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.

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    Illuminating the off-pathway nature of the molten globule folding intermediate of an a-ß parallel protein
    Lindhoud, S. ; Westphal, A.H. ; Borst, J.W. ; Mierlo, C.P.M. van - \ 2012
    PLoS ONE 7 (2012)9. - ISSN 1932-6203
    azotobacter-vinelandii apoflavodoxin - refractive-index - fluorescence depolarization - spectroscopic ruler - hydrogen-exchange - energy landscape - state - flavodoxin - aggregation - mechanism
    Partially folded protein species transiently form during folding of most proteins. Often, these species are molten globules, which may be on- or off-pathway to the native state. Molten globules are ensembles of interconverting protein conformers that have a substantial amount of secondary structure, but lack virtually all tertiary side-chain packing characteristics of natively folded proteins. Due to solvent-exposed hydrophobic groups, molten globules are prone to aggregation, which can have detrimental effects on organisms. The molten globule observed during folding of the 179-residue apoflavodoxin from Azotobacter vinelandii is off-pathway, as it has to unfold before native protein can form. Here, we study folding of apoflavodoxin and characterize its molten globule using fluorescence spectroscopy and Förster Resonance Energy Transfer (FRET). Apoflavodoxin is site-specifically labeled with fluorescent donor and acceptor dyes, utilizing dye-inaccessibility of Cys69 in cofactor-bound protein. Donor (i.e., Alexa Fluor 488) is covalently attached to Cys69 in all apoflavodoxin variants used. Acceptor (i.e., Alexa Fluor 568) is coupled to Cys1, Cys131 and Cys178, respectively. Our FRET data show that apoflavodoxin’s molten globule forms in a non-cooperative manner and that its N-terminal 69 residues fold last. In addition, striking conformational differences between molten globule and native protein are revealed, because the inter-label distances sampled in the 111-residue C-terminal segment of the molten globule are shorter than observed for native apoflavodoxin. Thus, FRET sheds light on the off-pathway nature of the molten globule during folding of an a-ß parallel protein
    Distant residues mediate picomolar binding affinity of a protein cofactor
    Bollen, Y.J.M. ; Westphal, A.H. ; Lindhoud, S. ; Berkel, W.J.H. van; Mierlo, C.P.M. van - \ 2012
    Nature Communications 3 (2012). - ISSN 2041-1723
    azotobacter-vinelandii apoflavodoxin - hydrogen-exchange - ligand-binding - oxidized flavodoxin - energy landscape - nmr relaxation - dynamics - kinetics - thermodynamics - topology
    Numerous proteins require cofactors to be active. Computer simulations suggest that cooperative interaction networks achieve optimal cofactor binding. There is a need for the experimental identification of the residues crucial for stabilizing these networks and thus for cofactor binding. Here we investigate the electron transporter flavodoxin, which contains flavin mononucleotide as non-covalently bound cofactor. We show that after binding flavin mononucleotide with nanomolar affinity, the protein relaxes extremely slowly (time constant ~5 days) to an energetically more favourable state with picomolar-binding affinity. Rare small-scale openings of this state are revealed through H/D exchange of N(3)H of flavin. We find that H/D exchange can pinpoint amino acids that cause tight cofactor binding. These hitherto unknown residues are dispersed throughout the structure, and many are located distantly from the flavin and seem irrelevant to flavodoxin's function. Quantification of the thermodynamics of ligand binding is important for understanding, engineering, designing and evolving ligand-binding proteins
    Interrupted hydrogen/deuterium exchange reveals the stable core of the remarkably helical molten globule of a-ß parallel protein flavodoxin
    Nabuurs, S.M. ; Mierlo, C.P.M. van - \ 2010
    Journal of Biological Chemistry 285 (2010)6. - ISSN 0021-9258 - p. 4165 - 4172.
