The four-electron reduction of dioxygen to water is the most exothermic non-photochemical reaction available to biology. A detailed molecular description of this reaction is needed to understand oxygen-based redox processes. Horseradish peroxidase (HRP) is a haem-containing redox enzyme capable of catalysing the reduction of dioxygen to water. We developed instrumentation and experimental methodology to capture and characterise by X-ray crystallography transient reaction intermediates in this reaction. An instrument was designed (“the vapour stream system”) to facilitate reaction initiation, monitoring and intermediate trapping. In combination with single crystal microspectrophotometry, it was used to obtain conditions for capturing a reactive dioxygen complex in HRP. X-ray studies on oxidised intermediates can be difficult for various reasons. Electrons re-distributed in the sample through the photoelectric effect during X-ray exposure can react with high-valency intermediates. In order to control such side reactions during data collection, we developed a new method based on an angle-resolved spreading of the X-ray dose over many identical crystals. Composite data sets built up from small chunks of data represent crystal structures which received different X-ray doses. As the number of electrons liberated in the crystal is dose dependent, this method allows us to observe and drive redox reactions electron-by-electron in the crystal, using X-rays. The methods developed here were used to obtain a three-dimensional movie on the X-ray-driven reduction of dioxygen to water in HRP. Separate experiments established high resolution crystal structures for all intermediates, showing such structures with confirmed redox states for the first time. Activity of HRP is influenced by small molecule ligands, and we also determined the structures of HRP in complex with formate, acetate and carbon monoxide. Other studies established conditions for successfully trapping the M-intermediate in crystals of mutant bacteriorhodopsin, but the poor diffraction quality of these crystals prevented high-resolution structural studies.

Structural biology has become an important part of researching various diseases and drug development. In this thesis, I provide details on how I worked with approaches to structural characterization of a heteromeric GABA(A)R. These pentameric ligand gated ion channels take part in regulating inhibition of action potentials in nerve cells by allowing the passage of Cl- ions when bound by gamma-aminobutyric acid (GABA). They are formed by the assembly of five subunits which can be of various different types, denoted by greek letters and a number. Much is still unknown about how GABA and several other ligands bind to these ion channels and how that impacts function. Obtaining a structure of these proteins can aid in closing those knowledge gaps. It is reasonable to screen the proteins you have before you study their structures by Cryo-EM in order to get the best result, a methodology for which is described here. I have followed this methodology to screen two heteromeric GABA$_A$R that we wish to determine the structure of, alpha 5 beta 3 and rho 1 gamma 2. Neither of the combinations of genes we used to express these proteins proved to produce the desired fully assembled heteromeric protein. In the case of alpha 5 beta 3, we only witnessed building blocks, with no fully assembled channels. In rho 1 gamma 2, we instead only witnessed fully formed homomers of the rho 1 subunit. These findings then exclude the gene constructs used from further structural study, and the methodology described will inform the next steps to be taken. / Strukturbiologi har blivit en viktig del av forskningen kring många sjukdomar samt utveckling av läkemedel. I denna uppsats delger jag hur jag arbetat med metoder för strukturell karakterisering av en heteromerisk GABA(A)R. Dessa pentameriska ligandstyrda jonkanaler deltar i regleringen av hämning av aktionspotentialer i nervceller genom att tillåta passagen av Cl- joner när gamma-aminosmörsyra (GABA) binder. Dessa består av fem subenheter som kan vara en av flera olika typer, vilka anges med en grekisk bokstav och en siffra. Mycket om hur GABA och andra ligander binder till dessa jonkanaler och hur det påverkar dess funktion är fortfarande okänt. Att hitta en struktur av dessa proteiner kan hjälpa oss att stänga kunskapsgapen. Det är klokt att undersöka om genen man ska använda för att uttrycka det sökta proteinet ger det man söker innan man sen börjar studera strukturen. Jag har beskrivit en metodologi för detta och följt den för två heteromeriska proteiner, alpha 5 beta 3 och rho 1 gamma 2. Ingen av kombinationerna av gener vi använt för att uttrycka dessa proteiner har producerat de sökta, fullt ihoppbyggda proteinerna. I fallet för alpha 5 \beta 3 så ser vi endast byggstenar och inga kompletta proteiner, och för rho 1 gamma 2 så ser vi endast homomeriska proteiner av rho 1. Dessa slutsatser exkluderar de genkonstruktioner vi använt från vidare strukturella studier, och stegen som bör tas härnäst beskrivs av den använda metodologin.

