TAILIEUCHUNG - Báo cáo khoa học: Structural features and dynamics of a cold-adapted alkaline phosphatase studied by EPR spectroscopy

EPR spectroscopy, performed after site-directed spin-labeling, was used to study structural dynamics in a cold-adapted alkaline phosphatase (EC ). Differences in the structural environment of six spin-labeled side chains allowed them to be classified (with reference to previously obtained mobility maps) as belonging to loop positions (either relatively surface exposed or in structural contact) or helix positions (surface exposed, in contact, or buried). | Structural features and dynamics of a cold-adapted alkaline phosphatase studied by EPR spectroscopy Pétur O. Heidarsson Snorri Th. Sigurdsson and Bjarni Asgeirsson Department of Biochemistry Science Institute University of Iceland Reykjavik Iceland Keywords alkaline phosphatase catalytic mechanism electron paramagnetic resonance protein dynamics site-directed spin-labeling Correspondence B. Asgeirsson Department of Biochemistry Science Institute University of Iceland Dunhaga 3 IS-107 Reykjavik Iceland Fax 354 552 8911 Tel 354 525 4800 E-mail bjarni@ Present address StructuralBiology and NMR Laboratory SBiN Lab University of Copenhagen Denmark Received 20 December 2008 revised 6 February 2009 accepted 9 March 2009 doi EPR spectroscopy performed after site-directed spin-labeling was used to study structural dynamics in a cold-adapted alkaline phosphatase EC . Differences in the structural environment of six spin-labeled side chains allowed them to be classified with reference to previously obtained mobility maps as belonging to loop positions either relatively surface exposed or in structural contact or helix positions surface exposed in contact or buried . The mobility map constructed in the present study provides structural information that is in broad agreement with the location in the crystal structure. All but one of the chosen serine-to-cysteine mutations reduced activity considerably and this coincided with improved thermal stability. The effect of spin-labeling on enzyme function ranged from nonperturbing to an almost complete loss of activity. In the latter case treatment with a thiol reagent reactivated the enzyme indicating relief of steric hindrance to the catalytic process. Two mutations of an active-site residue W274 K328 in Escherichia coli alkaline phosphatase known to reduce activity and increase stability of Vibrio alkaline phosphatase gave a coincidental reduction in mobility of a nearby .

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