Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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    We will mail you new results for this query: keywords==pas domain
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Structure of the Redox Sensor Domain of Azotobacter vinelandii NifL at Atomic Resolution: Signaling, Dimerization, and Mechanism.
Key, J. ; Hefti, M.H. ; Purcell, E.B. ; Moffat, K. - \ 2007
Biochemistry 46 (2007)12. - ISSN 0006-2960 - p. 3614 - 3623.
plant photoreceptor domain - hypoxia-inducible factor - nitrogen-fixation genes - binding protein nifa - pas domain - oxygen sensor - crystal-structure - escherichia-coli - in-vitro - adiantum phytochrome3
NifL is a multidomain sensor protein responsible for the transcriptional regulation of genes involved in response to changes in cellular redox state and ADP concentration. Cellular redox is monitored by the N-terminal PAS domain of NifL which contains an FAD cofactor. Flavin-based PAS domains of this type have also been referred to as LOV domains. To explore the mechanism of signal recognition and transduction in NifL, we determined the crystal structure of the FAD-bound PAS domain of NifL from Azotobacter vinelandii to 1.04 Å resolution. The structure reveals a novel cavity within the PAS domain which contains two water molecules directly coordinated to the FAD. This cavity is connected to solvent by multiple access channels which may facilitate the oxidation of the FAD by molecular oxygen and the release of hydrogen peroxide. The structure contains a dimer of the NifL PAS domain that is structurally very similar to those described in other crystal structures of PAS domains and identifies a conserved dimerization motif. An N-terminal amphipathic helix constitutes part of the dimerization interface, and similar N-terminal helices are identified in other PAS domain proteins. The structure suggests a model for redox-mediated signaling in which a conformational change is initiated by redox-dependent changes in protonation at the N5 atom of FAD that lead to reorganization of hydrogen bonds within the flavin binding pocket. A structural signal is subsequently transmitted to the -sheet interface between the monomers of the PAS domain
A His-tag based immobilization method for the preparation and reconstitution of apoflavoproteins
Hefti, M.H. ; Milder, F.J. ; Boeren, J.A. ; Vervoort, J.J.M. ; Berkel, W.J.H. van - \ 2003
Biochimica et Biophysica Acta. General subjects 1619 (2003)2. - ISSN 0304-4165 - p. 139 - 143.
para-hydroxybenzoate hydroxylase - pseudomonas-fluorescens - azotobacter-vinelandii - pas domain - flavin - riboflavin - protein - oxidase - nifl - chromatography
The NifL PAS domain from Azotobacter vinelandii is a flavoprotein with FAD as the prosthetic group. Here we describe a novel immobilization procedure for the large-scale preparation of apo NifL PAS domain and its efficient reconstitution with either 2,4a-C-13-FAD or 2,4a-C-13-FMN. In this procedure, the His-tagged holoprotein is bound to an immobilized metal affinity column and the flavin is released by washing the column with buffer containing 2 M KBr and 2 M urea. The apoprotein is reconstituted on-column with the (artificial) flavin cofactor, and then eluted with buffer containing 250 mM imidazole. Alternatively, the immobilized apoprotein can be released from the column matrix before reconstitution. The His-tag based immobilization method of preparing reconstituted (or apo) NifL PAS domain protein has the advantage that it combines a protein affinity chromatography technique with limited protein loss, resulting in a high protein yield with extremely efficient flavin reconstitution. This on-column reconstitution method can also be used in cases where the apoprotein is unstable. Therefore, it may develop as a universal method for replacement of flavin or other cofactors. (C) 2002 Elsevier Science B.V. All rights reserved.
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