Starting with the development of an enzymatic assay, where an enzyme in solution hydrolysed a solid-phase bound peptide, a model for the kinetics of enzyme action was introduced. This model allowed the estimation of kinetic parameters and enzyme activity for a system that has the peculiarity of not being saturable with the substrate, but with the enzyme. In a derivation of the model, it was found that the sensitivity of the signal to variations in the enzyme concentration had a transient increase along the reaction progress with a maximum at high substrate conversion levels. The same behaviour was derived for the sensitivity in classical homogeneous enzymatic assays and experimental evidence of this was obtained. The impact of the transient increase of the sensitivity on the error structure, and on the ability of homogeneous end-point enzymatic assays to detect competitive inhibition, came into focus. First, a non-monotonous shape in the standard deviation of progress curve data was found and it was attributed to the random dispersion in the enzyme concentration operating through the transient increase in the sensitivity. Second, a model for the detection limit of the quantity Ki/[I] (the IDL-factor) as a function of the substrate conversion level was developed for homogeneous end-point enzymatic assays. It was found that the substrate conversion level where the IDL-factor reached an optimum was beyond the initial velocity range. Moreover, at this optimal point not only the ability to detect inhibitors but also the robustness of the assays was maximized. These results may prove to be relevant in drug discovery for optimising end point homogeneous enzymatic assays that are used to find inhibitors against a target enzyme in compound libraries, which are usually big (>10000) and crowded with irrelevant compounds.

Many different chemicals are used in the everyday life, like detergents and pharmaceuticals. However, their production has a big impact on health and environment as much of the raw materials are not renewable and the standard ways of production in many cases includes toxic and environmentally hazardous components. As the population and as the life standard increases all over the planet, the demand for different important chemicals, like pharmaceuticals, will increase. A way to handle this is to apply the concept of Green chemistry, where biocatalysis, in the form of enzymes, is a very good alternative. Enzymes do not normally function in industrial processes and needs modifications through protein engineering to cope in such conditions. To be able to efficiently improve an enzyme, there is a need to understand the mechanism and characteristics of that enzyme. Acyloins (α-hydroxy ketones) are important building blocks in the synthesis of pharmaceuticals. In this thesis, the enzyme alcohol dehydrogenase A (ADH-A) from Rhodococcus ruber has been in focus, as it has been shown to display a wide substrate scope, also accepting aryl-substituted alcohols. The aim has been to study the usefulness of ADH-A as a biocatalyst towards production of acyloins and its activity with aryl-substituted vicinal diols and to study substrate-, regio-, and enantioselectivity of this enzyme. This thesis is based on four different papers where the focus of the first has been to biochemically characterize ADH-A and determine its mechanism, kinetics and its substrate-, regio-, and enantioselectivity. The second and third paper aims towards deeper understanding of some aspects of selectivity of ADH-A. Non-productive binding and its importance for enantioselectivity is studied in the second paper by evolving ADH-A towards increased activity with the least favored enantiomer through protein engineering. In the third paper, regioselectivity is in focus, where an evolved variant displaying reversed regioselectivity is studied. In the fourth and last paper ADH-A is studied towards the possibility to increase its activity towards aryl-substituted vicinal diols, with R-1-phenyl ethane-1,2-diol as the model substrate, and the possibility to link ADH-A with an epoxide hydrolase to produce acyloins from racemic epoxides.

