<p>The human glucose transporter GLUT1 facilitates glucose to be accumulated on the other side of the cell membrane. The functional state of GLUT1 is uncertain due to diversity of the reports. In this thesis, the activity of red blood cell GLUT1 was extensively studied to further characterize this protein.</p><p>The human red blood cell membranes were stripped to become vesicles with low-ionic alkaline solution in the presence or absence of dithioerithritol. The supernatant of partially solubilized membrane vesicles provided approximately 65% of the vesicle proteins. GLUT1 purified from this supernatant showed a little high-affinity cytochalasin B binding activity. On the other hand, the vesicles stripped with dithioerythritol provided mostly monomeric GLUT1 and those without dithioerythritol provided monomeric and oligomeric GLUT1. MALDI-ToF-MS detected variant GLUT1 fragments between the two preparations. Residual endogenous phospholipids per GLUT1 also showed difference. However, the equilibrium exchange of glucose was retained for both GLUT1 preparations. Cytochalasin B-binding activity of GLUT1 in streptoavidin-biotin-immobilized red blood cells showed that both dissociation constant and binding sites per GLUT1 fell between those of wheat germ lectin-immobilized red blood cells with or without polylysine coating, which indicated the switching of two cytochalasin B-binding states of GLUT1. It is concluded that GLUT1 in red blood cells contains approximately two equal portions, monomeric with high-affinity cytochalasin B-binding activity and oligomeric without high-affinity cytochalasin B-binding activity. In the partial solubilization of the membrane vesicles, GLUT1 which does not have high-affinity cytochalasin B-binding activity is pooled. This might provide a resolution to select oligomerically and functionally different GLUT1 for crystallization.</p><p>In addition a modified micro-Bradford assay with CaPE precipitation was developed to achieve a routine quantitation method for membrane proteins and the effects of cholesterol and PEG(5000)-DSPE on reconstituted GLUT1 were preliminarily determined.</p>

Control of protein immobilization at sensor surfaces is of great interest within various scientific fields, since it enables studies of specific biomolecular interactions. To achieve this, one must be able to immobilize proteins with retained native structure, while minimizing non-specific protein binding. The high affinity interaction between streptavidin (SA) and biotin is extensively used as a linker between a surface, where SA is immobilized, and the (biotinylated) molecule of interest. Self- assembled monolayers (SAMs) of poly- and oligo ethylene glycol (PEG and OEG) derivatives have been proven in literature to minimize non-specific protein binding, and biotin-exposing SAMs have been shown efficient for immobilization of SA. The aim of this master's thesis project was to develop biotinylated gold surfaces for quartz crystal microbalance with dissipation monitoring (QCM-D) applications through the self-assembly of mixed monolayers of thiolated OEG (or PEG) derivatives with or without a terminal biotin head group. For this, different thiol compounds were to be compared and evaluated. For the systems under study, the required biotin density for maximum specific SA immobilization was to be established, while keeping the non-specific serum adsorption at a minimum. Model experiments with biotinylated proteins immobilized to the SA-functionalized surfaces were to be performed to evaluate the possibilities for commercialization. A protocol for the preparation of a novel biotinylated surface was developed based on the immersion of gold substrates in an ethanolic incubation solution of dithiols with OEG chains (SS-OEG and SS-OEG-biotin, 99:1) and found to give reproducible results with respect to low non-specific protein binding and immobilization of a monolayer of SA. The modified surfaces allowed for subsequent immobilization of biotinylated bovine serum albumin (bBSA) and biotinylated plasminogen (bPLG). PLG was the subject of a challenging case study, using a combination of QCM-D and surface plasmon resonance (SPR), where the immobilized protein was subjected to low molecular weight ligands that were believed to induce conformational changes. The high control of the surface chemistry allowed for the interpretation of the increased dissipation shift upon ligand binding in terms of conformational changes. An obstacle before commercialization of the described biotinylated surfaces is that they do not seem stable for storage &gt; 7 days. The reasons for this have to be investigated further.

