<p>The information for mitochondrial and chloroplastic protein import and targeting generally resides in the N-terminal part of the protein, called a targeting peptide. The targeting peptide is cleaved off by the organellar processing peptidases after import of a precursor protein. Free targeting peptides generated inside the organelle after import are rapidly degraded by proteolysis as their accumulation can have toxic effects on the organelle. The aim of this thesis has been to investigate the newly identified targeting peptide degrading protease, the PresequenceProtease (PreP). We have shown that the two isoforms of <i>Arabidopsis </i>PreP (<i>At</i>PreP1 and <i>At</i>PreP1) are dually targeted and localized to both mitochondria and chloroplasts. Dual targeting of the <i>At</i>PreP1 is due to an ambiguous targeting peptide with a domain organization for mitochondrial and chloroplastic targeting. Both the <i>At</i>PreP1 and <i>At</i>PreP2 are expressed in <i>Arabidopsis</i> in an organ specific manner and they have distinct but overlapping substrate specificity. The crystal structure of the recombinant <i>At</i>PreP1 E80Q was solved at 2.1 Å resolution. The structure represents the first substrate bound, closed conformation of a protease from the pitrilysin family. The PreP polypeptide folds in a unique peptidasome structure, surrounding a huge cavity of more than 10 000 Å3 in which the active site resides. A novel mechanism for proteolysis is proposed involving hinge-bending motions in response to substrate binding. PreP in human mitochondria has a novel function: degradation of amyloid β-peptide (Aβ). We show that under circumstances when Aβ is present in mitochondria of Alzheimer’s patients, PreP is the protease responsible for degradation of this toxic peptide. </p>

<p>The synthesis and breakdown of sugars is one of the most important functions in Nature. Consequently, sugar structures are used both as energy storage and as building blocks to stabilise and protect the cell. The formation of these structures is performed by glycosyltransferases (GT), an enzyme group structurally conserved within all kingdoms. Until now, only two different folds have been discovered by crystallisation of GTs, i.e. GT-A and GT-B. A third fold family has however been proposed by fold predictions. In this thesis, a multivariate data analysis was successfully used in classifying and predicting both fold and reaction mechanism (inverting or retaining) of GTs. This method was also used to obtain information about the separating parameters for the reaction mechanism classification. This information could be traced back to the amino acid sequence. The method could as well be used to analyse and identify the properties of membrane binding regions of GTs, and subsequently distinguish soluble from membrane-associated enzymes. Most functionally characterised enzymes only use one substrate, synthesising one product. <i>Mycoplasma pneumoniae</i>, a common human pathogen with a small genome has only three proposed GTs. The bacterium was, however expected to have a greater number of GTs, due to its ability to make both glycolipids and capsule. Here we have determined the function of one of these enzymes, MPN483 and discovered its ability to both use different acceptors, and make elongated glycolipids with up to three galactose residues, with both DAG and ceramide as the base. Many of the synthesised glycolipids were also found to be immunogenic, hence showing their biological importance. The properties of lipids are known to be important for the function of a biological membrane. We have here shown that not only the charge but also the shape of the lipids are important for several protein mediated membrane processes in <i>Echerichia coli</i>, such as the function of the LacY.</p>

<p>In recent years, the use of alcohol and psychoactive drugs in combination withdriving has recieved increased attention. The lack of in-vehicle devices capable ofdetecting recent drug consumption and the difficulties associated with the breathbasedalcolocks in use today makes it interesting to investigate methods that areable to non-invasivelly measure analytes directly in the blood.</p><p>The assignment of this project, commissioned by Volvo Technology Corporationand Volvo Car Corporation, is to map substances that constitute a possible threatto traffic safety, identify suitable detection markers as a proof of administrationof these substances, and study possible non-invasive techniques to detect thesemarkers. The objective is to present for Volvo if and how to continue evaluatingand developing a non-invasive detection device.</p><p>The project has been carried out by performing an extensive literature study and averification experiment. From the literature review, a number of substances affectingdriving performance could be identified, and a metabolic study was performedfor each drug to map suitable biomarkers. Furthermore, two potential techniquesfor non-invasive detection, near-infrared Raman spectroscopy and near-infraredspectroscopy, were found and evaluated. The experiment was conducted usingnear-infrared Raman spectroscopy, with the aim of investigating the sensitivityand linearity of the method for ethanol detection.</p><p>Based on the theoretical evaluation, both near-infrared Raman spectroscopy andnear-infrared spectroscopy are expected to have potential for non-invasive detectionof ethanol. The experiment further proved the theoretical conclusionsmade for near-infrared Raman spectroscopy. However, neither of the techniquesis thought to have potential for drug detection.Altogether, we believe that non-invasive ethanol detection is possible, but suggestfurther experiments in order to determine which technique to be preferred.</p>