Ground state destabilization is a hypothesis to explain enzyme catalysis. The most popular interpretation of it is the entropic effect, which states that enzymes accelerate biochemical reactions by bringing the reactants to a favorable position and orientation and the entropy cost of this is compensated by enthalpy of binding. Once the enzyme-substrate complex is formed, the reaction could proceed with negligible entropy cost. Deamination of cytidine catalyzed by E.coli cytidine deaminase appears to agree with this hypothesis. In this reaction, the chemical transformation occurs with a negligible entropy cost and the initial binding occurs with a large entropy penalty that is comparable to the entropic cost of the uncatalyzed reaction. Our calculations revealed that this reaction occurs with different mechanisms in the cytidine deaminase and water. The uncatalyzed reaction involves a concerted mechanism and the entropy cost of this reaction appears to be dominated by the reacting fragments and first solvation shell. The catalyzed reaction occurs via a stepwise mechanism in which a hydroxide ion acts as the nucleophile. In the active site, the entropy cost of hydroxide ion formation is eliminated due to pre-organization of the active site. Hence, the entropic effect in this reaction is due to a pre-organized active site rather than ground state destabilization. In the second part of this thesis, we investigated peptide bond formation and peptidyl-tRNA hydrolysis at the peptidyl transferase center of the ribosome. Peptidyl-tRNA hydrolysis occurs by nucleophilic attack of a water molecule on the ester carbon of peptidyl-tRNA. Our calculations showed that this reaction proceeds via a base catalyzed mechanism where the A76 O2’ is the general base and activates the nucleophilic water. Peptide bond formation occurs by nucleophilic attack of the α-amino group of aminoacyl-tRNA on the ester carbon of peptidyl-tRNA. For this reaction we investigated two mechanisms: i) the previously proposed proton shuttle mechanism which involves a zwitterionic tetrahedral intermediate, and ii) a general base mechanism that proceeds via a negatively charged tetrahedral intermediate. Although both mechanisms resulted in reasonable activation energies, only the proton shuttle mechanism found to be consistent with the pH dependence of peptide bond formation.

Water is “the matrix of life” for its fascinating properties. The well-known simple water molecule consists of one oxygen atom and two hydrogen atoms, covering most of planet earth’ssurface. It is the most studied element in science; however, its properties are still not fully understood. Another essential building block of life is proteins, which manifest naturally in aqueous environments. The protein activity is controlled by the protein folding process that is dependent on the surrounding environment. It is hypothesized that the hydrogen bond network of water plays an important role in the folding process. Here, we investigate the protein lysozyme in liquid water as well as in the crystalline state ice Ih, exploring various temperatures, using FT-IR and XRD. Our main finding is that a transition occurs at approximately T=210 K, indicative of the hypothesised protein dynamic “glass” transitionobserved by previous studies in supercooled water at similar temperatures.

Due to global warming, our planet will experience more extreme weather conditions. Plants can protect themselves against these abiotic stress conditions with their stress response, which includes alternative splicing of certain genes. Alternative splicing is a post-transcriptional process where a single gene gives rise to different mRNAs, which in turn produces different proteins. In plants, this is usually done by intron retention. One type of protein that may be involved in this stress response are the non-specific lipid transfer proteins (LTPs). Indeed, evidence of intron retention has been found in the LTP genes in the liverwort Marchantia polymorpha, called MpLTPd. To investigate whether this alternative splicing is caused by abiotic stress or not, I subjected the moss to two different types of stress trials, drought and cold, and compared the general expression of the intron in MpLTPd2 and MpLTPd3 from the stressed samples to samples from a moss grown under normal conditions. I found that the expression of the intron did change in the stressed moss, but none of the differences were significant. This suggests that alterative splicing in MpLTPd2 and MpLTPd3 is not caused by cold and drought and that the intron-containing protein plays no role in the protection of M. polymorpha against abiotic stress.

Investigation of tumor growth has traditionally been studied focusing only on the cancer cells. However, tumors consist of a complex tissue organization where heterotypic signaling occurs between different cell types. The cross-talk between tumor cells and other surrounding cell types may ultimately prove to be as important for the tumor cell behavior as the internal signaling cascades in the tumor cell itself.Myeloid cells, such as granulocytes and monocytes, and thrombocytes play an important role in the tumor tissue, as a tumor can be compared to a wound healing process without the normal regulation mechanisms. Platelets are thought to facilitate tumor cell extravasation by binding to the tumor cell and recruiting myeloid cells that secrete factors aiding tumor migration through the endothelial cells. Studying the content of granules and vesicles of the platelets and myeloid cells can provide important knowledge about how the tumor interactions are mediated and which key proteins that controls these processes.Serglycin is an intracellular proteoglycan that attaches chains of negatively charged glycosaminoglycans. It is thought to have a function in retaining and storing proteins in hematipoietic cells. In this project the impact of the loss of serglycin on platelets and myeloid cells was investigated, using a spontaneous insulinoma serglycin knockout mouse model. The results suggests that serglycin does not affect the amount of neutrophil granulocytes and monocytes in peripheral blood, nor does it seem to affect the amount of platelets sequestered to the tumor tissue. A co-staining for platelets and MMP9 positive granulocytes was also performed in order to assess if granulocyte-platelet interactions in the tumor were affected by loss of serglycin. Interactions between these cells were observed in both genotypes. Von Willebrand factor levels in the tumor tissue also remained unchanged upon loss of serglycin. However, preliminary experiments indicated that serglycin seems to play a role in the intracellular amounts of vimentin and VEGFB in undifferentiated primary bone marrow derived monocytes.

