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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    Molecular mechanism of active photoprotein complex formation
    Eremeeva, E.V. - \ 2013
    Wageningen University. Promotor(en): Willem van Berkel; Ton Visser, co-promotor(en): E.S. Vyotski. - S.l. : s.n. - ISBN 9789461734587 - 194
    photo-eiwitten - obeline - moleculaire structuur - bioluminescentie - fluorescentie - photoproteins - obelin - molecular conformation - bioluminescence - fluorescence
    Strength, structure and stability of polyelectrolyte complex coacervates
    Spruijt, E. - \ 2012
    Wageningen University. Promotor(en): Martien Cohen Stuart, co-promotor(en): Jasper van der Gucht. - S.l. : s.n. - ISBN 9789461733542 - 291
    elektrolyten - bindingssterkte - elektrische dubbellaag - chemische structuur - moleculaire structuur - oppervlaktespanning - electrolytes - bond strength - electrical double layer - chemical structure - molecular conformation - surface tension - cum laude
    cum laude graduation (with distinction)
    Interplay between the bacterial nucleoid protein H-NS and macromolecular crowding in compacting DNA
    Wintraecken, C.H.J.M. - \ 2012
    Wageningen University. Promotor(en): Frans Leermakers, co-promotor(en): T. Odijk. - S.l. : s.n. - ISBN 9789461733023 - 124
    dna-bindende eiwitten - dna - bacteriën - chemische structuur - moleculaire structuur - dna binding proteins - dna - bacteria - chemical structure - molecular conformation

    In this dissertation we discuss H-NS and its connection to nucleoid compaction and organization. Nucleoid formation involves a dramatic reduction in coil volume of the genomic DNA. Four factors are thought to influence coil volume: supercoiling, DNA charge neutralization, macromolecular crowding and DNA deformation by NAPs. This study focuses mainly on the latter two factors, and on their interplay. We investigate both direct and indirect changes in DNA coil volume as a result of H-NS binding to DNA. H-NS / DNA binding is thought to be influenced by the self-association of H-NS, hence DNA self-association (both in bulk and on DNA) has also been investigated.
    Chapter 2 focuses on the known cooperative character of H-NS-DNA binding. The molecular origin of the cooperativity is poorly understood. High concentrations of H-NS are known to oligomerize extensively in the absence of DNA. Some models propose that cooperativity is caused by the same protein-protein interactions that cause oligomerization in solution, whereas others propose cooperativity may be induced by the DNA substrate. We have mutated some parts of H-NS we believed to be important in oligomerization to investigate the role of H-NS protein-protein interactions in cooperative DNA binding, and studied the oligomerization and DNA-binding properties of these mutants. The D68VD71V mutant has two aspartic acids in the linker region replaced by valines, making the linker significantly more hydrophobic. The double linker mutation D68VD71V dramatically enhances H-NS oligomerization in solution, and its temperature-dependence is changed as well in vitro. Yet there is only a moderate effect on DNA binding properties, which does point in the direction of enhanced cooperativity, as expected. This suggests that protein-protein interactions have a much larger effect on H-NS self-association in solution than on the DNA binding properties.
    Chapter 3 discusses the influence of the bacterial nucleoid protein H-NS on DNA coil sizes in solution, using Light Scattering, for both supercoiled and linear pUC18 DNA. We clearly find H-NS binding: the intensity of light scattered by the DNA coils increased upon the addition of H-NS. But, H-NS did not have a significant effect on the effective hydrodynamic radius of the coils. Our results suggest that under the conditions of our experiment (in particular the buffer conditions: 10mM Sodium Phosphate buffer, pH 7, 100mM NaCl), the H-NS proteins most likely did not cause extensive bridging of dsDNA, since this most certainly would have led to a significant effect on the DNA coil sizes. This absence of bridging in the absence of multivalent cations is consistent with single molecule DNA force measurements performed for similar buffer conditions by other authors. We also find that, although H-NS alone does not have a dramatic effect on DNA coil sizes in solution (for our solution conditions), it does have an interesting synergetic effect on polymer-induced condensation of DNA. Condensation of H-NS/DNA complexes was measured by their sedimentability in solutions of polyethylene glycol (PEG). In the absence of H-NS the critical concentration of PEG needed to condense DNA is approximately 15%, whereas the critical concentration is remarkably lower, about 3.5%, at near saturation concentrations of H-NS.
    Chapter 4 is concerned with the effect of binding of H-NS and macromolecular crowding on nucleoid compaction. An osmotic shock method using ampicillin was used to isolate the Escherichia coli nucleoids intact, disrupting the peptidoglycan layer. These nucleoids were stained with DAPI and photographed using confocal microscopy. This showed a decrease in the volume of the isolated nucleoids when polyethylene glycol (PEG) concentrations became higher. The addition of small amounts of H-NS appeared to enhance the compaction due to macromolecular crowding induced by PEG. Remarkably, in the absence of PEG, H-NS did not affect the compaction of the nucleoids even at higher concentrations. The results are consistent with previous experiments done on DNA-binding proteins HU and Sso7d by other research groups. Therefore, our results confirm a general enhancement of macromolecular crowding effect of cytoplasm by nucleoid-associated proteins binding.
    In Chapter 5 we focus on an archaeal NAP. Like bacteria, archaea have NAPs that bend DNA and form extended helical protein-DNA fibers. These do not condense the genomic DNA directly, but some NAPS strongly promote DNA condensation by macromolecular crowding, such as the bacterial HU. Using theoretical arguments, we show that this synergy can be explained by the larger diameter and lower net charge density of protein-covered DNA filaments compared to naked DNA. Therefore the effect should be nearly universal in prokaryotes. We illustrate this general effect by demonstrating that Sso7d, a 7 kDa basic DNA-bending protein from the archaeon Sulfolobus Solfataricus, does not significantly condense DNA by itself, using light-scattering to determine coil volumes. However, the Sso7d-coated DNA fibres are much more susceptible to macromolecular crowding-induced condensation. Clearly, if DNA-bending nucleoid proteins fail to condense DNA in dilute solution, this does not mean that they do not contribute to DNA condensation in the context of the crowded living cell.

    Visualisation and characterisation of apoflavodoxin folding
    Lindhoud, S. - \ 2012
    Wageningen University. Promotor(en): Willem van Berkel, co-promotor(en): Carlo van Mierlo. - S.l. : s.n. - ISBN 9789461733054 - 141
    flavoproteinen - moleculaire structuur - flavoproteins - molecular conformation
    Structural and biochemical characterization of 3-hydroxybenzoate 6-hydroxylase
    Montersino, S. - \ 2012
    Wageningen University. Promotor(en): Willem van Berkel, co-promotor(en): A. Mattevi. - [S.l.] : s.n. - ISBN 9789461732781 - 158
    aspecifiek mono-oxygenase - moleculaire structuur - biochemie - unspecific monooxygenase - molecular conformation - biochemistry

    The thesis deals with the characterization of a new flavoprotein hydroxylase 3 hydroxybenzoate 6-hydroxylase (3HB6H) from Rhodococcus jostii RHA1. 3HB6H is able to insert exclusively oxygen in para-position and the enzyme has been chosen to study the structural basis of such regioselectivity. As main result, functional mirror image active sites direct regioselective 3-hydroxybenzoate hydroxylation. Moreover, the nature and role of unprecedented phospholipid binding has been analyzed demonstrating a role in enzyme oligomerization and a possible protective role during catalysis. To conclude, the knowledge acquired improves our insight into the strategies of flavin-dependent regioselective hydroxylation and the results emerged in this thesis provide a foundation for further structural and kinetic studies on 3HB6H and related enzymes.

    The formation and deformation of protein structures with viscoelastic properties
    Riemsdijk, L.E. van - \ 2011
    Wageningen University. Promotor(en): Rob Hamer; Remko Boom, co-promotor(en): Atze Jan van der Goot. - S.l. : s.n. - ISBN 9789085858638 - 239
    eiwitten - nieuwe eiwitten - visco-elasticiteit - wei-eiwit - chemische structuur - moleculaire structuur - deeg - proteins - novel proteins - viscoelasticity - whey protein - chemical structure - molecular conformation - doughs

    This study describes the formation of a gluten substitute.

    Chapter 1 describes the properties that are necessary to obtain a gluten substitute.

    Chapter 2 describes the formation and properties of protein particle suspensions. Two proteins with different intrinsic properties, gelatin and whey protein, were selected as model materials.

    Chapter 3 describes the effects of simple shear flow on the formation and properties of gelatin particle suspensions. The application of well-defined simple shear flow during phase separation was used to control the protein particle size in a gelatin–dextran system.

    Chapter 4 describes the formation and properties of whey protein particle suspensions having different particle sizes and different abilities to form disulphide bonds. Application of shear during their formation was used.

    Chapter 5 describes a novel concept for making elastic dough through combining a whey protein particle suspension with native wheat starch. Three differently structured whey protein suspensions were evaluated.

    Chapter 6 discusses the use of the whey protein particle suspensions prepared and used in chapter 5 for baking bread.

    Chapter 7 describes the role of molecular properties on the final dough and bread that were discussed in chapters 5 and 6.

    Chapter 8 summarizes the main findings of the project on “The formation and deformation of protein structures with viscoelastic properties”.