The human glucose transporter GLUT1 facilitates glucose to be accumulated on the other side of the cell membrane. The functional state of GLUT1 is uncertain due to diversity of the reports. In this thesis, the activity of red blood cell GLUT1 was extensively studied to further characterize this protein. The human red blood cell membranes were stripped to become vesicles with low-ionic alkaline solution in the presence or absence of dithioerithritol. The supernatant of partially solubilized membrane vesicles provided approximately 65% of the vesicle proteins. GLUT1 purified from this supernatant showed a little high-affinity cytochalasin B binding activity. On the other hand, the vesicles stripped with dithioerythritol provided mostly monomeric GLUT1 and those without dithioerythritol provided monomeric and oligomeric GLUT1. MALDI-ToF-MS detected variant GLUT1 fragments between the two preparations. Residual endogenous phospholipids per GLUT1 also showed difference. However, the equilibrium exchange of glucose was retained for both GLUT1 preparations. Cytochalasin B-binding activity of GLUT1 in streptoavidin-biotin-immobilized red blood cells showed that both dissociation constant and binding sites per GLUT1 fell between those of wheat germ lectin-immobilized red blood cells with or without polylysine coating, which indicated the switching of two cytochalasin B-binding states of GLUT1. It is concluded that GLUT1 in red blood cells contains approximately two equal portions, monomeric with high-affinity cytochalasin B-binding activity and oligomeric without high-affinity cytochalasin B-binding activity. In the partial solubilization of the membrane vesicles, GLUT1 which does not have high-affinity cytochalasin B-binding activity is pooled. This might provide a resolution to select oligomerically and functionally different GLUT1 for crystallization. In addition a modified micro-Bradford assay with CaPE precipitation was developed to achieve a routine quantitation method for membrane proteins and the effects of cholesterol and PEG(5000)-DSPE on reconstituted GLUT1 were preliminarily determined.

HIV-1 has evolved with a great deal of functional complexity contained within a very small genome by encoding small, but critical viral proteins within larger viral genes and dividing the replication cycle into early and late phases to differentially produce all proteins leading to efficient replication and virion release. Early replication is restricted by the host spliceosome that processes HIV-1 vRNA transcripts so only the small intragenomic proteins are produced, one of which is Rev (Regulator of Virion Expression). Rev in turn governs the transition from early to late replication by interacting with a highly structured region of vRNA termed the Rev Response Element (RRE). The binding of Rev to the RRE is believed to cause a change in the vRNA tertiary structure and inhibition of splicing of the vRNA. Once, a Rev:RRE complex is formed, a nuclear export signal within Rev facilitates the export of partially spliced and unspliced vRNA to the cytoplasm. During late replication the partially spliced and unspliced vRNA is translated to polyproteins and is packaged into a budding virion where the viral aspartyl protease (HIV-1 PR) autocatalytically excises itself from the larger polyprotein and processes the remaining polyproteins to release all viral structural and functional proteins to form a mature and infectious virion. Since the vRNA salvaged by Rev is translated to the polyproteins containing HIV-1 PR, the inhibition of Rev function will reduce the amount of HIV-1 PR available and thereby reduce the amount of HIV-1 PR therapeutics required to elicit a clinical effect. Therfore a combination approach to HIV-1 treatment using suitably developed therapeutics that inhibit Rev and HIV-1 PR function represents an attractive synergistic approach to treating HIV-1 infection in vivo. The work of this thesis was divided into two parts, the first part was concerned with HIV-1 PR structural biology and addressing problems encountered with inhibitor design. A bicyclic peptide (based on inhibitors of analogous structure) was co-crystallised with active HIV-1 PR to develop an enzyme-product (E-P) complex and with a catalytically inactive mutant HIV-1 PR to provide an analogy to the enzyme-substrate (E-S) complex. Both structures of the E-P and E-S complexes were solved to 1.6Å resolution and were compared to a hydroxyethylamine isostere enzyme-inhibitor complex (E-I), highlighting the similarity of binding mode for all ligands. The inhibitor in the E-I complex was translated towards the S1 - S3 pockets of the substrate binding cleft relative to the substrate in the E-S complex due to the