    azotobacter-vinelandii apoflavodoxin - folding intermediate - hydrogen-exchange - unfolded molecules - energy landscape - on-pathway - aggregation - equilibrium - lactalbumin - apomyoglobin
    Kinetic intermediates that appear early during protein folding often resemble the relatively stable molten globule intermediates formed by several proteins under mildly denaturing conditions. Molten globules have a substantial amount of secondary structure but lack virtually all tertiary side-chain packing characteristics of natively folded proteins. Due to exposed hydrophobic groups, molten globules are prone to aggregation, which can have detrimental effects on organisms. The molten globule that is observed during folding of a-ß parallel flavodoxin from Azotobacter vinelandii is a remarkably non-native species. This folding intermediate is helical and contains no ß-sheet and is kinetically off-pathway to the native state. It can be trapped under native-like conditions by substituting residue Phe44 for Tyr44. To characterize this species at the residue level, in this study, use is made of interrupted hydrogen/deuterium exchange detected by NMR spectroscopy. In the molten globule of flavodoxin, the helical region comprising residues Leu110-Val125 is shown to be better protected against exchange than the other ordered parts of the folding intermediate. This helical region is better buried than the other helices, causing its context-dependent stabilization against unfolding. Residues Leu110–Val125 thus form the stable core of the helical molten globule of a-ß parallel flavodoxin, which is almost entirely structured. Non-native docking of helices in the molten globule of flavodoxin prevents formation of the parallel ß-sheet of native flavodoxin. Hence, to produce native a-ß parallel protein molecules, the off-pathway species needs to unfold
    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
    Topological switching between an a-ß parallel protein and a remarkably helical molten globule.
    Nabuurs, S.M. ; Westphal, A.H. ; Toorn, M. aan den; Lindhoud, S. ; Mierlo, C.P.M. van - \ 2009
    Journal of the American Chemical Society 131 (2009)23. - ISSN 0002-7863 - p. 8290 - 8295.
    azotobacter-vinelandii apoflavodoxin - pathway folding intermediate - structural-characterization - on-pathway - unfolded molecules - hydrogen-exchange - energy landscape - flavodoxin-ii - state - apomyoglobin
    Partially folded protein species transiently exist during folding of most proteins. Often these species are molten globules, which may be on- or off-pathway to native protein. Molten globules have a substantial amount of secondary structure but lack virtually all the tertiary side-chain packing characteristic of natively folded proteins. These ensembles of interconverting conformers are prone to aggregation and potentially play a role in numerous devastating pathologies, and thus attract considerable attention. The molten globule that is observed during folding of apoflavodoxin from Azotobacter vinelandii is off-pathway, as it has to unfold before native protein can be formed. Here we report that this species can be trapped under nativelike conditions by substituting amino acid residue F44 by Y44, allowing spectroscopic characterization of its conformation. Whereas native apoflavodoxin contains a parallel ß-sheet surrounded by a-helices (i.e., the flavodoxin-like or a-ß parallel topology), it is shown that the molten globule has a totally different topology: it is helical and contains no ß-sheet. The presence of this remarkably nonnative species shows that single polypeptide sequences can code for distinct folds that swap upon changing conditions. Topological switching between unrelated protein structures is likely a general phenomenon in the protein structure universe
    "Noncooperative formation of the off-pathway molten globule during folding of the a-ß parallel protein apoflavodoxin"
    Nabuurs, S.M. ; Westphal, A.H. ; Mierlo, C.P.M. van - \ 2009
    Journal of the American Chemical Society 131 (2009)7. - ISSN 0002-7863 - p. 2739 - 2746.
    azotobacter-vinelandii apoflavodoxin - nmr-spectroscopy - structural-characterization - different denaturants - unfolded molecules - hydrogen-exchange - energy landscape - transition-state - on-pathway - intermediate
    During folding of many proteins, molten globules are formed. These partially folded forms of proteins have a substantial amount of secondary structure but lack virtually all tertiary side-chain packing characteristic of native structures. Molten globules are ensembles of interconverting conformers and are prone to aggregation, which can have detrimental effects on organisms. Consequently, molten globules attract considerable attention. The molten globule that is observed during folding of flavodoxin from Azotobacter vinelandii is a kinetically off-pathway species, as it has to unfold before the native state of the protein can be formed. This intermediate contains helices and can be populated at equilibrium using guanidinium hydrochloride as denaturant, allowing the use of NMR spectroscopy to follow molten globule formation at the residue level. Here, we track changes in chemical shifts of backbone amides, as well as disappearance of resonances of unfolded apoflavodoxin, upon decreasing denaturant concentration. Analysis of the data shows that structure formation within virtually all parts of the unfolded protein precedes folding to the molten globule state. This folding transition is noncooperative and involves a series of distinct transitions. Four structured elements in unfolded apoflavodoxin transiently interact and subsequently form the ordered core of the molten globule. Although hydrophobic, tryptophan side chains are not involved in the latter process. This ordered core is gradually extended upon decreasing denaturant concentration, but part of apoflavodoxin's molten globule remains random coil in the denaturant range investigated. The results presented here, together with those reported on the molten globule of alpha-lactalbumin, show that helical molten globules apparently fold in a noncooperative manner
    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.
