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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    Non-native hydrophobic interactions detected in unfolded apoflavodoxin by paramagnetic relaxation enhancement
    Nabuurs, S.M. ; Kort, B.J. de; Westphal, A.H. ; Mierlo, C.P.M. van - \ 2010
    European Biophysics Journal 39 (2010)4. - ISSN 0175-7571 - p. 689 - 698.
    beta parallel protein - azotobacter-vinelandii apoflavodoxin - denatured state - folding mechanism - hydrogen-exchange - molten globule - flavodoxin-ii - native-like - pathway - nmr
    Transient structures in unfolded proteins are important in elucidating the molecular details of initiation of protein folding. Recently, native and non-native secondary structure have been discovered in unfolded A. vinelandii flavodoxin. These structured elements transiently interact and subsequently form the ordered core of an off-pathway folding intermediate, which is extensively formed during folding of this a–ß parallel protein. Here, site-directed spin-labelling and paramagnetic relaxation enhancement are used to investigate long-range interactions in unfolded apoflavodoxin. For this purpose, glutamine-48, which resides in a non-native a-helix of unfolded apoflavodoxin, is replaced by cysteine. This replacement enables covalent attachment of nitroxide spin-labels MTSL and CMTSL. Substitution of Gln-48 by Cys-48 destabilises native apoflavodoxin and reduces flexibility of the ordered regions in unfolded apoflavodoxin in 3.4 M GuHCl, because of increased hydrophobic interactions in the unfolded protein. Here, we report that in the study of the conformational and dynamic properties of unfolded proteins interpretation of spin-label data can be complicated. The covalently attached spin-label to Cys-48 (or Cys-69 of wild-type apoflavodoxin) perturbs the unfolded protein, because hydrophobic interactions occur between the label and hydrophobic patches of unfolded apoflavodoxin. Concomitant hydrophobic free energy changes of the unfolded protein (and possibly of the off-pathway intermediate) reduce the stability of native spin-labelled protein against unfolding. In addition, attachment of MTSL or CMTSL to Cys-48 induces the presence of distinct states in unfolded apoflavodoxin. Despite these difficulties, the spin-label data obtained here show that non-native contacts exist between transiently ordered structured elements in unfolded apoflavodoxin
    Extensive formation of off-pathway species during folding of an alpha-beta parallel protein is due to docking of (non)native structure elements in unfolded molecules
    Nabuurs, S.M. ; Westphal, A.H. ; Mierlo, C.P.M. van - \ 2008
    Journal of the American Chemical Society 130 (2008)50. - ISSN 0002-7863 - p. 16914 - 16920.
    azotobacter-vinelandii apoflavodoxin - nmr chemical-shifts - 8 m urea - secondary structure - denatured state - hydrogen-exchange - energy landscape - intermediate - topology - conformations
    Detailed information about unfolded states is required to understand how proteins fold. Knowledge about folding intermediates formed subsequently is essential to get a grip on pathological aggregation phenomena. During folding of apoflavodoxin, which adopts the widely prevalent ¿¿ß parallel topology, most molecules fold via an off-pathway folding intermediate with helical properties. To better understand why this species is formed, guanidine hydrochloride-unfolded apoflavodoxin is characterized at the residue level using heteronuclear NMR spectroscopy. In 6.0 M denaturant, the protein behaves as a random coil. In contrast, at 3.4 M denaturant, secondary shifts and 1H¿15N relaxation rates report four transiently ordered regions in unfolded apoflavodoxin. These regions have restricted flexibility on the (sub)nanosecond time scale. Secondary shifts show that three of these regions form ¿-helices, which are populated about 10% of the time, as confirmed by far-UV CD data. One region of unfolded apoflavodoxin adopts non-native structure. Of the ¿-helices observed, two are present in native apoflavodoxin as well. A substantial part of the third helix becomes ß-strand while forming native protein. Chemical shift changes due to amino acid residue replacement show that the latter ¿-helix has hydrophobic interactions with all other ordered regions in unfolded apoflavodoxin. Remarkably, these ordered segments dock non-natively, which causes strong competition with on-pathway folding. Thus, rather than directing productive folding, conformational preorganization in the unfolded state of an ¿¿ß parallel-type protein promotes off-pathway species formation.
    Tertiary structure in 7.9 M guanidinium chloride: the role of Glu-53 and Asp-287 in Pyrococcus furiosus endo-beta-1,3-glucanase
    Chiaraluce, R. ; Florio, R. ; Angelaccio, S. ; Gianese, G. ; Lieshout, J.F.T. van; Oost, J. van der; Consalvi, V. - \ 2007
    FEBS Journal 274 (2007). - ISSN 1742-464X - p. 6167 - 6179.
    denatured state - staphylococcal nuclease - protein stability - unfolded state - thermophilic proteins - residual structure - m-values - intermediate - thermodynamics - salt
    The thermodynamic stability of family 16 endo-ß-1,3-glucanase (EC from the hyperthermophilic archaeon Pyrococcus furiosus is decreased upon single (D287A, E53A) and double (E53A/D287A) mutation of Asp287 and Glu53. In accordance with the homology model prediction, both carboxylic acids are involved in the composition of a calcium binding site, as shown by titration of the wild-type and the variant proteins with a chromophoric chelator. The present study shows that, in P. furiosus, endo-ß-1,3-glucanase residues Glu53 and Asp287 also make up a calcium binding site in 7.9 m guanidinium chloride. The persistence of tertiary structure in 7.9 m guanidinium chloride, a feature of the wild-type enzyme, is observed also for the three variant proteins. The ¿GH2O values relative to the guanidinium chloride-induced equilibrium unfolding of the three variants are approximatelty 50% lower than that of the wild-type. The destabilizing effect of the combined mutations of the double mutant is non-additive, with an energy of interaction of 24.2 kJ·mol¿1, suggesting a communication between the two mutated residues. The decrease in the thermodynamic stability of D287A, E53A and E53A/D287A is contained almost exclusively in the m-values, a parameter which reflects the solvent-exposed surface area upon unfolding. The decrease in m-value suggests that the substitution with alanine of two evenly charged repulsive side chains induces a stabilization of the non-native state in 7.9 m guanidinium chloride comparable to that induced by the presence of calcium on the wild-type. These results suggest that the stabilization of a compact non-native state may be a strategy for P. furiosus endo-ß-1,3-glucanase to thrive under adverse environmental conditions.
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