    Self-organization of polymers in bulk and at interfaces
    Charlaganov, M. - \ 2009
    Wageningen University. Promotor(en): Frans Leermakers; Martien Cohen Stuart, co-promotor(en): O.V. Borisov. - [S.l. : S.n. - ISBN 9789085855026 - 129
    polymeren - moleculaire structuur - colloïdale eigenschappen - polymers - molecular conformation - colloidal properties
    Fully atomistic analysis of polymeric systems is computationally very demanding because the time and length scales involved span over several orders of magnitude. At the same time many properties of polymers are universal in the sense that they do not depend on the chemical nature of the comprising monomers. This makes coarse-grained methods, such as self-consistent field (SCF) modeling, an ideal tool for studying them. In this thesis we employ SCF modeling to study intra- and intermolecular self-organization organization of polymers and ordering of polymers near interfaces. Where possible, the results are compared to experiments and predictions of analytical theories.
    Modeling membrane protein structure through site-directed ESR spectroscopy
    Kavalenka, A.A. - \ 2009
    Wageningen University. Promotor(en): Herbert van Amerongen, co-promotor(en): Marcus Hemminga; J. Strancar. - [S.l. : S.n. - ISBN 9789085854241 - 119
    oppervlakte-eiwitten - moleculaire structuur - spectroscopie - paramagnetische elektronenresonantiespectroscopie - surface proteins - molecular conformation - spectroscopy - electron paramagnetic resonance spectroscopy
    Site-directed spin labeling (SDSL) electron spin resonance (ESR) spectroscopy is a
    relatively new biophysical tool for obtaining structural information about proteins. This
    thesis presents a novel approach, based on powerful spectral analysis techniques (multicomponent
    spectral simulations and evolutionary optimizations of ESR spectra) and
    modeling of the protein structure by calculating the restrictions of the conformational space
    of the attached spin label.
    First, the feasibility of the ESR spectral analysis was enhanced by speeding-up the
    spectrum optimization and by automation of the analysis routines to enable the handling of
    large sets of spectroscopic data (e.g., for the joint analysis of SDSL-ESR spectra from
    multiple sites of a spin-labeled protein). According to the testing examples a speed-up
    factor of 5-7 was achieved.
    Secondly, SDSL-ESR was used to study the topology of the long N-terminal domain
    of the photosynthetic light-harvesting complex CP29. Wild-type protein containing a single
    cysteine at position 108 and nine single cysteine mutants were produced, allowing to label
    different parts of the domain with a nitroxide spin label. In all cases the apoproteins were
    either solubilized in detergent, or they were reconstituted with their native pigments in
    vitro. The spin label ESR spectra were analyzed in terms of a multi-component spectral
    simulation approach. These results permit to trace the structural organization of the long Nterminal
    domain of CP29 leading to a structural model for its N-terminal domain.
    Thirdly, we proposed a novel way to translate the local structural constraints gained
    by SDSL-ESR data into a low-resolution structure of a protein by simulating the
    restrictions of the local conformational spaces of the spin label attached at different protein
    sites along the primary structure of the membrane-embedded protein. The proposed
    structural model takes into account the restricting effect of the protein backbone, amino
    acid side chains and lipid environment. We tested the sensitivity of this approach for
    artificial oligopeptides and then for membrane-embedded M13 major coat protein
    decorated with a limited number of strategically placed spin labels by employing highthroughput
    site-directed mutagenesis. We found a reasonably good agreement of the
    simulated and the experimental data taking a protein conformation close to an α-helix.
    Finally, by using an optimization algorithm we optimized the parameters of the
    protein-lipid model by improving the fit of the simulation data to the experimental
    conformational space data. The outcome of the optimization was a family of best-fit
    structures of membrane-embedded M13 protein, which not only agree with the available
    SDSL-ESR data, but also was consistent with a recent model based on site-directed
    fluorescence labeling.
    Therefore, the present method provides a challenging starting point for the
    development of a powerful methodology for the protein structure characterization, an
    alternative approach to conventional techniques.
    Biomolecular design elements : cortical microtubes and DNA-coated colloids
    Tindemans, S. - \ 2009
    Wageningen University. Promotor(en): Bela Mulder. - [S.l. : S.n. - ISBN 9789085853954 - 172
    microtubuli - celskelet - moleculaire structuur - macromoleculen - microtubules - cytoskeleton - molecular conformation - macromolecules
    This thesis deals with the self-organizing properties of systems of biomolecules.
    Why do alpha-beta parallel proteins, like flavodoxins, form misfolded off-pathway intermediates?
    Nabuurs, S.M. - \ 2009
    Wageningen University. Promotor(en): Sacco de Vries, co-promotor(en): Carlo van Mierlo. - [S.l.] : S.n. - ISBN 9789085853510 - 144
    eiwitten - moleculaire structuur - proteins - molecular conformation
    The question: “Why do α-β parallel proteins, like flavodoxins, form misfolded off-pathway
    intermediates?" is the main subject of this thesis. A. vinelandii apoflavodoxin is chosen as protein
    of interest as it is a representative of α-β parallel proteins, which are widely prevalent in nature. The
    folding behavior of A. vinelandii apo- and holoflavodoxin has been studied extensively during the
    past years. Both denaturant-induced equilibrium and kinetic (un)folding of apoflavodoxin have been
    characterized in detail using GuHCl as denaturant 1-8. An off-pathway intermediate plays a major role
    during apoflavodoxin folding and is also observed during the kinetic folding of other proteins with
    an α-β parallel topology of which the folding mechanism has been studied 9.
    Approximately 90% of folding molecules fold via off-pathway intermediate Ioff, which is a
    relatively stable species that needs to unfold to produce native protein and thus acts as a trap
    3. Residual structure in the unfolded state of apoflavodoxin probably facilitates formation of
    this species. In chapter 2 detailed information about unfolded apoflavodoxin is revealed by
    heteronuclear NMR spectroscopy. In 6.0 M GuHCl apoflavodoxin behaves as a random coil as is
    shown by far-UV CD and by 1H-15N R2 relaxation rates. Upon lowering denaturant concentration
    the amount of residual structure in apoflavodoxin increases. Chemical shift deviations between
    unfolded apoflavodoxin in 3.4 and 6.0 M GuHCl reveal in unfolded apoflavodoxin in 3.4 M GuHCl
    the presence of three transiently formed α-helices and of one structured region that is neither an
    α-helix nor a β-sheet. One of these transiently formed α-helices is non-native, and a part of this
    helix becomes a β-strand in native apoflavodoxin. Four regions with restricted flexibility on the
    (sub)nanosecond time scale are revealed by 1H-15N R2 relaxation rates of unfolded apoflavodoxin in
    3.4 M GuHCl. These four regions coincide with the ordered regions found by chemical shift analysis
    and match with regions of large AABUF (average area buried upon folding), which is correlated
    with hydrophobicity 10. Chemical shift deviations upon substitution of a glutamine residue with
    a more hydrophobic cysteine residue on position 48, in the middle of the non-native α-helix in
    unfolded apoflavodoxin, show that this non-native helix has hydrophobic interactions with all other
    ordered regions in unfolded apoflavodoxin. Formation of native and non-native helices in unfolded
    apoflavodoxin and subsequent docking of these helices leads to formation of a compact off-pathway
    intermediate.
    The formation of this off-pathway intermediate is discussed in chapter 3. Backbone amide
    resonances of unfolded apoflavodoxin are followed in a series of 1H-15N HSQC spectra acquired
    at concentrations of GuHCl between 4.05 M and 1.58 M. Analysis of cross peak disappearance of
    unfolded backbone amides made it possible to determine midpoints of unfolding of 68 backbone
    amides. Residues were grouped in five different groups according to their midpoint of unfolding.
    The group with the highest Cm value forms the folding core of the molten globule of apoflavodoxin in
    presence of GuHCl. This folding core roughly coincides with the regions with restricted flexibility in
    unfolded apoflavodoxin. The core is gradually extended upon decreasing denaturant concentration,
    but part of apoflavodoxin’s molten globule remains random coil in the denaturant range investigated.
    The formation of the off-pathway intermediate of apoflavodoxin is non-cooperative and involves
    a series of distinct transitions in contrast to the cooperative formation of native apoflavodoxin 7.
    In addition, chemical shifts of the amides of unfolded apoflavodoxin could be tracked over the
    denaturant range investigated. Analysis of the chemical shift changes shows that structure formation
    within virtually all parts of the unfolded protein precedes folding to the molten globule. The results
    presented in this chapter, together with those reported on the molten globule of α-lactalbumin 11,
    show that helical molten globules apparently fold in a non-cooperative manner.
    To investigate long-range interactions in unfolded apoflavodoxin that lead to formation of this
    off-pathway intermediate, in chapter 4 use is made of site-directed spin labeling. For this purpose,
    glutamine at position 48, which resides in a non-native α-helix of unfolded apoflavodoxin, is
    replaced by a cysteine. This replacement enables covalent attachment of two different nitroxide spin
    labels, MTSL and CMTSL. Due to this amino acid replacement stability of native apoflavodoxin
    against unfolding decreases and attachment of the nitroxide spin label MTSL leads to a further
    decrease in stability. Replacement of Gln48 by Cys48 decreased flexibility of the ordered regions in
    unfolded apoflavodoxin in 3.4 M GuHCl, due to increased hydrophobic interactions. Interactions
    are detected between the MTSL spin label attached to Cys69 and region Ser40 - Leu62 of unfolded
    apoflavodoxin in 6.0 M GuHCl. These non-specific hydrophobic interactions between nitroxide
    spin labels and hydrophobic patches of unfolded apoflavodoxin perturb the unfolded protein.
    Our observations show that in 6.0 M GuHCl spin-labeled apoflavodoxin is less random coil
    than C69A apoflavodoxin is. Thus, care needs to be taken in the use of spin labels for the study
    of the conformational and dynamic properties of unfolded proteins. In 3.4 M GuHCl the attached
    CMTSL spin label induces the presence of two distinct states in unfolded apoflavodoxin. In one of
    these states, the spin label attached to residue 48 has persistent contact with residue Leu78. The spin
    label data show that non-native contacts exist between transiently ordered structured elements in
    unfolded apoflavodoxin.
    Full population of the molten globule-like folding state of apoflavodoxin is possible through
    covalent introduction of just a single extra oxygen atom in the protein, achieved by replacing Phe44
    with Tyr44 through site-directed mutagenesis (chapter 5). This replacement leads to significant
    destabilization of native apoflavodoxin, as is demonstrated by GuHCl-induced equilibrium
    (un)folding and thermal unfolding experiments. Decreasing salt concentration destabilizes native
    apoflavodoxin even further. As a result, the native state of F44Y apoflavodoxin is hardly populated.
    Instead, in absence of denaturant, virtually all protein molecules exist as molten globule-like folding
    intermediate. Direct characterization of this intermediate by far-UV CD is possible, it is shown that
    the molten globule has a totally different topology: it is helical and lacks the parallel β-sheet of native
    apoflavodoxin.
    Full population of the molten globule state of F44Y apoflavodoxin enables use of H/D
    exchange for the characterization at the residue level by NMR spectroscopy of apoflavodoxin’s
    molten globule folding intermediate. In chapter 6, interrupted H/D exchange is used to detect the
    stable core of apoflavodoxin’s molten globule in absence of denaturant. Exchange rates could be
    determined for 68 backbone amides. Amide protons of residues Lys16 – Phe25 are poorly protected
    against exchange, and structure formed in this region is very unstable. In chapter 4 chemical shift
    data and Cm-values showed that these residues belong to the most unstable part of apoflavodoxin’s
    molten globule, as they remain random coil down to a GuHCl concentration of 1.58 M. Leu110 to
    Val125 have the highest protection factors against H/D exchange and form the single stable core of
    apoflavodoxin’s molten globule in absence of denaturant. The residues of this molten globule, which
    have the highest midpoints against unfolding by GuHCl, roughly coincide with those residues
    that are transiently ordered in unfolded apoflavodoxin. Only one of the four regions mentioned is
    significantly protected against exchange in this intermediate. This suggests that this helix is better
    buried in apoflavodoxin’s molten globule compared to the other helices. Hydrophobic interactions
    of this helix with the other ordered parts of the molten globule, although loose in nature, cause
    context-dependent stabilization of this helix against unfolding. The helical molten globule contains
    thus a single stable core. Non-native docking of helices in apoflavodoxin’s molten globule prevents
    formation of the parallel β-sheet of native apoflavodoxin. Hence, to produce native α-β parallel
    protein molecules, the off-pathway species needs to unfold.
    Discussion
    Formation of non-native secondary and tertiary structure in unfolded protein is the answer
    to the question: “Why do α-β parallel proteins, like flavodoxins, form misfolded off-pathway
    intermediates?” The presence of non-native secondary structure elements in unfolded proteins is
    probably a widespread phenomenon. However, subsequent formation of folding intermediates that
    contain these non-native structure elements is likely but rarely reported.
    In this thesis, it is proven for the first time that formation of native and non-native helices within
    an unfolded α-β parallel protein and subsequent non-native docking of these structured regions
    leads to formation of a compact helical off-pathway intermediate.
    One of the helices (residues Leu110 to Val125) forms a stable core in the molten globule
    in absence of denaturant. Hydrophobic interactions of this helix with the other ordered parts of
    the molten globule cause its context-dependent stabilization. Non-native docking of the helices
    prevents formation of the parallel β-sheet of native protein. To produce native α-β parallel protein
    molecules, the off-pathway species needs to unfold and as a result non-native interactions and nonnative
    secondary structure are disrupted.
    This thesis shows that acquisition of native-like topology is not necessarily the general result
    of the initial collapse in protein folding. Rather than directing productive folding, conformational
    pre-organization in the unfolded state of an α-β parallel type protein promotes off-pathway species
    formation. The data presented in this thesis indicate that especially proteins that contain domains
    with an α-β parallel topology seem susceptible to off-pathway intermediate formation.
    A single polypeptide sequence can code for monomeric protein folds that are largely different
    under native-like conditions. The amino acid sequence of apoflavodoxin codes for the α-β parallel
    topology of the native state, as well as for a helical protein species. Upon a mild change of conditions,
    topological switching between both folds occurs and a monomeric protein species with a distinct
    fold becomes energetically most favorable. Topological switching between unrelated protein
    structures is likely a general phenomenon in the protein structure universe.
    Structural and functional analysis of Eukaryal-like proteins from the hyperthermophilic archaeon Sulfolobus solfataricus
    Wu Hao, - \ 2007
    Wageningen University. Promotor(en): John van der Oost; Willem de Vos, co-promotor(en): Z. Rao. - [S.l.] : S.n. - ISBN 9789085048220 - 150
    eiwitten - moleculaire structuur - eiwitgebruik - alfa-galactosidase - archaea - proteins - molecular conformation - protein utilization - alpha-galactosidase - archaea
    The research presented in this thesis is aimed at applying technologies in bioinformatics, biochemistry, structural biology and cell biology to reveal the global regulation network in archaea, gain insights in the mechanism of archaeal signal transduction, and provide details on the evolution of the well-conserved archaeal-eukaryal information processing systems (i.e. transcription, translation, and replication). The global regulation network includes several novel core eukaryal-like proteins (e.g. MBF and SsGBP) that are predicted to operate in the regulation of transcription and/or translation in archaea. In addition, approaches are described to analyze the function of the predicted regulators that involved the development of antibiotic resistance marker for hyperthermophiles since the genetic modification of hyperthermophiles has been hampered, at least in part, by the lack of suitable selection markers.
    Structure and dynamics of an essential transmembrane segment of the proton translocation channel of V-ATPase
    Duarte, A.M. - \ 2007
    Wageningen University. Promotor(en): Herbert van Amerongen, co-promotor(en): Marcus Hemminga; Carlo van Mierlo. - [S.l.] : S.n. - ISBN 9789085047377 - 117
    transmembraaneiwitten - moleculaire structuur - micellen - biofysica - transmembrane proteins - molecular conformation - micelles - biophysics
    In the last decades osteoporosis has become a major subject on the field of drug discovery and design. One of the enzymes recently considered important to use as a target for theses drugs is the enzyme H+-VO-ATPase. This proton pump is located in the osteoclast cells, which are positioned at the bone surface. These enzymes control the proton flux to the bone surface and consequently bone resorption. One major task on drug design is the knowledge of the secondary and tertiary structure of the enzyme under study. The topology of the V-ATPase protein complex has been largely established, however, only the three-dimensional structure of some individual subunits is known up to the time this thesis was printed. The work presented in this thesis focuses on the proton translocation channel located in subunit a of the V-ATPase complex. For this purpose, we designed two peptides, consisting of 25 and 37 amino acid residues, representing the seventh transmembrane segment of subunit a that encompass the proton translocation channel as well as the region of interest for possible inhibitors. Using a combination of NMR (nuclear magnetic resonance) and CD (circular dichroism) spectroscopy we analysed the conformation of these V-ATPase peptides in different membrane-mimicking environments: aqueous solutions of SDS (sodium dodecyl sulphate) and amphipols, and the organic solvent DMSO (dimethylsulphoxide). The conformation of the V-ATPase peptides in SDS micelles was studied by CD spectroscopy, however, due to their low solubility NMR spectroscopy turned out to be impossible. The CD results showed that the size of the peptide can drastically alter the solubilization in SDS. For the 37-residue V-ATPase peptide the overall conformation was a-helical and not dependent on the SDS concentration. On the other hand, the conformation of the 25-residue V-ATPase peptide depended on the peptide to SDS ratio changing between an a-helix and β-sheet conformation. As an alternative solubilising agent for the peptides in aqueous solutions we tested amphipols, a new class of macromolecules that were designed to solubilise transmembrane proteins for NMR and X-ray studies. The CD and tryptophan fluorescence spectroscopy results showed that both peptides aggregated in a β-sheet conformation. The formation of these b‑sheets aggregates might result from the interaction of the arginine residue present in the V-ATPase peptides with the anionic polymer. Such an interaction could prevent the peptide from cross­ing the hydrophobic core of the particle, preventing the formation of an a‑he­lix. High-quality high-resolution NMR spectra of the V-ATPase peptides were obtained in DMSO enabling to analyse the atomic structure of the peptides. The use of DMSO on structural studies of transmembrane polypeptides always raised some debate in the literature. This fact motivated us to perform a molecular dynamics study to investigate the solvation of the 25-residue V-ATPase peptide by DMSO. From this work, we show that DMSO can provide both polar and apolar environments to the peptide, making it a good membrane-mimicking organic solvent. The NMR study of the 37-residues peptide enabled us to confirm the a-helical conformation and to predict that the transmembrane spanning region of the seventh transmembrane segment is larger than expected. Instead of 25 transmembrane residues, we propose a transmembrane region of 32 residues. Furthermore, the NMR study of the 25-residue peptide lead us to postulate the existence of a hinge region located near the cytoplasmic end of the channel. It is proposed that the presence of this hinge allows the opening and closing of the proton translocation channel and provides flexibility for the channel to act as a binding pocket for inhibitors. The resulting NMR data sets for the two V-ATPase peptides are deposited in the BioMagResbank (BMRB) under the access numbers 15025 and 6878, and the calculated structure ensemble for the 25-residue peptide is deposited in the PDB databank with entry 2NVJ.
    Distance constraints from site-directed spectroscopy as a tool to study membrane protein structure
    Vos, W.L. - \ 2007
    Wageningen University. Promotor(en): Herbert van Amerongen, co-promotor(en): Marcus Hemminga. - [S.l.] : S.n. - ISBN 9789085046257 - 106
    oppervlakte-eiwitten - moleculaire structuur - spectroscopie - surface proteins - molecular conformation - spectroscopy
    Membrane proteins are involved in nearly every process in the living cell. Their scientific importance cannot be overstated, and they account for nearly 60% of all prescribed drugs. Despite being an abundant and important class of proteins, high-resolution structural data on membrane proteins are relatively scarce. X-ray diffraction and NMR spectroscopy are routinely applied nowadays for the determination of structures of water-soluble proteins. However, for membrane proteins that require an amphipathic environment, there is not yet a well-defined strategy for obtaining the structure. For this reason, techniques based on site-directed labeling are being developed to study membrane proteins in their natural environment. In this work, we use two techniques based on the dipole-dipole interaction between two labels, electron spin resonance (ESR) and fluorescence (or Förster) resonance energy transfer (FRET) to obtain low-resolution (0.3-3 nm) distance information on the structure of membrane peptides. FRET is used to study the conformation of a reference membrane protein, i.e. M13 major coat protein, in fully hydrated vesicles. The FRET-derived distance constraints are used to refine the set of high-resolution structures that is available in the protein databank. We show that the coat protein adopts an extended conformation that is not very different from the conformation in the phage particle. In a separate part of this work, we use the FRET approach to monitor the conformation of the coat protein under conditions of hydrophobic mismatch. Although it was suggested that transmembrane protein domains can adapt their backbone conformation to different conditions of hydrophobic stress and that M13 coat protein is a flexible protein that can adapt to a multitude of environments, we show that the conformation of the coat protein in fact is similar under different conditions of hydrophobic mismatch. A parallel approach, based on ESR spin labeling, is used to study the conformation of a peptide that is derived from the crucial proton translocating domain of vacuolar ATPase. First we present a method to enhance the analysis for the determination of distances between two spin labels based on matrix-assisted laser desorption/ionization - time of flight mass spectrometry. Secondly, we use the data from the ESR experiments to study the structure of the peptide. Based on the combined results from the ESR experiments, molecular dynamics simulations and circular dichroism studies we conclude that the peptide forms a dynamica-helix when bound to SDS micelles. We discuss these findings in the light of the current models for proton translocation in the vacuolar ATPase.
    Supramolecular coordination polymers in water: rings, chains and networks
    Vermonden, T. - \ 2005
    Wageningen University. Promotor(en): Ernst Sudhölter, co-promotor(en): Ton Marcelis. - s.l. : S.n. - ISBN 9789085041481 - 128
    polymeren - moleculaire structuur - synthese - macromoleculen - supramoleculaire chemie - polymers - molecular conformation - synthesis - macromolecules - supramolecular chemistry
    Supramolecular polymers are polymers in which the monomers are held together by non-covalent interactions. In solution these polymers can break and recombine reversibly yielding polymers with an average degree of polymerization. This thesis is devoted to water-soluble coordination polymers, in which the bonds between the monomers are based on metal ion coordination. The most successful ligands used in this research project to construct reversible coordination polymers are based on pyridine-2,6-dicarboxylate groups connected by oligoethylene oxide spacers of different lengths. These ligands were used for the research described in chapters 2, 3 and 4.