    Macromolecular crowding compacts unfolded apoflavodoxin and causes severe aggregation of the off-pathway intermediate during apoflavodoxin folding
    Engel, R. ; Westphal, A.H. ; Huberts, D. ; Nabuurs, S.M. ; Lindhoud, S. ; Visser, A.J.W.G. ; Mierlo, C.P.M. van - \ 2008
    Journal of Biological Chemistry 283 (2008)41. - ISSN 0021-9258 - p. 27383 - 27394.
    fluorescence correlation spectroscopy - azotobacter-vinelandii apoflavodoxin - inclusion-body formation - protein stability - hydrogen-exchange - escherichia-coli - molecular chaperones - refractive-index - self-association - creatine-kinase
    To understand how proteins fold in vivo, it is important to investigate the effects of macromolecular crowding on protein folding. Here, the influence of crowding on in vitro apoflavodoxin folding, which involves a relatively stable off-pathway intermediate with molten globule characteristics, is reported. To mimic crowded conditions in cells, dextran 20 at 30% (w/v) is used, and its effects are measured by a diverse combination of optical spectroscopic techniques. Fluorescence correlation spectroscopy shows that unfolded apoflavodoxin has a hydrodynamic radius of 37 +/- 3 angstrom at 3M guanidine hydrochloride. Forster resonance energy transfer measurements reveal that subsequent addition of dextran 20 leads to a decrease in protein volume of about 29%, which corresponds to an increase in protein stability of maximally 1.1 kcal mol(-1). The compaction observed is accompanied by increased secondary structure, as far-UV CD spectroscopy shows. Due to the addition of crowding agent, the midpoint of thermal unfolding of native apoflavodoxin rises by 2.9 degrees C. Although the stabilization observed is rather limited, concomitant compaction of unfolded apoflavodoxin restricts the conformational space sampled by the unfolded state, and this could affect kinetic folding of apoflavodoxin. Most importantly, crowding causes severe aggregation of the off-pathway folding intermediate during apoflavodoxin folding in vitro. However, apoflavodoxin can be over expressed in the cytoplasm of Escherichia coli, where it efficiently folds to its functional native form at high yield without noticeable problems. Apparently, in the cell, apoflavodoxin requires the help of chaperones like Trigger Factor and the DnaK system for efficient folding.
    Tryptophan-Tryptophan energy migration as a tool to follow apoflavodoxin folding
    Visser, N.V. ; Westphal, A.H. ; Hoek, A. van; Mierlo, C.P.M. van; Visser, A.J.W.G. ; Amerongen, H. van - \ 2008
    Biophysical Journal 95 (2008). - ISSN 0006-3495 - p. 2462 - 2469.
    azotobacter-vinelandii apoflavodoxin - refractive-index - fluorescence depolarization - lipoamide dehydrogenase - glutathione-reductase - flavin fluorescence - hydrogen-exchange - backbone dynamics - protein-structure - flavodoxin-ii
    Submolecular details of Azotobacter vinelandii apoflavodoxin (apoFD) (un)folding are revealed by time-resolved fluorescence anisotropy using wild-type protein and variants lacking one or two of apoFD's three tryptophans. ApoFD equilibrium (un)folding by guanidine hydrochloride follows a three-state model: native unfolded intermediate. In native protein, W128 is a sink for Förster resonance energy transfer (FRET). Consequently, unidirectional FRET with a 50-ps transfer correlation time occurs from W167 to W128. FRET from W74 to W167 is much slower (6.9 ns). In the intermediate, W128 and W167 have native-like geometry because the 50-ps transfer time is observed. However, non-native structure exists between W74 and W167 because instead of 6.9 ns the transfer correlation time is 2.0 ns. In unfolded apoFD this 2.0-ns transfer correlation time is also detected. This decrease in transfer correlation time is a result of W74 and W167 becoming solvent accessible and randomly oriented toward one another. Apparently W74 and W167 are near-natively separated in the folding intermediate and in unfolded apoFD. Both tryptophans may actually be slightly closer in space than in the native state, even though apoFD's radius increases substantially upon unfolding. In unfolded apoFD the 50-ps transfer time observed for native and intermediate folding states becomes 200 ps as W128 and W167 are marginally further separated than in the native state. Apparently, apoFD's unfolded state is not a featureless statistical coil but contains well-defined substructures. The approach presented is a powerful tool to study protein folding.