<p><p>The adsorption of proteins to solid surfaces has been extensively investigated during the past 20-30 years. The knowledge can be applied in biotechnological applications in for example immunoassays and biosensors. Human carbonic anhydrase II is a widely studied protein and the CO₂-activity makes it an interesting candidate for biotechnological purposes. To make this possible, the factors affecting the adsorption of proteins have to be mapped. The stability of the protein is under great influence of the adsorption and the protein tends to undergo conformational changes leading to a molten globule like state upon adsorption. The stability of a protein also affects the extent of conformational changes and the nature of the adsorption. A more stable protein, adsorbs with less structural changes as a consequence of adsorption, and desorbs from the surface more rapidly than a less stable one. Also the hydrophobicity, charge and area of the surface are affecting the interaction with the protein. Still, the same adsorption pattern is noticed for the same protein at different surfaces, leading to the conclusion that the properties of the protein affect the interaction, rather than the properties of the surface. Biosensors containing carbonic anhydrase have been developed. These make measurement and detection of zinc ions possible. To be able to use carbonic anhydrase as a potential agent in biotechnology, attached to solid surfaces, the protein has to be biotechnologically engineered to get a more stable structure, or else the denaturation will destroy this possibility.</p></p>

<p>Heparan sulfates (HSs) are a class of anionic carbohydrate chains found at cell surfaces and in the extracellular matrix where they interact with a number of proteins. HS is characterized by extreme structural heterogeneity, and has been implicated in a number of biological phenomenon like embryogenesis, morphogen gradient formation and signalling of growth factors such as FGF, PDGF etc. Despite the characteristic structural heterogeneity, evidence from compositional studies show that the HS structure is expressed in a tightly regulated manner, implying a functional significance, which is most likely in the modulation of cell behaviour through HS-protein interactions. The lack of molecular tools has, however, hampered the understanding of HS structures with functional significance. This work therefore aims at characterizing the structural requirements on HS involved in the interaction with the anti-HS phage display antibodies HS4C3, AO4B08 and HS4E4 and a selected growth factor PDGF-BB. The characterization was done with the help of tailored oligosaccharide libraries generated from sources bearing structural resemblance to HS.</p><p>The work has thus made available tools that preferentially recognize certain structural features on the HS chain and will aid in the further study of HS structure and its regulation. Evidence is also provided to support the notion that HS protein interactions can occur in multiple manners, utilizing any of the structural features on the HS chain.</p>

Membrane proteins are essential for life, and roughly one-quarter of all open reading frames in sequenced genomes code for membrane proteins. Unfortunately, our understanding of membrane proteins lags behind that of soluble proteins, and is best reflected by the fact that only 0.5% of the structures deposited in the protein data-bank (PDB) are of membrane proteins. This discrepancy has arisen because their hydrophobicity - which enables them to exist in a lipid environment - has made them resistant to most traditional approaches used for procuring knowledge from their soluble counter-parts. As such, novel methods are required to facilitate our knowledge acquisition of membrane proteins. In this thesis a generic approach for rapidly obtaining information on membrane proteins from the classic bacterial encyclopedia Escherichia coli is described. We have developed a Green Fluorescent Protein C-terminal tagging approach, with which we can acquire information as to the topology and ‘expressibility’ of membrane proteins in a high-throughput manner. This technology has been applied to the whole E. coli inner membrane proteome, and stands as an important advance for further membrane protein research.

Protein aggregation, leading to the formation and depositions of amyloids, is a cause for a number of diseases such as Alzheimer’s and Creutzfeld-Jacob’s disease, systemic amyloidoses, type II diabetes and others . More than 20 proteins are associated with protein misfolding diseases and even a larger number of proteins can self-assemble into amyloid in vitro. Relating structural and functional properties of amyloid is of particular interest, as this will lead to the identification of the main factors and mechanisms involved in the process of protein misfolding and aggregation; consequently, this will provide a basis for developing new strategies to treat protein misfolding diseases. The aim of the thesis is to investigate structural aspects of amyloid formation and relate that to the functional properties of amyloid. The first paper describes the amyloid formation of equine lysozyme (EL). We have demonstrated that EL enters an amyloid forming pathways under conditions where the molten globule state is populated. We have found that the morphology of the amyloids depend on the calcium-binding to lysozyme, specifically the holo-protein assembles into short, linear protofilaments, while the apo-EL forms ring-shaped structures. The morphology of EL amyloid significantly differs from the amyloid fibrils of human and hen lysozymes. We have suggested that the stable alpha-helical core of EL, which remains structured in the molten globule intermediate, may obstruct the formation of fibrilar interface and therefore leads to assembly of short, curly fibrils and rings.In the second paper, we describe the cytotoxicity of EL amyloids. We have analysed the amyloid intermediates on the pathway towards amyloid fibrils. The sizes of amyloid oligomers were determined by atomic force microscopy (AFM) and the formation of cross-beta sheet was shown by thioflavin T (ThT) binding. The toxicity studies show that the oligomers formed during amyloid growth phase are toxic to a range of cell lines and cultures and the toxicity is size-dependant.The last manuscript describes a novel method for manufacturing of silver nanowires by the biotemplating using amyloid fibrils. The amyloid assembled from an abundant and cheap hen egg white lysozyme was used as a scaffold for casting ultrathin silver nanowires. We have manufactured nanowires with a diameter of 1.0-2.5 nm and up to 2 micrometers in length. Up to date, it is the thinnest silver nanowires produced by using biotemplating and at least one order of magnitude thinner than nanowires manufactured by chemical synthesis.

