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.

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