    Chapter 2 deals with the formation of water-soluble reversible coordination polymers of Zn 2+ ions with bifunctional ligands that differ in spacer length. Besides linear chains also rings are formed. Viscosity measurements were used to follow the formation of chains and rings as a function of the ratio between metal ions and ligands, the total ligand concentration, and the temperature. To explain the experimental results a theoretical model was developed that accounts for the formation of both chains and rings. At low concentrations and at a 1:1 metal to ligand ratio, a large fraction of the ligand monomers is incorporated in small rings, with a small contribution to the viscosity. Rings are less important at higher concentrations, or if one of the two components is in excess. Also the length of the bifunctional ligands determines the amount of rings that are formed. The largest fraction of rings is found for bifunctional ligands that are just long enough to form a monomer ring around one metal ion. The fractions of monomers in chains and rings could be estimated from 1 H NMR measurements and they are in good agreement with the model predictions. With increasing temperature, the fraction of monomers in rings decreases. As a result, the reduced viscosity increases with increasing temperature.

    In chapter 3, the formation of soluble supramolecular three-dimensional coordination polymers with Nd 3+ and La 3+ in aqueous solution is described for two bifunctional ligands that differ in spacer length.Neodymium(III) ions can bind three terdendate ligand groups.Viscosity measurements were used to monitor the network formation as a function of the ligand concentration and the ratio between metal ions and ligands. For corresponding conditions, solutionscontainingNd 3+ andligands with short spacersgave always much higher viscosities than solutionscontainingNd 3+ and ligands with longer spacers.The ligand with the longer spacer isflexible enough to bind with both chelating groups to only one metal ion (ring-formation). This causes the polymers to stop growing, resulting in smaller average sizes of the three-dimensional polymers. The ring-structures could be demonstrated by 1 H NMR spectroscopy using La 3+ at low concentrations.

    At very high concentrations of the three-dimensional polymers, viscoelastic materials are obtained. The rheology of these reversible coordination polymer networks in aqueous solution is described in chapter 4. The polymers are formed byneodymium(III) ions and bifunctional ligands. The rheological properties of the viscoelastic materials can be described with the Maxwell model. The scaling of the elastic modulus, relaxation time and zero-shear viscosity with concentration are in good agreement with the predictions of Cates' model that describes the dynamics of linear equilibrium polymers. This indicates that the networks have only few cross-links and can be described as linear equilibrium polymers. The gels are also thermo-reversible. At high temperatures, fast relaxation was found, resulting in liquid-like behavior. Upon cooling, the viscoelastic properties returned immediately. From the temperature dependence of the relaxation time,an activationenergy of 49 kJ/mol was determined for the breaking and reptation of the polymers.

    In chapter 5, the syntheses of four different ligand derivatives are described. These ligands are potential candidates for the construction and study of coordination polymers. The different 4-functionalised pyridine-based ligands were synthesized with aminomethyl, oxazolinyl, pyrazolyl and methylimidazolyl groups at the 2- and 6-position, respectively. The nitrogens of these groups together with the pyridine nitrogen can act as terdendate ligands for metal ions. Synthetic handles on the 4-position of the pyridine group were introduced via ether or ester bonds leading to monofunctional, bifunctional and amphiphilic ligands.