Trypanosoma brucei is a causative agent of African sleeping sickness. It is an extracellular parasite which circulates in the blood, lymph and eventually invades the central nervous system. There is a great need for new medicines against the disease and specific properties of nucleoside kinases in the pathogen can be exploited as targets for chemotherapy.  T. brucei contains a gene where two thymidine kinase sequences are fused into a single open reading frame. These types of tandem thymidine kinases were found only in different types of parasites, which made us to believe that it might be beneficial for them. Each thymidine kinase sequence in these tandem enzymes are here referred to as a domain. By cloning and expressing each domain from T. brucei separately, we found that domain 1 was inactive and domain 2 was as active as the full-length enzyme. T. brucei thymidine kinase phosphorylated the pyrimidine nucleosides thymidine and deoxyuridine and to some extent purine nucleosides like deoxyinosine and deoxyguanosine. Human thymidine kinase increases the affinity to its substrates when it forms oligomers. Similarly, the T. brucei two thymidine kinase sequences, which can be viewed as a pseudodimer, had a higher affinity to its substrates than domain 2 alone.  T. brucei lacks de novo purine biosynthesis and it is therefore dependent on salvaging the required purine nucleotides for RNA and DNA synthesis from the host. Purine salvage is considered as a target for drug development. It has been shown that in the presence of deoxyadenosine in the growth medium, the parasites accumulate high levels of dATP and the extensive phosphorylation of deoxyadenosine leads to depleted ATP pools. Initially, we wondered if deoxyadenosine could be used as a drug against T. brucei. However, we found that T. brucei is partially protected against deoxyadenosine because it was cleaved by the enzyme methylthioadenosine phosphorylase (MTAP) to adenine and ribose-1-phosphate. At higher concentration of deoxyadenosine, 3 the formed adenine was not efficiently salvaged into ATP and started to inhibit MTAP instead. The deoxyadenosine was then instead phosphorylated by adenosine kinase leading to accumulation of dATP. The MTAP reaction makes deoxyadenosine itself useless as a drug and instead we focused on finding analogues of deoxyadenosine or adenosine that were cleavage-resistant and at the same time good substrates of T. brucei adenosine kinase. Our best hit was then 9-(2-deoxy-2-fluoro-ß-D-arabinofuranosyl) adenine (FANA-A). An additional advantage of FANA-A as a drug was that it was taken up by the P1 nucleoside transporter family, which makes it useful also against multidrug resistant parasites that often have lost the P2 transporter function and take up their purines solely by the P1 transporter. In parallel with our study of nucleoside metabolism in T. brucei, we also have a collaboration project where we screen essential oils from plants which are used in traditional medicine. If the essential oils are active against the trypanosomes, we further analyze the different components in the oils to identify new drugs against African sleeping sickness. One such compound identified from the plant Smyrnium olusatrum is isofuranodiene, which inhibited T. brucei proliferation with an IC50 value of 3 μM.

