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 1provides 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 4focuses 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 7describes 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
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
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.