Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant strains. Carbohydrate substrate binding proteins (SBPs) represent viable targets for the development of protein-based vaccines and new antimicrobials...

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Bibliographic Details
Published in:Journal of visualized experiments 2017-10 (128)
Main Authors: Culurgioni, Simone, Tang, Minzhe, Hall, David R, Walsh, Martin A
Format: Article
Language:English
Subjects:
Bacterial Proteins - chemistry
Bacterial Proteins - metabolism
Biochemistry
Crystallography, X-Ray - methods
Molecular Structure
Protein Conformation
Receptors, Cell Surface - chemistry
Receptors, Cell Surface - metabolism
Streptococcus pneumoniae - chemistry
Streptococcus pneumoniae - metabolism
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Summary:Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant strains. Carbohydrate substrate binding proteins (SBPs) represent viable targets for the development of protein-based vaccines and new antimicrobials because of their extracellular localization and the centrality of carbohydrate import for pneumococcal metabolism, respectively. Described here is a rationalized integrated protocol to carry out a comprehensive characterization of SP0092, which can be extended to other carbohydrate SBPs from the pneumococcus and other bacteria. This procedure can aid the structure-based design of inhibitors for this class of proteins. Presented in the first part of this manuscript are protocols for biochemical analysis by thermal shift assay, multi angle light scattering (MALS), and size exclusion chromatography (SEC), which optimize the stability and homogeneity of the sample directed to crystallization trials and so enhance the probability of success. The second part of this procedure describes the characterization of the SBP crystals using a tunable wavelength anomalous diffraction synchrotron beamline, and data collection protocols for measuring data that can be used to resolve the crystallized protein structure.
ISSN:1940-087X
1940-087X
DOI:10.3791/56294