increased length of the hydroxylethylamine isostere compared to the peptide backbone, although the inhibitor "puckered" the isostere linkage and maintains a binding mode similar to the substrate with very little overall differences in the position of the ligands and surrounding protein. The similarity of the E-S, E-I and E-P complexes was attributed to the macrocyclic ligands ordering the surrounding protein environment, especially the protein -strand "flap" structures that form a roof over the ligands in the active site but were not found to close more tightly in any of the trapped catalytic states. The new structures allowed refinement of details of the mechanism of peptide hydrolysis. The mechanism relies on the optimal nucleophilic attack of a water molecule on the scissile amide bond with concerted acid-base catalysis of the active site aspartyl residues intitiated by D125. The alignment and intrinsic position of the N-terminus of the bicyclic substrate was interpreted as being critical to facilitate efficient electron transfer with the bicyclic substrate. An N-terminal cyclic inhibitor, similar to the N-terminal portion of the bicyclic substrate, was used to address a major problem in HIV-1 PR drug design termed "cooperativity," where the sequential optimisation of an inhibitor (or substrate) to individual pockets of the substrate binding cleft, can negatively impact on adjacent and downfield subsites and thereby alter the binding mode of the "optimised" inhibitor. The technique referred to here as "templating" uses the N-terminal cycle to lock the binding mode into a known conformation, probing the S1' and S2' pockets. The structure activity relationship suggested that by viewing the S1' - S3' pockets as a single trough, bulky aromatic groups attached to an N-alkyl sulfonamide could be directed along the line of the trough without adverse interactions with the tops of the S1' and S3' pockets, providing very potent inhibitors. It was also found that specificity and potency of an inhibitor can be maintained with smaller functionalities that carry their bulk low and close to the inhibitor backbone in the S2' pocket, making the P2 functionalities more substrate-like. The second part of the thesis was concerned with establishing suitable surface plasmon resonance assays for testing potential inhibitors of Rev function. Recombinant Rev and its minimal RNA aptamer target (stem loop II of the RRE termed RBE3), were expressed, purified, and used to develop BIAcore-based assays and test potential inhibitors of their interaction. The system was applied to screening of aminoglycoside antibiotics and other small molecules in a competitive assay, and also to quantitative assay of Neomycin and moderate sized analytes: Rev and three peptidic analogues of the high-affinity binding site of Rev - the native peptide, succinylated form of the peptide and a form incorporating a novel helix-inducing cap. The peptide and protein assay was undertaken to test the proposition that helix induction of the high-affinity binding site of Rev can increase affinity for the biologically important RNA target and thereby form the basis of a new class of inhibitors. The screen of small molecule antagonists found that Neomycin was the best inhibitor of the Rev:RBE3 interaction and that efficacy of other aminoglycosides was due to the neamine-base structure presenting charge to bind to the RNA and blocking interaction with Rev. The quantitative assay was optimised to reduce non-specific interactions of Rev protein to allow reliable studies of the analytes with RBE3 by the sytematic testing of buffers and modifiers. It was found that mutliple analytes bound to the RBE3 aptamer and a comparison of the KD values found that the native and capped peptides had similar affinity for RBE3 RNA (native slightly greater at 21 ± 7nM cf capped 41 ± 10nM) that was greater than the Rev protein (101 ± 19nM), however the succinylated peptide exhibited stronger binding with a KD ≤8nM and Neomycin had the lowest affinity (KD 13 ± 3M). The similarity of the native and capped peptides may be due to the high concentration of salt in the assay buffers and was necessary for the stability of the Rev protein, but is sufficient to influence secondary structure of the peptides. Therefore, it could not be stated that the helix-inducing cap increased the affinity of the native peptide for the biologically important therapeutic target. The work conducted in this thesis firmly establishes foundations for the continued development of inhibitors against both Rev and HIV-1 PR that play key roles in the HIV-1 replication strategy. It is envisaged this work could lead to a novel synergistic therapeutic approach to treating HIV-1 infection.

