Cohesin-dockerin Interactions within and between Clostridium josui and Clostridium thermocellum
ABSTRACT The cellulosome components are assembled into the cellulosome complex by the interaction between one of the repeated cohesin domains of a scaffolding protein and the dockerin domain of an enzyme component. We prepared five recombinant cohesin polypeptides of the Clostridium thermocellum scaffolding protein CipA, two dockerin polypeptides of C. thermocellum Xyn11A and Xyn10C, four cohesin polypeptides of Clostridium josui CipA, and two dockerin polypeptides of C. josui Aga27A and Cel8A, and qualitatively and quantitatively examined the cohesin-dockerin interactions within C. thermocellum and C. josui, respectively, and the species specificity of the cohesin-dockerin interactions between these two bacteria. Surface plasmon resonance (SPR) analysis indicated that there was a certain selectivity, with a maximal 34-fold difference in the K(D) values, in the cohesin-dockerin interactions within a combination of C. josui, although this was not detected by qualitative analysis. Affinity blotting analysis suggested that there was at least one exception to the species specificity in the cohesin-dockerin interactions, although species specificity was generally conserved among the cohesin and dockerin polypeptides from C. thermocellum and C. josui, i.e. the dockerin polypeptides of C. thermocellum Xyn11A exceptionally bound to the cohesin polypeptides from C. josui CipA. SPR analysis confirmed this exceptional binding. We discuss the relationship between the species specificity of the cohesin-dockerin binding and the conserved amino acid residues in the dockerin domains.
SourceAvailable from: Milica Ciric01/2014
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ABSTRACT: Dynamic fluoroimmunoassay with a flow-through system using optical fiber probes consisting of polystyrene was developed and applied to a quantitative detection of E. coli O157:H7. The system measures E. coli as fluorescence of sandwich-type immune complexes formed by capture antibodies immobilized on the surface of the probe, E. coli cells, and fluorescently labeled detection antibodies. Excitation was carried out using an evanescent wave from the probe. Resulting fluorescence recoupled into the probe was detected by a photodiode. The assay system was constructed with a flow cell which was available for sequential injection of experimental reagents. In vitro characterization was performed using the flow cell, and the calibration range of E. coli O157:H7 was from 10(3) to 10(7) cells/mL. The measurement for each sample was completed within 12 min. Furthermore, it was also possible to estimate the concentrations of E. coli O157:H7 by the increasing rate of fluorescence during binding reaction of detection antibodies to antigens. This minimized the time for measurement down to 6 min. The system is suitable for rapid and direct determination for microorganisms or bacteria in food, clinical, and environmental sources.03/2013; 3(1):120-31. DOI:10.3390/bios3010120
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ABSTRACT: Clostridium cellulovorans, produce multi-enzymatic complexes known as cellulosomes, which assemble via the interaction of a dockerin module in the cellulosomal subunit with one of several cohesin modules in the scaffolding protein, to degrade the plant cell wall polymer. An enhanced cohesin-dockerin interaction was demonstrated by modified certain cellulosomal enzymes with altered amino acid residues at the crucial binding site, 11(th) and 12(th) positions in dockerin module. In fluorescence intensity analyses using the cellulosome-based biomarker system, the modified cellulosomal enzymes (EngE SL to AI and EngH SM to AI) showed an increased intensity (1.4- to 2.2-fold) compared with the wild-type proteins. Conversely, modified ExgS (AI to SM) exhibited a reduced intensity (0.6- to 0.7-fold) compared with the wild type. In enzyme-linked and competitive enzyme-linked interaction assays, the some modified protein (EngE SL to AI and EngH SM to AI) showed their increased binding affinity toward the cohesins (Coh2 and Coh9). Surface plasmon resonance analysis quantitatively demonstrated the binding affinity of these two modified proteins toward cohesins showed similar or higher affinity comparing with its with wild type proteins. These results suggest the replacement of amino acid residues in the certain recognition site significantly affects the binding affinity of the cohesin-dockerin interaction. Copyright © 2015. Published by Elsevier B.V.International Journal of Biological Macromolecules 01/2015; 75. DOI:10.1016/j.ijbiomac.2015.01.018 · 3.10 Impact Factor