Heparan sulfates (HSs) are a class of anionic carbohydrate chains found at cell surfaces and in the extracellular matrix where they interact with a number of proteins. HS is characterized by extreme structural heterogeneity, and has been implicated in a number of biological phenomenon like embryogenesis, morphogen gradient formation and signalling of growth factors such as FGF, PDGF etc. Despite the characteristic structural heterogeneity, evidence from compositional studies show that the HS structure is expressed in a tightly regulated manner, implying a functional significance, which is most likely in the modulation of cell behaviour through HS-protein interactions. The lack of molecular tools has, however, hampered the understanding of HS structures with functional significance. This work therefore aims at characterizing the structural requirements on HS involved in the interaction with the anti-HS phage display antibodies HS4C3, AO4B08 and HS4E4 and a selected growth factor PDGF-BB. The characterization was done with the help of tailored oligosaccharide libraries generated from sources bearing structural resemblance to HS. The work has thus made available tools that preferentially recognize certain structural features on the HS chain and will aid in the further study of HS structure and its regulation. Evidence is also provided to support the notion that HS protein interactions can occur in multiple manners, utilizing any of the structural features on the HS chain.

The information for mitochondrial and chloroplastic protein import and targeting generally resides in the N-terminal part of the protein, called a targeting peptide. The targeting peptide is cleaved off by the organellar processing peptidases after import of a precursor protein. Free targeting peptides generated inside the organelle after import are rapidly degraded by proteolysis as their accumulation can have toxic effects on the organelle. The aim of this thesis has been to investigate the newly identified targeting peptide degrading protease, the PresequenceProtease (PreP). We have shown that the two isoforms of Arabidopsis PreP (AtPreP1 and AtPreP1) are dually targeted and localized to both mitochondria and chloroplasts. Dual targeting of the AtPreP1 is due to an ambiguous targeting peptide with a domain organization for mitochondrial and chloroplastic targeting. Both the AtPreP1 and AtPreP2 are expressed in Arabidopsis in an organ specific manner and they have distinct but overlapping substrate specificity. The crystal structure of the recombinant AtPreP1 E80Q was solved at 2.1 Å resolution. The structure represents the first substrate bound, closed conformation of a protease from the pitrilysin family. The PreP polypeptide folds in a unique peptidasome structure, surrounding a huge cavity of more than 10 000 Å3 in which the active site resides. A novel mechanism for proteolysis is proposed involving hinge-bending motions in response to substrate binding. PreP in human mitochondria has a novel function: degradation of amyloid β-peptide (Aβ). We show that under circumstances when Aβ is present in mitochondria of Alzheimer’s patients, PreP is the protease responsible for degradation of this toxic peptide.

The synthesis and breakdown of sugars is one of the most important functions in Nature. Consequently, sugar structures are used both as energy storage and as building blocks to stabilise and protect the cell. The formation of these structures is performed by glycosyltransferases (GT), an enzyme group structurally conserved within all kingdoms. Until now, only two different folds have been discovered by crystallisation of GTs, i.e. GT-A and GT-B. A third fold family has however been proposed by fold predictions. In this thesis, a multivariate data analysis was successfully used in classifying and predicting both fold and reaction mechanism (inverting or retaining) of GTs. This method was also used to obtain information about the separating parameters for the reaction mechanism classification. This information could be traced back to the amino acid sequence. The method could as well be used to analyse and identify the properties of membrane binding regions of GTs, and subsequently distinguish soluble from membrane-associated enzymes. Most functionally characterised enzymes only use one substrate, synthesising one product. Mycoplasma pneumoniae, a common human pathogen with a small genome has only three proposed GTs. The bacterium was, however expected to have a greater number of GTs, due to its ability to make both glycolipids and capsule. Here we have determined the function of one of these enzymes, MPN483 and discovered its ability to both use different acceptors, and make elongated glycolipids with up to three galactose residues, with both DAG and ceramide as the base. Many of the synthesised glycolipids were also found to be immunogenic, hence showing their biological importance. The properties of lipids are known to be important for the function of a biological membrane. We have here shown that not only the charge but also the shape of the lipids are important for several protein mediated membrane processes in Echerichia coli, such as the function of the LacY.