Therapeutic antibodies dominate the pharmaceutical market and improve the lives of millions of people annually. One important step when developing new medicines is to kinetically characterise the drug candidates. For antibodies this is difficult since many antibody reactions are extremely slow. By combining a mathematical formula that was recently published with the well-established technology from Gyros Protein Technologies, a new method for full kinetic characterization was developed and tested in this master thesis. The method provided precise data for five antibodies while also proving to be highly reproducible. By using small sample volumes, unlabelled reagents and having the reaction proceed in solution, this method offers advantages compared to many conventional approaches.

<p>Diiron carboxylate proteins constitute an important class of metall-containing enzymes. These proteins perform a multitude of reactions in biological systems that normally involve activation of molecular oxygen at the diiron site.</p><p>During activation and functioning of these proteins their diiron sites undergo redox changes in a rather wide range: from diferrous (FeII-FeII) to high potential intermediate Q(FeIV-FeIV). Two of these redox states are paramagnetic: (FeIV-FeIII), called high potential intermediate X, and (FeII-FeIII), called mixed-valent state of the diiron carboxylate proteins. In the present work it has been shown that these redox states are of functional relevance in two proteins with different functions.</p><p>Ribonucleotide reductase (RNR) from the human parasite<i> Chlamydia trachomatis</i> is a class I RNR. It is typical for class I RNR to initiate the enzymatic reaction on its large subunit, protein R1, by activation from a stable tyrosyl free radical in its small subunit, protein R2. This radical, in its turn, is formed through oxygen activation by the diiron center. In C. trachomatis the tyrosine residue is replaced by phenylalanine, which cannot form a radical. We have shown in the present work, that active <i>C. trachomatis</i> RNR uses the FeIII-FeIV state of the diiron carboxylate cluster in R2 instead of a tyrosyl radical to initiate the catalytic reaction.</p><p>The alternative oxidase (AOX) is a ubiquinol oxidase found in the mitochondrial respiratory chain of plants. The existence of the diiron carboxylate center in this protein was predicted on the basis of a conserved sequence motif consisting of the proposed iron ligands, four glutamate and two histidine residues. In experiments modeling the conditions of the enzyme catalytic cycle, i.e. reduction and reoxygenation of the overexpressed AOX in <i>Escherichia coli</i> membranes we were able to generate an EPR signal characteristic of a mixed-valent Fe(II)/Fe(III) binuclear iron center. The alternative oxidase is the first membrane protein where the existence of the diiron carboxylate center has been shown experimentally.</p>

<p>Experimentally derived biomolecular structures were determined by Nuclear Magnetic Resonance (NMR). The properties of selected peptides and proteins in solution and in membrane mimicking micelles were observed by circular Dichroism (CD), mass spectrometry (MS), and other spectroscopic techniques.</p><p>The mDpl(1-30) peptide (30 residues) of the mouse Doppel protein was found to be positioned as an α-helix in a DHPC micelle. The same peptide can disrupt and cause leakage in small unilamellar vesicles.</p><p>Single D-amino acid isomers of Trp-cage (20 residues), the smallest peptide with a protein-like fold, were analyzed by CD spectroscopy and were found to have different secondary structures and melting temperatures. They were compared against MS measurements specially designed to reveal the secondary structure of proteins.</p><p>We studied a novel protein in E. coli of unknown structure that is encoded by the putative transcription factor ORF: ygiT (131 residues). This protein comprises a helix-turn-helix (HTH) domain in the C-terminus and contains two CxxC motives in the N-terminal domain, which binds Zn. This protein was named 2CxxC. We succeeded in overexpressing and purifying 2CxxC in E. coli with enough yield for a 13C, 15N uniformly labeled NMR sample. The chemical shift assignment was completed and the NMR structure was calculated in reducing, slightly acidic conditions (1mM DTT, pH 5.5). The determined HTH domain shows good similarity with structures predicted by a homology search, while the N-terminal domain has no other homologous structure in the Protein Data Bank (PDB).</p><p>The structure of the paddle region (27 residues) of the HsapBK(233-260) voltage and Ca+2 activated potassium channel, in DPC micelles, was determined by NMR. It shows a helix-turn-helix loop, which agrees well with the expected structure and could help to verify the proposed models of the voltage gating mechanism.</p><p>The C-repressor (dimer of 99 residues) of bacteriophage P2 was analyzed by NMR. We assigned the chemical shifts and NMR structure determination is under way.</p>