    From the four synthesized bifunctional ligands described in chapter 5, only two were stable and soluble enough to study the coordination polymer properties with Zn 2+ . In water, a bifunctional ligand with two 2,6-bis(aminomethyl)pyridine groups as complexing groups was used. In an organic solvent (chloroform/acetonitrile), a bifunctional ligand with two 2,6-bis(methylimidazolyl)pyridine groups was used. The reversibility of the coordination bonds in these two coordination polymers was compared in the solvents mentioned. Viscosity measurements were used to follow the formation and breaking of the polymers as a function of the molar ratio for the ligands with 2,6-bis(methylimidazolyl)pyridine groups in an organic solvent. The breaking of the polymers made of the water-soluble ligands with 2,6-bis(aminomethyl)pyridine groups was shown by addition of monofunctional ligands. Viscosity measurements of the coordination polymers in water showed fast equilibration upon changes in concentration. In an organic solvent no changes in the size of the structures were found upon dilution. 1 H NMR measurements were used to monitor the ring-chain equilibrium of the coordination polymers containing the ligands with 2,6-bis(methylimidazolyl)pyridine groups in a chloroform/acetonitrile mixture. The coordination polymers in the organic solvent showed some exchange, but much slower than in the water-based system. Therefore, to prepare coordination polymers whose properties can be tuned rapidly by means of external changes, water is a more appropriate solvent than non-coordinating organic solvents. 

    Since the best material properties for water-soluble coordination polymers in this thesis are obtained by using metal ions that can act as branch points, further research may be directed to the use of multifunctional ligand molecules as alternative. A first attempt in that direction is described in chapter 7. Two trifunctional ligand molecules with pyridine-2,6-dicarboxylate groups are synthesized and the viscosity of solutions containing mixtures of bifunctional and trifunctional ligands was studied as a function of molar ratio. To increase the viscosity in water by increasing the percentage of trifunctional ligand a very polar but also rather flexible trifunctional ligand is necessary.
    Fundamentals of unfolding, refolding and aggregation of food proteins
    Broersen, K. - \ 2005
    Wageningen University. Promotor(en): Rob Hamer; Fons Voragen, co-promotor(en): Harmen de Jongh. - Wageningen : - ISBN 9789085042501 - 230
    eiwitten - voedsel - aggregatie - chemische structuur - moleculaire structuur - eiwittechnologie - proteins - food - aggregation - chemical structure - molecular conformation - protein engineering
    Protein functionality in food products strongly relies on the fact that proteins can undergo intermolecular interactions, called aggregation. It was found that very subtle dynamics inherent to the protein of interest can have consequences for the functional properties of proteins. The aim of this thesis is to explore structural features of proteins of importance to the generation of aggregation prone protein molecules. The approach selected involves chemical engineering in which functional groups of the protein are converted into a chemical group with different properties. This led to a detailed description of the structural impact of the modifications in relation to aggregate formation. It was found that the various modifications applied interact with the aggregation process in a rather diverse (but predictable) manner. The accumulation of data from this work in combination with results from literature was used to significantly improve the understanding of factors relevant to aggregation and to develop a model to predict aggregation propensity. This model can be used within the food and pharmaceutical industry to determine the aggregation propensity of proteins used in formulae and medication.
    Mean-field stationary diffusion: polymers in steady-state systems
    Scheinhardt-Engels, S.M. - \ 2004
    Wageningen University. Promotor(en): Gerard Fleer, co-promotor(en): Frans Leermakers. - [s.I.] : S.n. - ISBN 9789085040613 - 165
    polymeren - moleculaire structuur - membranen - diffusiecoëfficiënt - diffusieweerstand - polymers - molecular conformation - membranes - diffusivity - diffusion resistance
    Surfing the free energy landscape of flavodoxin folding
    Bollen, Y.J.M. - \ 2004
    Wageningen University. Promotor(en): Sacco de Vries, co-promotor(en): Carlo van Mierlo. - [S.l.] : S.n. - ISBN 9789085040644 - 165
    eiwitten - moleculaire structuur - azotobacter vinelandii - proteins - molecular conformation - azotobacter vinelandii

    The research described in this thesis has been carried out to obtain a better understanding of the fundamental rules describing protein folding. Protein folding is the process in which a linear chain of amino acids contracts to a compact state in which it is active. Flavodoxin from Azotobacter vinelandii is chosen as the representative of the group of OC-? parallel proteins. Flavodoxins are small monomeric proteins that contain a non-covalently bound FMN cofactor. The ?-? parallel topology is characterised by a five-stranded parallel ?-sheet surrounded by ?-helices at either side of the sheet. The doubly-wound topology is a rather popular fold: it belongs to the five most common observed folds, together with the ???-barrel, Rossman, thiamin-binding and P-loop hydrolase folds. In contrast to most protein folds, this topology is shared by many (i.e. nine) protein superfamilies. These nine superfamilies exhibit little or no sequence similarity and comprise a broad range of unrelated proteins with different functions like catalases, chemotactic proteins, lipases, esterases, and flavodoxins. By studying the folding behaviour of ?. vinelandii flavodoxin insight can be gained into how this large group of proteins folds.

    First, the equilibrium (un)folding of apoflavodoxin from ?. vinelandii (i.e. flavodoxin in the absence of the FMN cofactor) is investigated. Apoflavodoxin is structuraUy identical to holoflavodoxin except for some dynamic disorder in the flavin-binding region. A molten globule-like intermediate is shown to populate during denaturant-induced equilibrium unfolding of apoflavodoxin (Chapter 2).

    Subsequently, the folding and unfolding kinetics of the 179-residue A. vinelandii apoflavodoxin have been followed by stopped-flow experiments monitored by fluorescence intensity and anisotropy (Chapter 2). The denaturant concentration dependence of the folding kinetics is complex. Under strongly unfolding conditions, the kinetics can be described by a single rate constant. When this unfolding rate constant is plotted against the denaturant concentration, a change in the slope is observed. This, together with the absence of an additional unfolding process reveals the presence of two consecutive transition states on a linear pathway that surround a high-energy on-pathway intermediate.

    Under refolding conditions, two folding processes are observed. The slowest of these two processes is the one that is populated most, and it becomes faster with increasing denaturant concentration. This means that an unfolding step is rate-limiting for folding of the majority of apoflavodoxin molecules. This, together with the absence of a 1ag in the formation of native molecules, means that the intermediate that populates during refolding is off-pathway.

    The experimental data obtained on apoflavodoxin folding are consistent with the linear four-state folding mechanism I1 "=>unfolded apoflavodoxin t=>I2<=>native apoflavodoxin. The off-pathway intermediate I1 is the one that populates during refolding and that also populates during denaturant-induced equilibrium unfolding of apoflavodoxin. I2 is the unstable intermediate that is observed during kinetic unfolding.

    The presence of such on-pathway and off-pathway intermediates in the folding kinetics of proteins with an ?-? parallel topology is predicted from simulations of Go-like protein models. In addition, two kinetic folding intermediates, one on-pathway and the other off-pathway, seern to be present under specific experimental conditions during the folding of all proteins with an ?-? parallel topology that have been investigated. The appearance of folding intermediates in this class of proteins is apparently governed by protein topology (Chapter 3).

    Next, the local dynamics of apoflavodoxin have been studied by hydrogen deuterium exchange detected by heteronuclear NMR spectroscopy (Chapter 4). The use of native state hydrogen deuterium exchange detected by NMR spectroscopy leads to the identification of four partially unfolded forms (PUFs) of apoflavodoxin in which some non-native interactions apparently play a role. The rates of interconversion of these PUFs with native apoflavodoxin are determined. These rates are inconsistent with the PUFs being on a direct folding route between native and globally unfolded apoflavodoxin. PUFl and PUF2 are on an unfolding route starting from native apoflavodoxin that does not 1ead to the globally unfolded state of the protein. PUF3 and PUF4 are on a non-productive folding route starting from globally unfolded apoflavodoxin. A common free energy barrier separates both PUF3 and PUF4 from unfolded apoflavodoxin. This barrier has the same height as the one determined from stopped-flow kinetic folding studies that separates the known off-pathway apoflavodoxin folding intermediate I1 from the productive folding route. Therefore a single energy barrier is proposed to separate both PUF3 and PUF4 as well as I1 from the productive folding route. All three species thus need to unfold before productive folding of apoflavodoxin can occur (Chapter 4).

    The influence of the presence of the non-covalently bound flavin mononucleotide (FMN) cofactor on the global stability and on the kinetic folding of A. vinelandii holoflavodoxin (i.e. flavodoxin in presence of the FMN co-factor) are reported in Chapter 5. The denaturant-induced equilibrium (un)folding data of flavodoxin in the presence and absence of FMN are excellently described by a model in which only native apoflavodoxin binds to FMN. As the intermediate I1 populates during apoflavodoxin equilibrium (un)folding, the holoflavodoxin equilibrium (un)folding model consists of four species: unfolded apoflavodoxin, the apoflavodoxin folding intermediate I1, native apoflavodoxin and holoflavodoxin molecules. Cofactor binding to apoflavodoxin is shown to affect the protein stability in a theoretically predictable manner.

    Despite that many proteins require the binding of a ligand to be functional, the kinetic role of ligand-binding during folding is poorly understood. FMN binding to native apoflavodoxin occurs with two kinetically observable rate constants at all denaturant and protein concentrations studied, as is shown in Chapter 5. These two rate constants arise from two conformationally differing apoflavodoxin species, which most likely exist due to the binding of inorganic phosphate to the FMN phosphate binding site of a fraction of the A. vinelandii apoflavodoxin molecules.

    In Chapter 5 it is also shown that excess FMN does not accelerate flavodoxin folding, and FMN does not act as a nucleation site for flavodoxin folding. During kinetic folding of holoflavodoxin formation of native apoflavodoxin precedes ligand binding. Even under strongly denaturing conditions, global unfolding of holoflavodoxin occurs only after release of its FMN. The model that describes A. vinelandii apoflavodoxin kinetic folding, which includes the stable off-pathway intermediate I1 and a high-energy on-pathway intermediate I2, can now be extended to describe kinetic holoflavodoxin folding: I, + FMN<=>unfolded apoflavodoxin + FMN<=>I2 + FMN<=>native apoflavodoxin + FMN<=>holoflavodoxin (Chapter 5).

    Finally, in Chapter 6 native state WD exchange combined with NMR spectroscopy is used to probe the influence of FMN binding on the stability of A, vinelandii flavodoxin against local, subglobal and global unfolding. Almost the entire flavodoxin backbone is substantially more rigid in holoflavodoxin than in apoflavodoxin. No areas are detected in flavodoxin where FMN binding results in an increase of the local dynamics. Occasional release of FMN from holoflavodoxin results in the population of apoflavodoxin. Until FMN is rebound, these apoflavodoxin molecules behave as described in Chapter 4. Consequently, they will adopt the previously described partially unfolded forms (PUFs). At least three out of the four partially unfolded forms that apoflavodoxin occasionally adopts under native conditions are inaccessible to holoflavodoxin. Holoflavodoxin can form these partially unfolded conformations only when FMN is released.

    AIl observations described in this thesis are used to create a schematic free energy landscape of folding ofA. vinelandii flavodoxin. This schematic energy landscape provides insight into how a protein molecule that adopts the ?-? parallel topology surfs from its unfolded state to its characteristic folded state in which it is active.

    Outlook

    As described in Chapter 3 of this thesis, the appearance of both on- and off-pathway intermediates during the folding ofA. vinelandii apoflavodoxin appears to be governed by its ?-? parallel topology. Folding kinetics of other ?-? parallel proteins than the ones mentioned in this thesis need to be determined to verify this hypothesis.

    An interesting question is why intermediate I1 that A. vinelandii apoflavodoxin populates during its denaturant-mduced equilibrium (un)folding is off the direct folding route. This question may be resolved by studying the structure of intermediate I1 using among others multidimensional NMR experiments. In addition, the investigation of possible residual structure within unfolded apoflavodoxin can inform about the origin of the kinetic partitioning of folding apoflavodoxin molecules into two routes, one leading to native apoflavodoxin, the other one leading to the molten globule-like intermediate I1.