<p>Specific and non-specific interactions of solutes with immobilized biomembranes were studied using chromatographic methods. Liposomes, proteoliposomes and red blood cell (RBC) membrane vesicles were immobilized by a freeze-thawing procedure, whereas whole RBCs were adsorbed in the gel beds using electrostatic interaction, binding to wheat germ agglutinin (WGA) or the streptavidin-biotin interaction. </p><p>Superporous agarose gel with coupled WGA was the most promising matrix for RBC adsorption and allowed frontal chromatographic analyses of the cells for about one week. Dissociation constants for the binding of cytochalasin B and glucose to the glucose transporter GLUT1 were determined under equilibrium conditions. The number of cytochalasin B-binding sites per GLUT1 monomer was calculated and compared to corresponding results measured on free and immobilized membrane vesicles and GLUT1 proteoliposomes. This allowed conclusions about the protein´s binding state <i>in vitro</i> and <i>in vivo</i>. </p><p>Partitioning of drugs into biomembranes was quantified and the system was suggested as a screening method to test for possible intestinal absorption of drug candidates. We also studied how membrane partitioning of drugs is affected by the presence of integral membrane proteins or of charged phospholipids.</p><p>An attempt to combine the theory for specific binding and membrane partitioning of solutes in a single equation is briefly presented. </p>

<p>The human glucose transporter GLUT1 is abundant in red blood cells, the blood-brain barrier and epithelial cells, where it mediates the transport of the energy metabolite, glucose. In the present work some properties of GLUT1, including affinity binding of both substrates and inhibitors, transport rates as well as permeabilities of aromatic amino acids and drug-membrane interactions were analyzed by chromatographic methods.</p><p>Reconstitution by size-exclusion chromatography on Superdex 75 from a detergent with a low CMC that provides monomeric GLUT1 was examined regarding D-glucose- and CB binding as well as D-glucose transport. Upon steric immobilization in Superdex 200 gel beads, residual detergent could be washed away and dissociation constants in the same range as reported for binding to GLUT1 reconstituted from other detergents were obtained. The transport rate into the GLUT1 proteoliposomes was low, probably due to residual detergent. Binding to GLUT1 at different pH was analyzed and the affinity of glucose and GLUT1 inhibitors was found to decrease with increasing pH (5–8.7). The average number of cytochalasin B-binding sites per GLUT1 monomers was, in most cases, approximately 0.4. GLUT1 may work as a functional monomer, dimer or oligomer. To determine whether GLUT1 was responsible for the transport of the aromatic amino acids tyrosine and tryptophan, uptake values and permeabilities of these amino acids into liposomes and GLUT1 proteoliposomes were compared to the permeabilities of D- and L- glucose in the same systems. Dihydrocytochalasin B was identified to be a new inhibitor of tyrosine and tryptophan transport into red blood cells. Ethanol turned out to inhibit the specific binding between CB and GLUT1 and also to decrease the partitioning of CB and drugs into lipid bilayers. A capacity factor for drug partitioning into membranes that allows comparison between columns with different amount of immobilized lipids was validated, and turned out to be independent of flow rate, amount of lipids and drug concentration in the ranges tested.</p>

The human glucose transporter GLUT1 is abundant in red blood cells, the blood-brain barrier and epithelial cells, where it mediates the transport of the energy metabolite, glucose. In the present work some properties of GLUT1, including affinity binding of both substrates and inhibitors, transport rates as well as permeabilities of aromatic amino acids and drug-membrane interactions were analyzed by chromatographic methods. Reconstitution by size-exclusion chromatography on Superdex 75 from a detergent with a low CMC that provides monomeric GLUT1 was examined regarding D-glucose- and CB binding as well as D-glucose transport. Upon steric immobilization in Superdex 200 gel beads, residual detergent could be washed away and dissociation constants in the same range as reported for binding to GLUT1 reconstituted from other detergents were obtained. The transport rate into the GLUT1 proteoliposomes was low, probably due to residual detergent. Binding to GLUT1 at different pH was analyzed and the affinity of glucose and GLUT1 inhibitors was found to decrease with increasing pH (5–8.7). The average number of cytochalasin B-binding sites per GLUT1 monomers was, in most cases, approximately 0.4. GLUT1 may work as a functional monomer, dimer or oligomer. To determine whether GLUT1 was responsible for the transport of the aromatic amino acids tyrosine and tryptophan, uptake values and permeabilities of these amino acids into liposomes and GLUT1 proteoliposomes were compared to the permeabilities of D- and L- glucose in the same systems. Dihydrocytochalasin B was identified to be a new inhibitor of tyrosine and tryptophan transport into red blood cells. Ethanol turned out to inhibit the specific binding between CB and GLUT1 and also to decrease the partitioning of CB and drugs into lipid bilayers. A capacity factor for drug partitioning into membranes that allows comparison between columns with different amount of immobilized lipids was validated, and turned out to be independent of flow rate, amount of lipids and drug concentration in the ranges tested.

