Barley alpha-amylase/subtilisin inhibitor: structure, biophysics and protein engineering

P.K. Nielsen, Birgit Christine Bønsager, Kenji Fukuda, Birte Svensson

    Research output: Contribution to journalJournal articleResearchpeer-review

    Abstract

    Bifunctional alpha-amylase/subtilisin inhibitors have been implicated in plant defence and regulation of endogenous alpha-amylase action. The barley alpha-amylase/subtilisin inhibitor (BASI) inhibits the barley alpha-amylase 2 (AMY2) and subtilisin-type serine proteases. BASI belongs to the Kunitz-type trypsin inhibitor family of the beta-trefoil fold proteins. Diverse approaches including site-directed mutagenesis, hybrid constructions, and crystallography have been used to characterise the structures and contact residues in the AMY2/BASI complex. The three-dimensional structure of the AMY2/BASI complex is characterised by a completely hydrated Ca2+ situated at the protein interface that connects the three catalytic carboxyl groups in AMY2 with side chains in BASI via water molecules. Using surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC), we have recently demonstrated Ca2+-modulated kinetics of the AMY2/BASl interaction and found that the complex formation involves minimal structural changes. The modulation of the interaction by calcium ions makes it unique among the currently known binding mechanisms of proteinaceous alpha-amylase inhibitors.
    Original languageEnglish
    JournalBBA General Subjects
    Volume1696
    Pages (from-to)157-164
    ISSN0304-4165
    Publication statusPublished - 2004

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