    Stability of globular proteins in H2O and in D2O
    Efimova, Y.M. ; Haemers, S. ; Wierczinsky, B. ; Norde, W. ; Well, A.A. van - \ 2007
    Biopolymers 85 (2007)3. - ISSN 0006-3525 - p. 264 - 273.
    bovine plasma albumin - n-f transition - thermodynamic parameters - neutron-scattering - thermal-stability - hydrogen-exchange - mass-spectrometry - deuterated water - serum albumin - heavy-water
    In several experimental techniques D2O rather then H2O is often used as a solvent for proteins. Concerning the influence of the solvent on the stability of the proteins, contradicting results have been reported in literature. In this paper the influence of H2O-D2O solvent substitution on the stability of globular protein structure is determined in a systematic way. The differential scanning calorimetry technique is applied to allow for a thermodynamic analysis of two types of globular proteins: hen's egg lysozyme (LSZ) with relatively strong internal cohesion (hard globular protein) and bovine serum albumin (BSA), which is known for its conformational adaptability (soft globular protein). Both proteins tend to be more stable in D2O compared to H2O. We explain the increase of protein stability in D2O by the observation that D2O is a poorer solvent for nonpolar amino acids than H2O, implying that the hydrophobic effect is larger in D2O. In case of BSA the transitions between different isomeric forms, at low pH values the Nm and F forms, and at higher pH values Nm and B, were observed by the presence of a supplementary peak in the DSC thermogram. It appears that the pH-range for which the Nm form is the preferred one is wider in D2O than in H2O
    Probing solvent accessibility of transthyretin amyloid by solution NMR spectroscopy
    Olofsson, A. ; Ippel, J.H. ; Wijmenga, S.S. ; Lundgren, E. ; Ohman, A. - \ 2004
    Journal of Biological Chemistry 279 (2004)7. - ISSN 0021-9258 - p. 5699 - 5707.
    hydrogen-exchange - tetramer dissociation - subunit interface - x-ray - fibrils - core - intermediate - proteins - variants - mutants
    The human plasma protein transthyretin (TTR) may form fibrillar protein deposits that are associated with both inherited and idiopathic amyloidosis. The present study utilizes solution nuclear magnetic resonance spectroscopy, in combination with hydrogen/deuterium exchange, to determine residue-specific solvent protection factors within the fibrillar structure of the clinically relevant variant, TTRY114C. This novel approach suggests a fibril core comprised of the six beta-strands, A-B-E-F-G-H, which retains a native-like conformation. Strands C and D are dislocated from their native edge region and become solvent-exposed, leaving a new interface involving strands A and B open for intermolecular interactions. Our results further support a native-like intermolecular association between strands F-F' and H-H' with a prolongation of these beta-strands and, interestingly, with a possible shift in beta-strand register of the subunit assembly. This finding may explain previous observations of a monomeric intermediate preceding fibril formation. A structural model based on our results is presented.
    Conformation and orientation of a protein folding intermediate trapped by adsorption
    Engel, M.F.M. ; Visser, A.J.W.G. ; Mierlo, C.P.M. van - \ 2004
    Proceedings of the National Academy of Sciences of the United States of America 101 (2004)31. - ISSN 0027-8424 - p. 11316 - 11321.
    bovine alpha-lactalbumin - hydrogen-exchange - stability - surface - binding - nmr
    Although adsorption-induced conformational changes of proteins play an essential role during protein adsorption on interfaces, detailed information about these changes is lacking. To further the current understanding of protein adsorption, in this study, the orientation, conformation, and local stability of bovine a-lactalbumin (BLA) adsorbed on polystyrene nanospheres is characterized at the residue level by hydrogen/deuterium exchange and 2D NMR spectroscopy. Most of the adsorbed BLA molecules have conformational properties similar to BLA molecules in the acid-induced molten globule state (A state). A folding intermediate of BLA is thus induced and trapped by adsorption of the protein on the hydrophobic interface. Several residues, clustered on one side of the adsorbed folding intermediate of BLA, have altered amide proton exchange protection factors compared to those of the A state of BLA. This side preferentially interacts with the interface and includes residues in helix C, the calcium binding site, and part of the beta-domain. Local unfolding of this interacting part of the adsorbed protein seems to initiate the adsorption-induced unfolding of BLA. Adsorption-induced protein unfolding apparently resembles more the mechanical unfolding of a protein than the global unfolding of a protein as induced by denaturant, pH, or pressure. 2D macromolecular crowding prevented the minority of adsorbed BLA molecules, which arrived late at the interface, to unfold to the A state. Protein adsorption is a novel and challenging approach to probe features of the free energy landscapes accessible to unfolding proteins.
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