    The kinetic model for A. vinelandii apoflavodoxin folding presented in this thesis implies that apoflavodoxin molecules once they have formed the intermediate I1 need to unfold before folding to the native state can proceed. Studying the folding behaviour of single A. vinelandii apoflavodoxin molecules using sensitive fluorescence techniques can reveal to what extent an apoflavodoxin molecule has to unfold in the latter process. To date, only the folding kinetics of small proteins that fold in one step have been studied by single molecule detection techniques. Studying the folding ofindividual apoflavodoxin molecules can reveal the general dynamics involved in the partitioning of individual protein molecules into two separate folding trajectories.

    Finally, it will be highly interesting to study the folding behaviour of proteins in their natural environment. As pointed out in the first chapter of this thesis, the high concentration of biomacromolecules in cells is bound to influence the latter behaviour. Therefore, studying the influence macromolecular crowding agents have on folding flavodoxin molecules will be of great interest. In this thesis, a solid, and strongly necessary, basis is laid for the future perspective of the in vivo investigation of flavodoxin folding in the living cell.

    Detailed characterization of adsorption-induced protein unfolding
    Engel, M.F.M. - \ 2004
    Wageningen University. Promotor(en): Ton Visser; Sacco de Vries, co-promotor(en): Carlo van Mierlo. - [S.l.] : S.n. - ISBN 9789085040019 - 125
    runderserumalbumine - alfa-lactalbumine - moleculaire structuur - grensvlak - fysische eigenschappen - adsorptie - spectroscopie - bovine serum albumin - alpha-lactalbumin - molecular conformation - interface - physical properties - adsorption - spectroscopy
    Physical Modeling of microtubule force generation and self-organization
    Tanase, C. - \ 2004
    Wageningen University. Promotor(en): Bela Mulder, co-promotor(en): M. Dogeterom. - [S.l.] : S.n. - ISBN 9789085040217 - 158
    microtubuli - chemische structuur - moleculaire structuur - fysicochemische eigenschappen - microtubules - chemical structure - molecular conformation - physicochemical properties

    Biological systems are complex heterogeneous and far from equilibrium systems. The fundamental questions posed by the physics of such systems are what the force generation mechanisms are, and how energy is processed and distributed among the components inside them. In answering these questions we can understand how motion is generated and how the system is organized, which means a significant step toward grasping these systems in their full complexity. A systematic program means first the identification of the components, and studying its properties and interplay with other components. How these components integrates into a higher level of organization, comes as a secondary step.

    The cytoskeleton is a key ingredient of the living cell. The cytoskeleton is a complex of biopoly-mers which self-assembles and organize inside the living cells. There are many important functions that cytoskeleton fulfills. One is to give shape and rigidity to the cell, another is that cytoskeletal biopolymers serves as tracks for material transport across the cell. The examples could continue with the locomotion of cell, which is possible only due to the rearrangement of the cytoskeleton.

    This thesis is concerned with the physical aspects of microtubules, which represent a part of the cytoskeleton. Microtubules are tubular protein aggregates, which are particularly stiff. These biopolymers were originally discovered as the scaffold of the mitotic spindle, which is the cell division apparatus that separates the genetic material among the daughter cells. An important property of microtubules is the alternation between growing and shrinking states, a behavior which is termed as dynamic instability and make microtubules unique in the realm of polymers. It is precisely this property that makes possible for microtubules to be involved in multi scale dynamics, i.e. assembly-disassembly and organization.

    In making the time scale separation, some particular aspects of microtubule assembly and organization are presented and analyzed in two different parts of this thesis. The attention is focused on growing microtubules only, i.e. the dynamic instability does not play any role in the processes that are considered.

    In the first part of this thesis, it is investigated in detail the microtubule force production mechanism during self-assembly. In general, any polymer can generate force during polymerization. If the seed of the polymer is fixed, then polymer can push against an arbitrary object, if the insertion of subunits are allowed due to gap opening between the tip of the polymer and the corresponding object. The required gap openings are possible due to the thermal fluctuations, and it is due to this reason that the object that generates force by exploiting the thermal fluctuations is called Brownian ratchet. This particular type of motor does not contradicts the second law of thermodynamics, which forbids work production in isothermal systems. The problem is avoided as the system is out of equilibrium. In our example of the polymerization ratchet, the dynamics is driven by the chemical polymerization energy, which is simply converted into work by the Brownian ratchet mechanism itself. Microtubules that work as Brownian ratchets can be regarded as a particular type of a molecular (nano-)motor.

    In Chapter 3, the concept of Brownian ratchet is applied to microtubules. The main feature which is incorporated to this concept is the collective character of the microtubule growth, since these polymers are composed of many filaments. One important question is to investigate what is the maximum force that this particular type of molecular motor can generate. A second question is to see how the velocity of growth depends on the opposing force that an external object can exert. Does the velocity of growth depend on the relative arrangements of microtubule protofilaments inside the assembly? In other words, given its internal structure is there a optimal way that the microtubule can grow under load condition? The way that the investigation is carried out is that the model details are extracted with the help of computer simulations, and compared directly with experimental data.

    In Chapter 4, different regimes of microtubule growth are considered. Quantitative comparisons with available experimental data are successful in all cases, but a large number of free parameters justifies the need for different experiments. However, some qualitative aspects, such as the microtubule end structure can limit the number of possibilities, since end details were already observed in experiments. More exactly, cryo-electron microscope images show that microtubuls develop open sheets like structures at their end during growth. The disappearance of these structures is correlated in experiments with a hypothetical switch mechanism that triggers dynamic instabilities. Therefore, it appears natural to expect that a realistic growth model should reproduce such end structures. The model suggests that there is a sensitive relationship between the size of these structures and both the kinetic rates and the strength of the lateral bonds between protofilaments. Although the comparison with experiments is not fully quantitative, the analysis suggests that it is likely that the lateral bonding between the protofilaments is relatively week, Le. a couple of thermal energies kg T per subunit.

    In the second part of the thesis, I discuss some physical aspects regarding the organization of microtubules. In general, not referring only to microtubules, the importance of understanding the cytoskeleton organization is manifold. From physical point of view, the questions that are addressed in this thesis belong to the much broader context of pattern formation in far from equilibrium systems. Here, the fundamental problem is to find the relationship between the macroscopic properties of organized dissipative systems and their microscopic details that drive the sys-tem out of thermodynamic equilibrium. From the biological point of view, the investigation of the cytoskeleton organization is tightly related to understanding the biological functional role that different biopolymer arrangements assume in living cells.

    In Chapter 5, the attention is focused on the microtubule organization in higher plant cells. Particularly, the microtubule arrangements that appear in interphase cells or prior to their division have received a lot of attention from biologists in the past, but still little is known about the driving organization mechanism. In interphase plant cells, the microtubules organize on the cortex of the cell in a parallel array, which is oriented transversely to the main axis of the cell. Just before the onset of the division, this array narrows to a preprophase band which marks on the cortex the location of the separation wall between the daughter cells. From physical point of view, in this chapter is addressed the question if it is possible that passive factors could be responsible for such organized arrays. One possibility in this respect is the nematic transition driven by excluded volume interaction, which is a well known phenomenology from the physics of liquid crystals. This implies a direct relationship between the degree of ordering and the density of microtubules. A second possibility is that bending elasticity of microtubules is the driving factor for organizing microtubules on the cortex. Since the bending elasticity is an intrinsic property of microtubules, the organization in this case can be termed more exactly as se/f-organization.

    Active factors are the best candidates in driving large scale patterns in filamentous systems. In the past, the ability of motor proteins to organize filaments is demonstrated in both experiments and computer simulations. However, understanding the phase diagram remains an open theoretical problem. Based on phase diagram analysis, a minimal set of conditions can be derived in order to reproduce a particular phenomenology. In the last two chapters, two different approaches are adopted. In Chapter 6, a mean field Landau type theory is developed. In this case, the phenomenology of filamentous systems is described with no reference to microscopic details and the basic constrains, which are imposed, are the symmetries that the physical system is supposed to fulfill. This generic method reproduce the possibility of a transverse stripe that closely resembles the preprophase band in plant cells. This encouraging result suggests that cytoskeletal array like those observed in plant cells can be described by a mean field theory.

    In Chapter 7, a microscopic model is introduced, and based on this I derive the macroscopic evolution equations. The procedure is meant to meet the results that are derived in the generic approach, which is presented in Chapter 6, Besides the active components, I introduced also the passive interaction due to steric exclusion between filaments. The passive components alone are responsible for isotropic-nematic instabilities at high density, which drive the system to a liquid crystalline ordered phase. However, the active components can drive pattern formation in this system at densities that are below the critical value that corresponds to passive driven instabilities. The study in this chapter is limited at the level of linear stability analysis. However, the obtained results suggest that the stable arrays might be homogeneous nematic polar patterns, vortices, and asters. These features are consistent with the results obtained from other methods, like computer simulations or in vitro experiments, which are present in the literature. A full understanding of the emergent patterns requires the consideration of non-linear terms in evolution equations, which is the objective of future projects.

    Mechanical and conformational aspects of protein layers on water
    Martin, A.H. - \ 2003
    Wageningen University. Promotor(en): Martien Cohen Stuart, co-promotor(en): M.A. Bos; Ton van Vliet. - [S.I.] : S.n. - ISBN 9789058088109 - 126
    bovenlagen - moleculaire structuur - schuim - eiwitten - schuifsterkte - reologie - surface layers - molecular conformation - foams - proteins - shear strength - rheology

    Keywords: protein film, protein conformation, air/water interface, network formation, foam formation, foam stability, interfacial rheology, fracture behaviour.

    The aim of this thesis was to obtain systematic information on the importance of mechanical and conformational aspects for the formation of a visco-elastic protein network at the air/water interface. Such a protein network is formed upon adsorption at the interface and is assumed to play a role in the formation and stabilisation of emulsions and foams. To understand the formation of a visco-elastic layer with specific mechanical properties, one has to study the molecular processes occurring at the interface, namely protein adsorption, conformational changes that occur upon adsorption and the interactions between the adsorbed proteins. A series of proteins was studied with a tertiary structure varying from random coil (flexible) to rigid (globular):b-casein,b-lactoglobulin, ovalbumin and (soy) glycinin. Glycinin has only been studied preliminary in the past but, being an interesting substitute for animal proteins, it was investigated quite extensively in this thesis. The conformation of glycinin was found to be pH-dependent and this change in conformation strongly affected the adsorption behaviour and rheological properties of interfacial glycinin layers. The monomeric glycinin form present at pH 3 behaved as a good foaming agent whereas at pH 6.7 (hexamer form) no foam could be formed. Infrared Reflection Absorption Spectroscopy (IRRAS) showed that only minor changes occurred in the secondary structure of a protein upon adsorption at the interface. Ovalbumin andb-lactoglobulin showed a 10% loss ofb-sheet structures whereas glycinin (pH 3) formed intermolecular anti-parallelb-sheets. The latter is an indication for interfacial aggregation. Mechanical properties were determined by deformation in shear and dilation. Upon large deformations most protein films were found to exhibit fracture behaviour. The differences observed for ovalbumin,b-lactoglobulin and glycinin indicated a transition from a more yielding behaviour to a more brittle fracture behaviour. A correlation was found between several mechanical properties of adsorbed protein films and the stability against disproportionation of foams made with the corresponding proteins. Furthermore, correlations between macroscopic film properties and molecular properties of the proteins in terms of molecular dimensions and secondary structure were studied. It was discovered that the molecular area at the onset of surface pressure per unit protein molecular weight was strongly correlated to the steady-state shear stress of a saturated protein film. This means that protein 'hardness' largely determines the film properties but a quantitative model is yet to be developed. Practical relevance of the mechanical properties of adsorbed protein layers for the stability of emulsions and foams is discussed.