Specific and non-specific interactions of solutes with immobilized biomembranes were studied using chromatographic methods. Liposomes, proteoliposomes and red blood cell (RBC) membrane vesicles were immobilized by a freeze-thawing procedure, whereas whole RBCs were adsorbed in the gel beds using electrostatic interaction, binding to wheat germ agglutinin (WGA) or the streptavidin-biotin interaction. Superporous agarose gel with coupled WGA was the most promising matrix for RBC adsorption and allowed frontal chromatographic analyses of the cells for about one week. Dissociation constants for the binding of cytochalasin B and glucose to the glucose transporter GLUT1 were determined under equilibrium conditions. The number of cytochalasin B-binding sites per GLUT1 monomer was calculated and compared to corresponding results measured on free and immobilized membrane vesicles and GLUT1 proteoliposomes. This allowed conclusions about the protein´s binding state in vitro and in vivo. Partitioning of drugs into biomembranes was quantified and the system was suggested as a screening method to test for possible intestinal absorption of drug candidates. We also studied how membrane partitioning of drugs is affected by the presence of integral membrane proteins or of charged phospholipids. An attempt to combine the theory for specific binding and membrane partitioning of solutes in a single equation is briefly presented.

Caractériser la fonction des protéines est crucial pour notre compréhension des mécanismes moléculaires de la vie, des maladies, et aussi pour inspirer de nouvelles applications en bionanotechnologie. Pour y arriver, il est nécessaire de caractériser la structure et la dynamique de chaque état occupé par la protéine durant sa fonction. La caractérisation expérimentale des états transitoires des protéines représente encore un défi majeur parce que les techniques à haute résolution structurelle, telles que la spectroscopie RMN et la cristallographie aux rayons X, peuvent difficilement être appliquées à l’étude des états de courte durée. De plus, les techniques à haute résolution temporelle, telles que la spectroscopie de fluorescence, nécessitent généralement une chimie complexe pour introduire des fluorophores à des endroits spécifiques dans la protéine. Dans cette thèse nous introduisons l’utilisation des nanoantennes fluorescentes en tant que nouvelle stratégie pour détecter et signaler les changements de conformation des protéines via des interactions non covalentes entre des fluorophores spécifiques et la surface de la protéine. En utilisant des expériences et des simulations moléculaires, nous démontrons que des fluorophores chimiquement divers peuvent se lier et être utilisés pour sonder différentes régions d’une enzyme modèle, la phosphatase alcaline (PA). Ces nanoantennes peuvent être fixées directement aux protéines ou utilisées à l'aide du système de fixation simple et modulaire, le complexe biotine-streptavidine (SA), qui permet un criblage rapide et efficace de la nanoantenne optimale tant dans sa composition que sa longueur. Dans le cas de la PA, nous montrons que nos nanoantennes permettent la détection et la caractérisation des conformations distinctes incluant les changements conformationnels nanoscopiques produisant durant la catalyse du substrat. Nous démontrons également que les signaux fluorescents émis par la nanoantenne peuvent également permettre de caractériser la cinétique enzymatique d’une protéine en une seule expérience tout en incluant la détermination des paramètres « Michaelis-Menten » de ses substrats et inhibiteurs. Nous avons également exploré l'universalité de la stratégie ces nanoantennes fluorescentes en utilisant une autre protéine modèle, la Protéine G et son interaction avec les anticorps, et avons démontré son utilité pour mettre au point un essai permettant de détecter les anticorps. Ces nanoantennes simples et faciles à utiliser peuvent être appliquées pour détecter et analyser les changements conformationnels de toutes tailles et nos résultats suggèrent qu'elles pourraient être utilisées pour caractériser n’importe quel type de fonction. / The characterisation of protein function is crucial to understanding the molecular mechanisms of life and disease, and inspires new applications in bionanotechnology. To do so, it is necessary to characterise the structure and dynamics of each state that proteins adopt during their function. Experimental study of protein transient states, however, remains a major challenge because high-structural-resolution techniques, including