formed at the C-terminal end of the barrel. Substrate binding experiments have led to the location of two distinct maltose-binding sites; one lies in the active-site cleft, covering subsites −2 and −1; the other is located in a pocket adjacent to the active-site cleft. The structure of MTase, together with the conservation of active-site residues among family 13 glycoside hydrolases, are consistent with a common double-displacement catalytic mechanism for this enzyme. Analysis of maltose binding in the active site reveals that the transfer of dextrinyl residues longer than a maltosyl unit is prevented by termination of the active-site cleft after the −2 subsite by the side-chain of Lys151 and the stretch of residues 314–317, providing an explanation for the strict transfer specificity of MTase.