    Self-consistent-field theory for chain molecules: extensions, computational aspects, and applications
    Male, J. - \ 2003
    Wageningen University. Promotor(en): Gerard Fleer, co-promotor(en): Frans Leermakers. - [S.l.] : s.n. - ISBN 9789058087799 - 184
    polymeren - moleculaire structuur - thermodynamica - polymers - molecular conformation - thermodynamics - cum laude
    cum laude graduation (with distinction)
    Exploring conformational dynamics of flavoenzymes with flavin fluorescence relaxation spectroscopy
    Berg, P.A.W. van den - \ 2002
    Wageningen University. Promotor(en): A.J.W.G Visser; N.C.M. Laane. - S.l. : S.n. - ISBN 9789058086945 - 223
    amine oxidoreductasen - fluorescentie-emissiespectroscopie - moleculaire structuur - amine oxidoreductases - fluorescence emission spectroscopy - molecular conformation

    Research described in this thesis was aimed at gaining more insight into the active-site dynamics of dimeric flavoproteins by means of fluorescence relaxation spectroscopy. Three flavoproteins for which crystallographic data have suggested different types of functionally important motions were chosen as central systems; E. coli glutathione reductase, which displays a local conformational change in the protein environment; E. coli thioredoxin reductase, for which a major domain rotation was proposed to be essential for catalysis; and P. fluorescens p- hydroxybenzoate hydroxylase, in which the isoalloxazine ring of the flavin cofactor itself is mobile during catalysis. For interpretation of fluorescence data in terms of dynamic events in the proteins, explicit attention was paid to the photophysical and dynamic characteristics of the flavin cofactor.

    Chapter 1 provides a general introduction into the enzyme systems and into the principles and mechanisms of conformational dynamics and fluorescence relaxation spectroscopy.

    In Chapter 2, the dynamic properties of wild-type E. coli glutathione reductase (GR) are studied in comparison with those of the mutant enzymes GR Y177F and GR Y177G. Emphasis is laid on the relations between fluorescence lifetime patterns, protein dynamics and the mechanisms for fluorescence quenching in proteins. Experimental evidence is provided for the multiple quenching sites model.

    The implications of the comparative study on the gluthatione reductase enzymes for the interpretation of time-resolved fluorescence anisotropy decays are described in Chapter 3 , where a new mechanism for flavin fluorescence depolarization is proposed.

    Chapter 4 focuses on the conformational dynamics of E. coli thioredoxin reductase (TrxR) and the mutant enzyme TrxR C138S. Two catalytically important conformational states of the enzyme are detected and characterized by (sub)picosecond time-resolved and spectrally resolved fluorescence techniques. Flavin fluorescence relaxation experiments are combined with steady-state optical techniques to gain insight into the dynamic properties of the enzyme and the conformational equilibrium. The importance of enlarging the time window for the fluorescence detection of dynamic events is discussed.

    The mobile flavin in p- hydroxybenzoate hydroxylase (PHBH) is subject of a time-resolved fluorescence investigation in Chapter 5 . Different binary (mutant) enzyme/substrate (analogue) complexes are used to direct the conformation of the cofactor. The chapter reflects on possibilities and limitations of ensemble fluorescence lifetime data for studying protein dynamics.

    In Chapter 6 , a link is created between time-resolved fluorescence data of ensembles of molecules and the molecular dynamics of single molecules as retrieved from molecular dynamics (MD) simulations. Hereto, the system of investigation is simplified to the FAD cofactor, which can exist in both 'open' and 'closed' conformations. MD simulations provide insight into the dynamic behaviour of the free cofactor and into pathways for conformational transitions.

    Chapter 7 describes the first steps into the world of single-molecule detection through natural flavin fluorescence. Fluorescence Correlation Spectroscopy studies on FAD, FMN and lipoamide dehydrogenase provide a first glance into the future perspectives of detecting single flavoproteins and give an understanding of the specific obstacles that need to be overcome.

    The thesis is concluded by a summarizing discussion reflecting on the research described in this thesis in relation to developments in the field.

    The relationship between the molecular structure and ion adsorption on goethite
    Rietra, R.P.J.J. - \ 2001
    Wageningen University. Promotor(en): W.H. van Riemsdijk. - S.l. : S.n. - ISBN 9789058085030 - 117
    goethiet - adsorptie - ionen - moleculaire structuur - goethite - adsorption - ions - molecular conformation

    Keywords:

    Ion adsorption modeling, goethite, iron oxide, CD-MUSIC, phosphate, arsenate, vanadate, molybdate, tungstate, sulfate, selenate.

    A study is presented on the adsorption of inorganic ions on goethite with emphasis on the adsorption of oxyanions. Experimental results for a range of oxyanions (PO4, AsO4, VO4, WO4, MoO4, CrO4, SeO3, SeO4, SO4, Cl, NO3, ClO4) and Ca are presented and interpreted using the CD-MUSIC model. For some of these ions the coordination and structure of the adsorbed ions on goethite are known from spectroscopy (SO4, SeO4, PO4, AsO4, SeO3). Ideally, surface complexes derived from spectroscopy correspond with those resulting from the modeling of macroscopic adsorption data. This would assure that the mechanistic description of ion binding scales from the microscopic molecular structure to the macroscopic adsorption behavior. In the CD-MUSIC model it is assumed that the charge of the adsorbed ions is distributed at the interface as a function of the coordination and structure of the adsorbed ions and that this distribution of charge can be estimated using the bond valence concept of Pauling. In this study it is found that the macroscopic proton-ion adsorption stoichiometry is almost solely determined by the interfacial charge distribution of adsorbed complexes. It is shown that the experimentally determined proton-ion adsorption stoichiometry can be predicted on the basis of the spectroscopically identified structures of sulfate, selenite, phosphate and arsenate on goethite. By doing so a direct relationship is demonstrated between the molecular structure of adsorbed ions and macroscopic adsorption phenomena. By using this knowledge it is in principle possible to identify the structure and coordination of adsorbed complexes from the macroscopic adsorption data and vice versa. It is found that the spectroscopically suggested differentiation between inner- and outersphere complexes of sulfate and selenate, and the differentiation between bidenate and monodentate phosphate can be modeled satisfactory with the CD-MUSIC approach although the differentiation cannot be established solely from the available adsorption data. It is also found that the proton adsorption on goethite decreases in electrolyte solutions of NaCl, NaNO3 and NaClO4 (below the PZC) in the order Cl>NO3>ClO4 while sulfate and phosphate adsorption is lower in the order Cl<NO3<ClO4. These results can be explained well by assuming outersphere complexes of the electrolyte anions on the goethite surface with different intrinsic affinities.

    Enzymatic hydrolysis of [beta]-casein and [beta]-lactoglobulin : foam and emulsion properties of peptides in relation to their molecular structure
    Caessens, P.W.J.R. - \ 1999
    Agricultural University. Promotor(en): A.G.J. Voragen; H. Gruppen; S. Visser. - S.l. : S.n. - ISBN 9789058080073 - 133
    hydrolyse - caseïne - lactoglobulinen - colloïdale eigenschappen - moleculaire structuur - hydrolysis - casein - lactoglobulins - colloidal properties - molecular conformation

    Peptides derived fromβ-casein (βCN) andβ-lactoglobulin (βLg) were analysed for their foam- and emulsion-forming and -stabilising properties (further denoted functional properties) and for their structural characteristics in order to elucidate structure-function relationships.

    βCN was hydrolysed by plasmin and subsequent fractionation of the hydrolysate resulted in various hydrophilic, amphipathic and hydrophobic peptide fractions with clear differences in functional properties. The highly-charged N-terminal part of the amphipathic peptides appeared to be important for the emulsion-stabilising properties ofβCN peptides. The main secondary structure element ofβCN(-peptides) in solution was the unordered random coil, but upon adsorption onto an hydrophobic interfaceα-helix was induced. The hydrophobic C-terminal part ofβCN accounted for the high maximum surface load on the interface, while the N-terminal part ofβCN seemed to be responsible for theα-helix induction upon adsorption. No clear relation between the secondary structure and the functionality was observed in this system but a relation between a high surface load and good stabilising properties seemed to exist.

    BovineβLg was hydrolysed by the action of trypsin, plasmin and Staphylococcus aureus V8 protease. Overall, the plasmin hydrolysate had the best functional properties at pH 6.7, compared to the other hydrolysates and was investigated further. DuringβLg/plasmin hydrolysis significant SH/SS-exchange has taken place yielding a large number of different peptides. The peptides present were (1) peptides composed of a single amino acid chain lacking a cysteine residue, (2) peptides composed of a single amino acid chain containing intramolecular disulphide bonds and (3) peptides composed of 2 amino acid chains linked by an intermolecular disulphide bond. The occurrence of the SH/SS exchange and the homogeneous distribution of charge and hydrophobicity hinder an efficient fractionation of the hydrolysate.

    In conclusion, the production of specific peptides and peptide fractions is more complicated forβLg than forβCN, mainly because of the differences in primary structure (such as the distribution of charge and hydrophobicity) between the proteins. The foam- and emulsion-forming properties of peptides can be superior to those of intact proteins, as long as they have both charged and hydrophobic areas. The foam- and emulsion-stabilising properties of peptides depend highly on the amount of repulsion they can produce (either by a strong amphipathicity or by a high surface load).

    Function, mechanism and structure of vanillyl-alcohol oxidase
    Fraaije, M.W. - \ 1998
    Agricultural University. Promotor(en): N.C.M. Laane; W.J.H. van Berkel. - S.l. : Fraaije - ISBN 9789054858287 - 182
    oxidoreductasen - chemische structuur - moleculaire structuur - reactiemechanisme - oxidoreductases - chemical structure - molecular conformation - reaction mechanism

    Lignin is a heterogeneous aromatic polymer formed by all higher plants. As the biopolymer lignin is a major constituent of wood, it is highly abundant. Lignin biodegradation, an essential process to complete the Earth's carbon cycle, is initiated by action of several oxidoreductases excreted by white-rot fungi. The resulting degradation products may subsequently be used by other microorganisms. The non-lignolytic fungus Penicillium simplicissimum can grow on various lignin metabolites. When this ascomycete is grown on veratryl alcohol, a major lignin metabolite, production of an intracellular aryl alcohol oxidase is induced. Purification and initial characterization revealed that this enzyme is able to oxidize vanillyl alcohol into vanillin and was therefore named: vanillyl-alcohol oxidase (VAO). Furthermore, it was found that VAO is a homooctamer of about 500 kDa with each subunit containing a covalently bound 8a-( N3-histidyl)-FAD redox group. As VAO showed some interesting catalytical and structural features, a PhD-project was started in 1993 with the aim of elucidating its reaction mechanism.

    In the initial stage this PhD-project, it was found that VAO has a rather broad substrate specificity. However, it was unclear which substrates are of physiological relevance. In a recent study, evidence was obtained that 4-(methoxymethyl)phenol represents a physiological substrate (Chapter 2). When the fungus is grown on 4-(methoxymethyl)phenol, VAO is expressed in large amounts, while the phenolic compound is fully degraded. HPLC analysis showed that VAO catalyzes the first step in the degradation pathway of 4-(methoxymethyl)phenol (Fig. 1).

    Figure 1. Degradation pathway of 4-methoxymethyl)phenol in Penicillium simplicissimum.

    This type of reaction (breakage of an ether bond) is new for flavoprotein oxidases. Furthermore, 4-(methoxymethyl)phenol has never been described in the literature as being present in nature. Yet, it can be envisaged that this phenolic compound is formed transiently during the biodegradation of lignin, a biopolymer of phenolic moieties with many ether bonds.

    Concomitant with the induction of VAO a relatively high level of catalase activity was observed. A further investigation revealed that P. simplicissimum contains at least two hydroperoxidases both exhibiting catalase activities: an atypical catalase and a catalase-peroxidase (Chapter 3). Purification of both enzymes showed that the periplasmic atypical catalase contains an uncommon chlorin-type heme as cofactor. The intracellular catalase-peroxidase represents the first purified dimeric eucaryotic catalase-peroxidase. So far, similar catalase-peroxidases have only been identified in bacteria. These procaryotic hydroperoxidases show some sequence homology with cytochrome c peroxidase from yeast which is in line with their peroxidase activity. EPR experiments revealed that the catalase-peroxidase from P. simplicissimum contains a histidine as proximal heme ligand and thereby can be regarded as a peroxidase-type enzyme resembling the characterized procaryotic catalase-peroxidases.