Solute carriers (SLCs) are membrane-bound transporter proteins, important for nutrient, ion, drug and metabolite transport across membranes. A quarter of the human genome codes for membrane-bound proteins, and SLCs make up the largest group of transporter proteins. Due to their ability to transport a large repertoire of substances across, not just the plasma membrane, but also the membrane of internal organelles, they hold a key position in maintaining homeostasis affecting metabolic pathways. Unfortunately, some of the more than 400 identified SLCs are still not fully characterized, even though a quarter of these are associated with human disease. In addition, there are about 30 membrane-bound proteins with strong resemblance to SLCs, of which very little is known. The aim of this thesis is to characterize some of these putative SLCs, focusing on their localization and function in the central nervous system. Since many of the known SLCs play a vital part in metabolism and related pathways, the response to different nutritional conditions has been used as a key method. MFSD14A and MFSD14B, characterized in Paper I, are putative SLCs belonging to the Major Facilitator Superfamily (MFS) and found to be neuronal, differentially expressed in the mouse central nervous system and transiently upregulated in mouse embryonic cortex cultures due to amino acid deprivation. They were also altered in areas of the mouse brain after starvation as well as after high fat diet. In Paper II, the effect on gene regulation due to complete amino acid starvation was monitored in a mouse hypothalamic cell line and 47 different genes belonging to SLCs, or putative SLCs, were found to be affected. Of these, 15 genes belonged to already known amino acid transporters, whereas 32 were putative SLCs with no known function or SLCs not known to react to amino acids. The three SV2 proteins, SV2A, SV2B and SV2C, were studied in Paper III using human neuroblastoma cell lines. The high metabolic state of cancers often result in an upregulation and alteration of transporter proteins, and alterations of the SV2 proteins were found following different treatments performed in this study. Paper IV focused on putative SLCs of MFS type and their role in glucose metabolism. Mouse embryonic cortex cultures were subjected to glucose starvation and the gene expression of 19 putative transporters were analyzed. All but four of the putative transporters were affected either at 3h or 12h of glucose deprivation. In conclusion, several SLCs and putative SLCs studied in this thesis are strongly affected by alteration in metabolism, either due to amino acids or glucose or both. This makes the putative SLCs dynamic membrane-bound proteins, possibly transporters, highly affected by nutritional status and most likely regulated to maintain homeostasis.

Proteins are the most abundant biomolecules within a cell and are involved in all biochemical cellular processes ultimately determining cellular function. Therefore, to develop a complete understanding of cellular processes, obtaining knowledge about protein function and interaction at a molecular level is critical. Consequently, the investigation of proteins in their native environment or in partially purified mixtures is a major endeavour in modern life sciences. Due to their high chemical similarity, the inherent problem of studying proteins in complex mixtures is to specifically differentiate one protein of interest from the bulk of other proteins. Site-specific protein functionalisation strategies have become an indispensable tool in biochemical- and cell biology studies. This thesis presents the development of a new enzymatic site-specific protein functionalisation strategy that is based on the reversible covalent phosphocholination of short amino acid sequences in intact proteins. A synthetic strategy has been established that allows access to functionalised CDP-choline derivatives carrying fluorescent reporter groups, affinity tags or bioorthogonal handles. These CDP-choline derivatives serve as co-substrates for the bacterial phosphocholinating enzyme AnkX from Legionella pneumophila, which transfers a phosphocholine moiety to the switch II region of its native target protein Rab1b during infection. We identified the octapeptide sequence TITSSYYR as the minimum recognition sequence required to direct the AnkX catalysed phosphocholination and demonstrated the functionalisation of proteins of interest carrying this recognition tag at the N- or C-terminus as well as in internal loop regions. Moreover, this covalent modification can be hydrolytically reversed by the action of the Legionella enzyme Lem3, which makes the labeling strategy the first example of a covalent and reversible approach that is fully orthogonal to current existing methodologies. Thus, the here presented protein functionalisation approach holds the potential to increase the scope of possible labeling strategies in complex biological systems. In addition to the labeling of tagged target proteins, a CDP-choline derivative equipped with a biotin affinity-tag was synthesised and used in pull-down experiments to investigate the substrate scope of AnkX and to elucidate the role of protein phosphocholination during Legionella pneumophila infection. / Proteiner utgör huvudbeståndsdelen av alla biomolekyler i en cell. Dessa är involverade i alla cellulära processer som bestämmer cellens egenskaper. För att förstå de cellulära processerna är det nödvändigt att förstå proteinernas funktion på molekylär nivå. Att studera proteiner i deras naturliga omgivning, det vill säga inuti en cell eller i ett cellextrakt, är en stor utmaning i dagens livsvetenskaper. Eftersom proteiner är kemiskt lika varandra så är det svårt att skilja ett från tusentals andra. Att specifikt märka proteiner för att skilja ut dem från bakgrunden har blivit ett viktigt arbetssätt i modern biokemi och cellbiologi. Avhandlingen beskriver utvecklandet av en ny metod för reversibel och kovalent enzymatisk märkning baserat på fosfokolinering/defosfokolinering av en kort aminosyrasekvens i intakta proteiner. En syntesmetod för att framställa onaturliga CDP-kolinderivat har etablerats vilket tillåter oss att framställa CDP-kolin som bär en funktionalitet, vilket kan vara ett färgämne eller en affinitetstagg. Dessa onaturliga CDP-kolinderivat accepteras som co-substrat av enzymet AnkX från Legionella pneumophila vilket transfererar den funktionaliserade delen av CDP-kolinderivatet till en kort aminosyrasekvens baserad på AnkX’s naturliga substrat vid infektion, det lilla GTPaset Rab1. Under avhandlingsarbetets gång identifierades den kortaste aminosyrasekvensen som känns igen av AnkX, endast de åtta aminosyrorna TITSSYYR är nödvändiga för igenkänning av AnkX. Dessa åtta aminosyror kan genetiskt infogas i början, slutet eller mitt i ett protein för igenkänning och funktionalisering via AnkX och våra syntetiska CDP-kolinderivat. Vid Legionellainfektion i eukaryota celler klyvs fosfokolineringen efter en viss tid, eftersom Legionella pneumophila producerar ett fosfodiesteras, Lem3, som tar bort de fosfokolineringar som AnkX har installerat när de inte längre behövs. Vi har använt Lem3 för att ta bort märkning i sekvensen TITSS(PC)YYR, vilket gör vår strategi helt reversibel. Vi har kunnat demonstrera att AnkX-Lem3 systemet accepterar ett brett spektrum av CDP-kolinderivat, vilket gör metoden till den första av sitt slag, eftersom den är fullt reversibel. Vi har vidare undersökt vilka proteiner AnkX reagerar med inuti celler, vi använde oss av ett CDP-kolinderivat funktionaliserat med biotin, vilket har tillåtit oss att fiska ut alla de proteiner som fosfokolineras av AnkX. Förutom de små GTPaserna i Rab-familjen så identifierade vi även IMPDH2, ett enzym som reglerar det hastighetsbestämmande steget i syntesen av guanosin-nukleotider. Detta är mycket intressant, eftersom det leder till frågan ifall Legionella pneumophila manipulerar sin värdcell genom att förändra mängden GTP i förhållande till ATP.

