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The Rapid Screening and Optimisation of Crystallisation Conditions of the Bacterial Periplasmic Binding Protein, HbpA and the Elucidation of its Role as a Glutathione Import Protein

Published: Monday, September 12, 2011
Last Updated: Monday, September 12, 2011
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A recent study reported in PNAS employed TTP LabTech’s mosquito® crystal for the identification of a bacterial periplasmic lipoprotein in H. influenzae Rd.

The bacterial periplasmic protein was previously thought to be a heme-binding protein A (HbpA) that specifically binds both reduced glutathione (GSH) and oxidised glutathione (GSSG) with physiologically relevant affinity.

Vergauwen and colleagues1 employed a combination of structural, biochemical and cellular studies in establishing the physiological role of this protein, as a periplasmic binding protein that primes glutathione import via its cognate ABC-like dipeptide transporter DppBCDF.

In this study, purified recombinant GbpA from H. Parasuis, was used to carry out an extensive crystallisation screen using TTP LabTech’s mosquito® Crystal.  This approach allowed the rapid and efficient screening of nearly 2000 unique crystallisation conditions with a limited amount of protein sample (~1 mg purified GbpA).

The application of state-of-the-art crystallisation robotic platforms has emerged as a powerful technical advancement in structural biology in the post-genomic era.   Savvas Savvides, Professor of Structural Biology at Ghent University (Belgium) and senior author of the study,commented that  “mosquito® Crystal proved to be a tremendously efficient tool in setting up close to 2000 crystallisation trials using a limited amount of sample. The speed, less than 2 minutes per plate and reproducibility in pipetting 50 nL droplets, without sample cross-contamination offered a clear experimental advantage”.  

Savvas highlighted that “the defining experimental undertaking that helped us to dispel any doubts about the proposed function of GbpA resulted from crystallographic studies of GbpA in complex with glutathione”. This study provided the first snapshot of a physiologically relevant complex of a GbpA-family protein with its ligand cargo, providing the missing link for understanding at the atomic level how GbpA-family proteins could serve as glutathione-binding platforms for priming their cognate dipeptide permeases.  Accordingly, HbpA has now been renamed as Glutathione-binding protein A (GbpA) to reflect its biological function accurately and to facilitate the correct annotation of GbpA-like proteins in bacterial genomes .

1. Vergauwen, B., Elegheert, J., Dansercoer, A., Devreese, B. & Savvides, S.N. (2010) Glutathione import in Haemophilus influenzae Rd is primed by the periplasmic heme-binding protein HbpA. PNAS 107: pp13270-5.



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