    In Chapter 4, the subcellular localization of both VAO and catalase-peroxidase in P. simplicissimum was studied by immunocytochemical techniques. It was found that VAO and catalase-peroxidase are only partially compartmentalized. For both enzymes, most of the label was found in the cytosol and nuclei, while also some label was observed in the peroxisomes. The similar subcellular distribution of both oxidative enzymes suggests that catalase-peroxidase is involved in the removal of hydrogen peroxide formed by VAO. The VAO amino acid sequence revealed no clear peroxisomal targeting signal (PTS). However, the C-terminus consists of a tryptophan-lysine-leucine (WKL) sequence which resembles the well-known PTS1 which is characterized by a C-terminal serine-lysine-leucine (SKL) consensus sequence.

    Soon after the start of the project, it was discovered that, aside from aromatic alcohols, VAO also converts a wide range of other phenolic compounds, including aromatic amines, alkylphenols, allylphenols and aromatic methylethers (Chapter 5). Based on the substrate specificity (Fig. 2) and results from binding studies, it was suggested that VAO preferentially binds the phenolate form of the substrate. From this and the relatively high pH optimum for turnover, it was proposed that the vanillyl-alcohol oxidase catalyzed conversion of 4-allylphenols proceeds through a hydride transfer mechanism involving the formation of a p -quinone methide intermediate.

    Figure 2. Reactions catalyzed by VAO.

    In Chapter 6, the kinetic mechanism of the oxidative demethylation of 4-(methoxymethyl)phenol was studied in further detail using the stopped-flow technique. It was established that the rate-limiting step during catalysis is the reduction of the flavin cofactor by the aromatic substrate (Fig. 3). Furthermore, it was found that during this step a binary complex is formed between the reduced enzyme and a product intermediate. Spectral analysis revealed that the enzyme-bound intermediate is the p -quinone methide form of 4-(methoxymethyl)phenol. Upon reaction of this complex with molecular oxygen, the final product is formed and released in a relatively fast process. Using H218O, we could demonstrate that, upon flavin reoxidation, water attacks the electrophilic quinone methide intermediate to form the aromatic product 4-hydroxybenzaldehyde.

    Figure 3. Reaction mechanism for the oxidative demethylation of 4-(methoxymethyl)phenol.

    In Chapter 7, the enantioselectivity of VAO was investigated. VAO catalyzes the enantioselective hydroxylation of 4-ethylphenol, 4-propylphenol and 2-methoxy-4-propylphenol with an ee of 94% for the R-enantiomer. Isotope labeling experiments confirmed that the oxygen atom incorporated into the alcoholic products is derived from water. During the VAO-mediated conversion of short-chain 4-alkylphenols, 4-alkenylic phenols are produced as well. The reaction of VAO with 4-alkylphenols also results in minor amounts of phenolic ketones which is indicative for a consecutive oxidation step.

    Also the kinetic mechanism of VAO with 4-alkylphenols was studied (Chapter 8). For the determination of kinetic isotope effects, Ca-deuterated analogues were synthezised. Interestingly, conversion of 4-methylphenol appeared to be extremely slow, whereas 4-ethyl- and 4-propylphenol were rapidly converted. With these latter two substrates, relatively large kinetic deuterium isotope effects on the turnover rates were observed indicating that the rate of flavin reduction is rate-limiting. With all three 4-alkylphenols, the process of flavin reduction was reversible with the rate of reduction being in the same range as the rate of the reverse reaction. With 4-ethylphenol and 4-propylphenol, a transient intermediate is formed during the reductive half-reaction. From this and based on the studies with 4-(methoxymethyl)phenol, a kinetic mechanism was proposed which obeys an ordered sequential binding mechanism. With 4-ethylphenol and 4-propylphenol, the rate of flavin reduction determines the turnover rate, while with 4-methylphenol, a step involved in the reoxidation of the flavin seems to be rate limiting. The latter step might be involved in the decomposition of a flavin N5 adduct.

    During crystallization experiments it was found that VAO crystals are highly sensitive towards mercury and other heavy atom derivatives. Therefore, the reactivity of VAO towards mercury in solution was studied (Chapter 9). Treatment of VAO with p -mercuribenzoate showed that one cysteine residue reacts rapidly without loss of enzyme activity. Subsequently, three sulfhydryl groups react leading to enzyme inactivation and dissociation of the octamer into dimers. From this, it was proposed that subunit dissociation accounts for the observed sensitivity of VAO crystals towards mercury compounds.

    Recently, the crystal structure of VAO was solved (Chapter 10). The VAO structure represents the first crystal structure of a flavoenzyme with a histidyl bound FAD. The VAO monomer comprises two domains (Fig. 4).

    Figure 4. Crystal structure of VAO at 0.25 nm resolution.

    The larger domain forms a FAD-binding module while the other domain, the cap domain, covers the reactive part of the FAD cofactor. By solving the binding mode of several inhibitors, the active site of VAO could be defined. This has clarified several aspects of the catalytic mechanism of this novel flavoprotein. Three residues, Tyr108, Tyr503and Arg504, are involved in substrate activation by stabilizing the phenolate form of the substrate. This is in line with the proposed formation and stabilisation of the p -quinone methide intermediate and the substrate specificity of VAO. The structure of the enzyme 4-heptenylphenol complex revealed that the shape of the active-site cavity controls substrate specificity by providing a 'size exclusion mechanism'. Furthermore, the active site cavity has a rigid architecture and is solvent-inaccessible. A major role in FAD binding is played by residues 99-110, which form the so-called 'PP loop'. This loop contributes to the binding of the adenine portion of FAD and compensates for the negative charge of the pyrophosphate moiety of the cofactor. The crystal structure also established that the C8-methyl group of the isoalloxazine ring is linked to the Ne2 atom of His422. Intriguingly, this residue is located in the cap domain.

    From the crystallographic data and sequence alignments, we have found that VAO belongs to a new family of structurally related flavin-dependent oxidoreductases (Chapter 11). In this study, 43 sequences were found, which show moderate homology with the VAO sequence. As sequence homology was mainly found in the C-terminal and N-terminal parts of the proteins, it could be concluded that the homology is indicative for the conservation of a novel FAD-binding domain as was found in the crystal structure of VAO (Fig. 5). This structurally related protein family includes flavin-dependent oxidoreductases isolated from (archae)bacteria, fungi, plants, animals and humans, indicating that this family is widespread. Furthermore, the sequence analysis predicts that many members of this family are covalent flavoproteins containing a histidyl bound FAD.

    Figure 5. Schematic drawing of the structural fold of the newly discovered flavoprotein family.

    Some of the VAO-mediated reactions are of relevance for the flavour and fragrance industry. For example, reactions of VAO with vanillyl alcohol, vanillylamine or creosol all result in the formation of vanillin, the major constituent of the well-known vanilla flavour. Furthermore, as shown in Chapter 7, VAO is able to enantioselectively hydroxylate phenolic compounds resulting in the production of interesting synthons for the fine-chemical industry. Because of its versatile catalytic potential and as VAO does not need external cofactors, but only uses molecular oxygen as a cheap and mild oxidant, VAO may develop as a valuable tool for the biotechnological industry. Furthermore, the recent cloning of the VAO gene and the available crystal structure will allow protein engineering to redesign the catalytic performance of VAO, which is of main interest for biotechnological applications. Therefore, like glucose oxidase andD-amino acid oxidase, VAO can be placed among an emerging group of flavoprotein oxidases, that catalyze transformations of industrial relevance.

    Biocatalysis in non-conventional media : kinetic and thermodynamic aspects
    Vermuë, M. - \ 1995
    Agricultural University. Promotor(en): J. Tramper. - S.l. : S.n. - ISBN 9789054854623 - 177
    biokatalyse - enzymen - moleculaire structuur - grenslaag - oppervlakteverschijnselen - biocatalysis - enzymes - molecular conformation - boundary layer - surface phenomena

    During the past decade biocatalysis in non-conventional media has gained a lot of interest. Especially in the field of bio-organic synthesis, where poorly water-soluble substrates and products are involved, these media are very attractive.

    Non-conventional media generally consist of an apolar solvent phase and an aqueous phase. In this thesis, mixtures of water with water-miscible organic solvents, or water- immiscible organic solvents or (near-)supercritical solvents are described. The conventional aqueous phase contains the cellular or enzymic biocatalyst. The aqueous phase can vary from a dilute aqueous solution, with a thermodynamic water activity a w close to 1, to a dried enzyme particle with only a monolayer of adsorbed water molecules (a w < 1).

    In non-conventional media biocatalytic processes are governed by the presence of a phase boundary when two phases are involved. This phase boundary not only influences the rate of the bioconversion (kinetics), but also the yield of the reaction (thermodynamic equilibrium). In this thesis, several factors are described which affect the (kinetics), and thermodynamics of biocatalytic porcessen in non-conventional media.

    Chapter 2 gives an overview of the recent developments in the field of medium engineering for biocatalysis in non-conventional media. In this chapter a few basic design rules for the rational design are formulated. These rules may serve as useful tools for optimization of biocatalytic processes in non-conventional media.

    A typical example of a non-conventional reaction medium is the mixture of water and water-immiscible organic solvent. Especially for this type of reaction media the liquid-impelled loop reactor has been developed. This reactor has been used for the bioconversion of tetralin, a very toxic apolar compound. In Chapter 3 the general strategy for the selection of a suitable solvent for the bioconversion of such toxic apolar compounds in the liquid-impelled loop reactor is given, where the tetralin conversion is used as a typical example. The water-immiscible solvents should be non-toxic and nonbiodegradable. Additionally, they should reduce the toxicity of the apolar substrate and they must be practical for use in the liquid-impelled loop reactor. All the steps in the selection procedure proved to be essential. Among the 57 solvents tested, only FC-40 proofs to be suitable for bioconversion of tetralin in the liquid-impelled loop reactor. In addition, the cellular biocatalyst needs to be immobilized, to reduce emulsion formation inside the bioreactor.

    For the bioconversion of tetralin in the liquid-impelled loop reactor oxygen is needed. Chapter 4 describes the mass transfer of tetralin and oxygen in the liquidimpelled loop reactor from the apolar solvent phase to the aqueous phase, where the bioconversion occurs. It is found that in case of mass-transfer limitation, tetralin is the rate-limiting substrate and not oxygen.

    One of the selection criteria of a suitable solvent for bioconversion of apolar substates is its non-toxicity for the biocatalyst. The log Poctanol , which describes the hydrophobicity of the solvent, is a good measure for the toxicity of the solvent in a twoliquid phase system. The toxicity of a water-immiscible solvent for cellular biocatalyst is caused by two factors, i.e. the presence of a phase boundery (phase toxicity) and by the solvent molecules that are dissolved in the aqueous phase (molecular toxicity). Chapter 5 describes these effects separately. When the solvent concentration in the membrane of the cellular biocatalyst reaches a critical concentration, the solvent becomes toxic. The toxic concentration in the membrane is constant and independent of the solvent used. It is directly related via the partition coefficient over the membrane and water, to the solvent concentration in the aqueous phase. This is in turn directly related to the log Poctanol of the solvent. If the critical membrane concentration of a certain microorganism is known, the toxicity of any solvent can be predicted with the
    log Poctanol .

    Apart from the log Poctanol , also the Hildebrand solubility parameter δcan be used as a measure of the hydrophobicity of the solvent. In Chapter 6 this parameter has been used successfully as an indicator of the solubility of apolar compounds in near-supercritical carbon dioxide (SCCO 2 ). In addition, the effect of this parameter on the transesterification rate of Lypozyme in this non-aqueous reaction medium has been studied. The change in δof near-supercritical carbon dioxide hardly influences the reaction rate. The water content of the medium influences the kinetics much more.