The Central Dogma of molecular biology describes a framework for how genetic information is transferred in cells, placing RNA as a messenger between DNA and translated proteins. During the last years, interest in RNA research has grown tremendously due to the increasing understanding and recognition of the importance of RNA in regulation of gene expression, biochemical catalysis, and genome integrity surveillance. Most importantly, RNA content, unlike DNA, changes constantly, fine-tuning the cellular response to match the environmental conditions. There is a clear potential for RNA biomarkers, reflecting both the natural and pathological conditions in vivo. Various methods have been developed to study RNA, of which the most common tools and techniques are described in this thesis. Since many of these gold standard methods are based on detecting RNA derivative (cDNA), there is a wide scope for efficient alternative tools directly targeting RNA. In Paper I, the spatiotemporal expression of human adenovirus-5 mRNA in epithelial and blood cells infected with the virus has been studied. For this, padlock probes and rolling circle amplification (RCA) were used to visualize, quantify and analyse both viral and host cell cDNAs in different infection scenarios, at single cell level. In Paper II, direct RNA detection fidelity has been evaluated using padlock probes. A novel type of probe (iLock) that is activated on RNA via invasive cleavage mechanism, prior to RCA was developed in this approach. Using iLocks, a substantial improvement of direct RNA sensing fidelity has been observed. In Paper III, RNA modifications were introduced in otherwise DNA iLock probes to enhance the probes’ efficiency on miRNAs. Using chimeric iLock probes, multiplexed differentiation of conserved miRNA family members were performed with next- generation sequencing-by-ligation readout. Efficient replication of chimeric probes used in Paper III implies that the Phi29 DNA polymerase readily accepts RNA-containing circles as amplification substrates. In Paper IV, real-time RCA monitoring for measurement of amplification rates and analysis of amplification patterns of various RNA-containing circles was achieved. Moreover, the RCA products were sequenced as a proof for the reverse-transcriptase activity of the Phi29 DNA polymerase. This thesis effectively contributes to a better understanding of mechanisms influencing RNA detection with, but not limited to, padlock probes. It expands the available RNA analyses toolkit with novel strategies and solutions, which can be potentially adapted for RNA-focused research, in general and molecular diagnostics, in particular. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.</p>