title: Confirmation of a Protein-Protein Interaction in the Pantothenate Biosynthetic Pathway by Using Sortase-Mediated Labelling.
abstract: High-throughput studies have been widely used to identify protein-protein interactions; however, few of these candidate interactions have been confirmed in vitro. We have used a combination of isothermal titration calorimetry and fluorescence anisotropy to screen candidate interactions within the pantothenate biosynthetic pathway. In particular, we observed no interaction between the next enzyme in the pathway, pantothenate synthetase (PS), and aspartate decarboxylase, but did observe an interaction between PS and the putative Nudix hydrolase, YfcD. Confirmation of the interaction by fluorescence anisotropy was dependent upon labelling an adventitiously formed glycine on the protein N-terminal affinity purification tag by using Sortase. Subsequent formation of the protein-protein complex led to apparent restriction of the dynamics of this tag, thus suggesting that this approach could be generally applied to a subset of other protein-protein interaction complexes.
authors.affiliation: School of Chemistry and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK.
authors.forename: Philip M
authors.initials: PM
authors.lastname: Morrison
authors.affiliation: School of Chemistry and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK.
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Balmforth
authors.affiliation: School of Chemistry and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK.|Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK.
authors.forename: Samuel W
authors.initials: SW
authors.lastname: Ness
authors.affiliation: School of Chemistry and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Williamson
authors.affiliation: School of Chemistry and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK.|John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, UK.
authors.forename: Michael D
authors.identifier: http://orcid.org/0000-0003-3920-7079
authors.initials: MD
authors.lastname: Rugen
authors.affiliation: School of Chemistry and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK.
authors.forename: W Bruce
authors.identifier: http://orcid.org/0000-0002-7352-0360
authors.initials: WB
authors.lastname: Turnbull
authors.affiliation: School of Chemistry and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK. m.e.webb@leeds.ac.uk.
authors.forename: Michael E
authors.identifier: http://orcid.org/0000-0003-3574-4686
authors.initials: ME
authors.lastname: Webb
mesh_terms: D019881:Aminoacyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D053898:Biosynthetic Pathways; D003546:Cysteine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D005454:Fluorescence Polarization; D015394:Molecular Structure; D010205:Pantothenic Acid / Q000096:biosynthesis* / Q000737:chemistry; D010453:Peptide Synthases / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation
chemical_list: D001426:Bacterial Proteins; D010205:Pantothenic Acid; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases; D010453:Peptide Synthases; C022318:pantothenate synthetase
keywords: fluorescence anisotropy; pantothenate biosynthesis; sortase labeling
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