    Water not only affects the kinetics of a synthetic reaction, but it also affects the equilibrium yield of these reactions. When the thermodynamic water activity a w is decreased, water-dependent side-reactions such as in transesterification reactions are suppressed (Chapter 6). In esterification reactions, a shift in equilibrium towards synthesis is expected upon decreasing the a w .

    Chapter 7 describes a new method to control the a w during esterification reactions. With this a w -control method the a w can be maintained at an optimal value, at which the biocatalyst still shows sufficient activity while a high thermodynamic product yield can be obtained.

    This thesis actually covers two central themes in biocatalysis in non-conventional media: kinetics and thermodynamics. In Chapter 8 a general discussion highlights how thermodynamics can be used as a basic tool to reveal the processes that govern biocatalysis in non-conventional media.

    Molecular structure and interfacial behaviour of polymers
    Lent, B. van - \ 1989
    Agricultural University. Promotor(en): G.J. Fleer; J.M.H.M. Scheutjens. - S.l. : van Lent - 104
    kunststoffen - industrie - oppervlakten - grensvlak - vloeistofmechanica - capillairen - oppervlaktespanning - polymeren - oppervlakteverschijnselen - grenslaag - moleculaire structuur - plastics - industry - surfaces - interface - fluid mechanics - capillaries - surface tension - polymers - surface phenomena - boundary layer - molecular conformation

    The aim of this study was to investigate the influence of the molecular structure on the interfacial behaviour of polymers. Theoretical models were developed for three different systems. All these models are based on the self-consistent field theory of Scheutjens and Fleer for the adsorption of homopolymers.

    This self-consistent field theory is a lattice model. All possible polymer conformations on the lattice are taken into account. The potential of a conformation is sum of the local potentials of the segments of the molecule. In each layer a mean field approximation is used to calculate the mixing energy. The volume fraction profile is determined by the segmental potentials and vice versa. A numerical method is used to solve the obtained set of equations.

    In chapter 2 the influence of association of block copolymers on adsorption is considered. In order to model spherical aggregates (micelles), the planar lattice, as used for modelling planar aggregates (membranes) and adsorption on flat surfaces, is replaced by a spherical lattice. The equilibrium solution concentration in a micellar solution is determined by a small system thermodynamics argument. The adsorption of diblock copolymers with long lyophobic and short lyophilic blocks shows strongly cooperative effects. A single molecular layer is present if the lyophobic block adsorbs. The adsorption isotherm shows an S-shape at the onset of adsorption. A strong increase of the adsorbed amount occurs near the cmc and above the cmc the adsorbed amount is almost constant. A bilayer at the surface can be formed if the lyophilic block adsorbs. Adsorption of the lyophilic blocks would expose the insoluble blocks to the solvent. Therefore, a second layer of molecules adsorbs with their lyophobic block towards the molecules attached to the surface. The influence of the interaction energies and the block sizes on these trends is described. The results obtained show good qualitative agreement with experimental results on surfactant adsorption.

    The adsorption of random copolymers from solution is described in chapter 3. Experimentally, random copolymers are usually very polydisperse, both in chain length and in primary structure. Random copolymers which are only polydisperse in primary structure are considered here. They can be prepared experimentally by random chemical modification of monomer units of monodisperse homopolymers. The sequence distribution of random copolymers is determined by the fractions of the segment types in the polymer and the correlation factors between them. For random copolymers consisting of two different segment types, a blockiness parameter B is defined. The extremes of this parameter are -1 and 1, where the lower limit depends on the fractions of the different segment types. A value of B = -1 represents an alternating copolymer, whereas B = 1 stands for a mixture of two homopolymers. The complete statistical sequence distribution is implemented into the theory. In the results section random copolymers with two different segment types are studied. Chains with a higher than average content of adsorbing segments are preferentially adsorbed from the bulk solution. Only in the first few layers near the surface this preferential effect plays a role. In the remainder of the profile the segment types are more randomly mixed. The adsorption behaviour of these random copolymers is remarkably different from the adsorption of diblock copolymers. In the latter case, the chains have their adsorbing segments mainly in the layers near the surface, whereas further away from the surface long dangling tails of nonadsorbing segments are found. Random copolymers cannot spacially separate their segments so easily. Much higher adsorbed amounts are found for diblock copolymers than for random copolymers with the same fraction of adsorbing segments. The adsorption of random copolymers is less than that of homopolymer of equal length and consisting of the same type of adsorbing segments. Only for very high adsorption energies the adsorbed amounts are essentially the same. An increase in the blockiness parameter of the chains gives an higher adsorbed amount, but it is always below the adsorbed amount of the homopolymer. Analytical expressions have been derived which relate the interaction parameters of purely random copolymer and homopolymer.

    In chapter 4 the interactions between surfaces coated with grafted polymer (also called hairy plates or soft surfaces) in the presence of nonadsorbing polymer is studied. The interaction free energy between the surfaces is obtained from the partition function. which is rederived for this more general case. For hard plates the interaction is fully determined by the osmotic pressure of the bulk solution and the depletion layer thickness. However. It turns out that In the case of soft surfaces the hairs have an attractive contribution to the free energy of interaction at a plate separation just below twice the hydrodynamic layer thickness of the grafted layer. The hairs mix mutually more easily than with free polymer. At a larger overlap of hairs the interaction becomes repulsive. In contrast with bare planar surfaces, the free energy of interaction between hairy surfaces shows a minimum as a function of the concentration of free polymer in the bulk solution. At a certain (very low) surface coverage the attraction is minimal. For even lower and for larger grafting densities the plates become more attractive. Increasing the repulsion between the hairs and free polymer makes the attraction stronger. The solvencies of grafted and free polymer have a less pronounced effect. Without free polymer, the interaction between the hairy surfaces becomes attractive if the solvency becomes worse than theta conditions.

    It can be concluded that the self-consistent field theory has been successfully extended to three rather complex but technologically relevant systems. In this way a better understanding of the behaviour of polymers near interfaces has been obtained.

    Studies on 2-oxoacid dehydrogenase multienzyme complexes of Azotobacter vinelandii
    Bosma, H.J. - \ 1984
    Landbouwhogeschool Wageningen. Promotor(en): C. Veeger, co-promotor(en): A. de Kok. - Wageningen : Bosma - 127
    azotobacter vinelandii - oxidoreductasen - synthese - moleculaire structuur - azotobacter vinelandii - oxidoreductases - synthesis - molecular conformation
    In this thesis, some studies on the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes of Azotobacter vinelandii are described; the emphasis strongly lies on the pyruvate dehydrogenase complex.

    A survey of the literature on 2-oxoacid dehydrogenase complexes is given in chapter 1. It appears that the A.vinelandii pyruvate dehydrogenase complex resembles the complexes from other gram-negative bacteria with respect to its composition and working mechanism. The A.vinelandii complex is however much smaller than the pyruvate dehydrogenase complexes isolated from other sources.

    Chapter 2 describes the procedure that has been optimized for the isolation of the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes (PDC and OGDC respectively) from A.vinelandii. In comparison to the previous isolation procedure, several advantages exist. The A.vinelandii PDC is obtained as an essentially pure three-component complex, in a high yield (40-50%). 80% of the losses can be accounted for by discarded side-fractions, which indicates that the complex is hardly inactivated during its purification. The specific activity of the final preparation is about two times higher (15-19 U/mg) than previously could be obtained. From these observations we conclude that the formerly observed "fourth component" of A.vinelandii PDC was a mere contaminant. With the revised procedure, the 2-oxoglutarate dehydrogenase complex (OGDC) is obtained in a high yield (40-50%), free from contaminants. In the "old" procedure this complex was irreversibly inactivated by the action of protamine sulfate.

    In chapter 3 some observations on the A.vinelandii OGDC are reported. The molecular mass of this complex is of the order of 2.4 to 3.2 MDa, as determined by laser light-scattering measurements. The three component enzymes have the same molecular masses as have been reported for the OGDC's of Escherichia coli and pig-heart. The activity of the complex is regulated by its substrates in an analogues way as has been reported for the E.coli complex, and we therefore conclude that the A.vinelandii complex probably strongly resembles the OGDC of E.coli. In this chapter, an isolation procedure for the lipoamide dehydrogenase component is described, and it is shown that the lipoamide dehydrogenase components of the A.vinelandii PDC and OGDC probably are identical.

    The association behaviour of the A.vinelandii pyruvate dehydrogenase complex is described in chapter 4. From sedimentation and light-scattering studies we conclude that a monomer-dimer equilibrium exists for this complex; the molecular mass of the monomer has been estimated that 800 kDa. In this thesis, this monomer-dimer mixture is referred to as the 18 S form of the complex. Upon addition of polyethylene glycol 6000 and MgCl 2 , the 18 S form of the complex aggregates into a large structure, resembling the pyruvate dehydrogenase complex of E.coli with respect to its sedimentation, coefficient (56 S) and its appearance on electron micrographs. The isolated dihydrolipoyl transacetylase component of A.vinelandii PDC has a molecular mass of 2 MDa, and on electron micrographs it resembles the dihydrolipoyl acetyltransferase component of E.coli. It is concluded that this large structure probably is composed of 32 subunits. Upon the binding of the pyruvate dehydrogenase and lipoamide dehydrogenase components, this large particle dissociates into the smaller structures that are characteristic for the intact A.vinelandii complex. The small (18 S) and the large (56 S) forms of the (sub)complexes are in slow equilibrium, and this equilibrium can be perturbed by high hydrostatic pressure. From light-scattering measurements at varying pressures it is concluded that the 56 S form of the complex probably is an octamer of the 800 kDa monomers.

    The measurements concerning the chain-stoichiometry of A.vinelandii PDC are described in chapter 5. A novel method for the determination of chain-ratios was developed, based on the covalent modification of lysine residues in the three component enzymes with trinitrobenzene sulfonic acid. With this technique, an average chain ratio of 1.3:1:0.5 (pyruvate hydrogenase: dihydrolipoyl acetyl transferase:lipoamide dehydrogenase) was found for the isolated A.vinelandii PDC. In combination with the results of chapter 4, it is concluded that A.vinelandii PDC is based on a tetrameric dihydrolipoyl acetyltransferase core, to which the periferal components are bound in a non-covalent way. The complex can be reconstituted from its individual components, and from these reconstitution experiments it follows that the complex has maximal activity when three pyruvate dehydrogenase dimers and one lipoamide dehydrogenase dimer are bound to the dihydrolipoyl transacetylase tetramer.

    In chapter 6, the results of acetylation experiments are given. It is shown that the reductive acetylation of the lipoyl groups probably is the rate-limiting step in the reaction sequence of the A.vinelandii pyruvate dehydrogenase complex. In so-called servicing experiments, an extensive exchange of acetyl groups between individual (monomeric) pyruvate dehydrogenase complex particles is found. This phenomenon (inter-core transacetylation) has until now only been observed for the A.vinetandii complex. It is shown that the inter-core transacetylation occurs when two monomeric particles are associated. Although the transacetylation reactions show large effects in the servicing experiments, these reactions are however too slow to be of physiological importance. The servicing experiments also show that the large " E.coli -like" isolated dihycirolipoyl acetyltransferase component is composed of rather independently operating tetramers, i.e. the large form of the A.vinelandii PDC does not function as a large entity.

    In chapter 7, the results of the three preceding chapters are summarized and translated into a three-dimensional model of the molecular organisation of the A.vinelandii PDC. The merits of this model are discussed in relation to the generally accepted model for the pyruvate dehydrogenase complex of E.coli. It is suggested that the pyruvate of Azotobacter vinelandii could represent the morphological subunit of the larger structure that is found in Escherichia coli and perhaps in other gramnegative bacteria. It is concluded that further experiments have to be performed, in which the complexes of the two organisms are directly compared. to establish whether such a unifying model does exist.

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