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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    Time-resolved fluorescence analysis of the mobile flavin cofactor in p-hydroxybenzoate hydroxylase.
    Berg, P.A.W. van den; Grever, K. ; Hoek, A. van; Berkel, W.J.H. van; Visser, A.J.W.G. - \ 2007
    Journal of Chemical Sciences 119 (2007)2. - ISSN 0974-3626 - p. 123 - 133.
    maximum-entropy method - pseudomonas-fluorescens - crystal-structure - 4-hydroxybenzoate hydroxylase - glutathione-reductase - effector specificity - adenine-dinucleotide - protein nanospace - nadph binding - wild-type
    Conformational heterogeneity of the FAD cofactor in p-hydroxybenzoate hydroxylase (PHBH) was investigated with time-resolved polarized flavin fluorescence. For binary enzyme/substrate (analogue) complexes of wild-type PHBH and Tyr222 mutants, crystallographic studies have revealed two distinct flavin conformations; the ‘in’ conformation with the isoalloxazine ring located in the active site, and the ‘out’ conformation with the isoalloxazine ring disposed towards the protein surface. Fluorescence-lifetime analysis of these complexes revealed similar lifetime distributions for the ‘in’ and ‘out’ conformations. The reason for this is twofold. First, the active site of PHBH contains various potential fluorescence-quenching sites close to the flavin. Fluorescence analysis of uncomplexed PHBH Y222V and Y222A showed that Tyr222 is responsible for picosecond fluorescence quenching free enzyme. In addition, other potential quenching sites, including a tryptophan and two tyrosines involved in substrate binding, are located nearby. Since the shortest distance between these quenching sites and the isoalloxazine ring differs only little on average, these aromatic residues are likely to contribute to fluorescence quenching. Second, the effect of flavin conformation on the fluorescence lifetime distribution is blurred by binding of the aromatic substrates: saturation with aromatic substrates induces highly efficient fluorescence quenching. The flavin conformation is therefore only reflected in the small relative contributions of the longer lifetimes
    Flavin fluorescence dynamics of p-hydroxybenzoate hydroxylase in relation to flavin mobility.
    Berg, P.A.W. van den; Grever, K. ; Hoek, A. van; Berkel, W.J.H. van; Visser, A.J.W.G. - \ 1997
    In: Fifth International Conference on Methods and Applications of Fluorescence Spectroscopy Berlin, Germany (1997)
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