NMR spectroscopy and X-ray crystallography, can often not be directly applied to study short-lived protein states. On the other hand, high-temporal-resolution techniques, such as fluorescence spectroscopy, typically require complicated site-specific labelling chemistry. This thesis introduces the use of fluorescent nanoantennas as a new strategy for sensing and reporting on protein conformational changes through noncovalent dye-protein interactions driven by a high local concentration. Using experiments and molecular simulations, we first demonstrate that chemically diverse dyes can bind and be used to probe different regions of a model enzyme, intestinal alkaline phosphatase (AP). These nanoantennas can be attached directly to proteins or employed using the simple and modular biotin-streptavidin (SA) attachment system, which enables rapid and efficient screening for high sensitivity by tuning their length and composition. We show that these nanoantennas enable the detection and characterisation of distinct conformational changes of AP, including nanoscale conformational changes that occur during substrate catalysis. We also show that the fluorescent signal emitted by the nanoantenna enables complete characterisation of enzyme kinetics in one experiment, including determination of Michaelis-Menten parameters of substrates and inhibitors of AP. We then explored the universality of the nanoantenna strategy by using a different model protein system. Protein G was shown to interact with antibodies, using a rapid screening strategy for antibody detection. These effective and easy-to-use nanoantennas could potentially be employed to monitor various conformational changes, and our results offer potential for characterising various protein functions.

Cell separation based on antibody-targeted magnetic beads has been widely used in a number of applications in immunology, microbiology, oncology and more recently, in the isolation of circulating tumour cells (CTCs) in cancer patients. Although other cell separation techniques such as size based cell filtration and Fluorescence Activated Cell Sorting have also been in popular use, immunomagnetic cell isolation possesses the advantages of high throughput, good specificity and reduced cell stress. However, certain fundamental features of the cell-bead interface are still unknown. In this study, some of the key features of the cell-bead synapse were investigated in an effort to improve the efficiency of immunomagnetic cell isolation and reduce its dependence on high expressing cell surface markers. A clinically relevant antibody fragment (Fab) against tyrosine kinase receptor HER2 was applied to study the immunomagnetic isolation of HER2 expressing cancer cells. First, the minimum number of target proteins required on a cell for it to be isolated was determined. Second, the importance of the primary antibody affinity was investigated, using a series of Fab mutants with known kinetics and it was shown that despite starting with sub-nanomolar affinity, improving Fab affinity increased cell isolation. Third, the influence of the connection between the primary antibody and the bead was studied by comparing Fab bridged to the magnetic bead via a secondary antibody, Protein L or streptavidin; the high affinity biotin-streptavidin linkage increased isolation sensitivity by an order of magnitude. Fourth, the effect of manipulating cytoskeletal polymerization and cell membrane fluidity using small molecules was tested; cholesterol depletion decreased isolation and cholesterol loading increased cell isolation. The insights from these observations were then applied to isolate a panel of cell lines expressing a wide range of surface HER2. While the standard approach isolated less than 10% of low HER2 expressing cancer cells from spiked rabbit and human blood, our enhanced approach with the optimized cholesterol level, antibody affinity and antibody-bead linkage could specifically isolate more than 80% of such cells. The final part of this work focussed on developing an antibody clamp that could physically restrict the antigen within its binding site on the Fab and prevent antigen dissociation, using the HER2-Fab complex and the anti-myc peptide antibody 9E10. Work from this thesis provides useful insights into the molecular and cellular parameters guiding immunomagnetic cell isolation and can be used to extend the range of target receptors and biomarkers for tumour cell isolation and other types of cell separation, thereby enhancing the power and capacity of this approach.