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

DOI: 10.3791/56294 DOI Help

Authors: Simone Culurgioni (Diamond Light Source; Research Complex at Harwell) , Minzhe Tang (Diamond Light Source; Research Complex at Harwell) , David R. Hall (Diamond Light Source) , Martin Walsh (Diamond Light Source)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Journal Of Visualized Experiments

State: Published (Approved)
Published: October 2017

Open Access Open Access

Abstract: 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.

Subject Areas: Biology and Bio-materials, Chemistry


Instruments: I03-Macromolecular Crystallography , I04-Macromolecular Crystallography

Added On: 16/10/2017 14:52

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jove-protocol-56294-biochemical-structural-characterization-carbohydrate-transport.pdf

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