The complexity of multi cellular organisms demands systems that facilitate communicationbetween cells. The neurons in our brains for instance are specialized in this cell-cellcommunication. The flow of ions, through their different ion channels, across the membrane, isresponsible for almost all of the communication between neurons in the brain by changing theneurons membrane potentials. Voltage-gated ion channels open when a certain thresholdpotential is reached. This change in membrane potential is detected by voltage-sensors in the ionchannels. In this licentiate thesis the Homo sapiens voltage- and calcium-gated BK potassiumchannel (HsapBK) has been studied. The NMR solution structure of the voltage-sensor ofHsapBK was solved to shed light upon the voltage-gating in these channels. Structures of othervoltage-gated potassium channels (Kv) have been determined by other groups, enablingcomparison among different types of Kv channels. Interestingly, the peptide-lipid interactions ofthe voltage-sensor in HsapBK are crucial for its mechanism of action.Uni cellular organisms need to sense their environment too, to be able to move towardsmore favorable areas and from less favorable ones, and to adapt their gene profiles to currentcircumstances. This is accomplished by the two-component system, comprising a sensor proteinand a response regulator. The sensor protein transfers signals across the membrane to thecytoplasm. Many sensor proteins contain a HAMP domain close to the membrane that isinvolved in transmitting the signal. The mechanism of this transfer is not yet revealed. Ourstudies show that HAMP domains can be divided into two groups based on the membraneinteraction of their AS1 segments. Further, these two groups are suggested to work by differentmechanisms; one membrane-dependent and one membrane-independent mechanism.Both the voltage-gating mechanism and the signal transduction carried out by HAMPdomains in the membrane-dependent group, demand peptide-lipid interactions that can be readilymodulated. This modulation enables movement of peptides within membranes or within thelipid-water interface. These conditions make these peptides especially suitable for NMR studies.

Diiron carboxylate proteins constitute an important class of metall-containing enzymes. These proteins perform a multitude of reactions in biological systems that normally involve activation of molecular oxygen at the diiron site. During activation and functioning of these proteins their diiron sites undergo redox changes in a rather wide range: from diferrous (FeII-FeII) to high potential intermediate Q(FeIV-FeIV). Two of these redox states are paramagnetic: (FeIV-FeIII), called high potential intermediate X, and (FeII-FeIII), called mixed-valent state of the diiron carboxylate proteins. In the present work it has been shown that these redox states are of functional relevance in two proteins with different functions. Ribonucleotide reductase (RNR) from the human parasite Chlamydia trachomatis is a class I RNR. It is typical for class I RNR to initiate the enzymatic reaction on its large subunit, protein R1, by activation from a stable tyrosyl free radical in its small subunit, protein R2. This radical, in its turn, is formed through oxygen activation by the diiron center. In C. trachomatis the tyrosine residue is replaced by phenylalanine, which cannot form a radical. We have shown in the present work, that active C. trachomatis RNR uses the FeIII-FeIV state of the diiron carboxylate cluster in R2 instead of a tyrosyl radical to initiate the catalytic reaction. The alternative oxidase (AOX) is a ubiquinol oxidase found in the mitochondrial respiratory chain of plants. The existence of the diiron carboxylate center in this protein was predicted on the basis of a conserved sequence motif consisting of the proposed iron ligands, four glutamate and two histidine residues. In experiments modeling the conditions of the enzyme catalytic cycle, i.e. reduction and reoxygenation of the overexpressed AOX in Escherichia coli membranes we were able to generate an EPR signal characteristic of a mixed-valent Fe(II)/Fe(III) binuclear iron center. The alternative oxidase is the first membrane protein where the existence of the diiron carboxylate center has been shown experimentally.

Experimentally derived biomolecular structures were determined by Nuclear Magnetic Resonance (NMR). The properties of selected peptides and proteins in solution and in membrane mimicking micelles were observed by circular Dichroism (CD), mass spectrometry (MS), and other spectroscopic techniques. The mDpl(1-30) peptide (30 residues) of the mouse Doppel protein was found to be positioned as an α-helix in a DHPC micelle. The same peptide can disrupt and cause leakage in small unilamellar vesicles. Single D-amino acid isomers of Trp-cage (20 residues), the smallest peptide with a protein-like fold, were analyzed by CD spectroscopy and were found to have different secondary structures and melting temperatures. They were compared against MS measurements specially designed to reveal the secondary structure of proteins. We studied a novel protein in E. coli of unknown structure that is encoded by the putative transcription factor ORF: ygiT (131 residues). This protein comprises a helix-turn-helix (HTH) domain in the C-terminus and contains two CxxC motives in the N-terminal domain, which binds Zn. This protein was named 2CxxC. We succeeded in overexpressing and purifying 2CxxC in E. coli with enough yield for a 13C, 15N uniformly labeled NMR sample. The chemical shift assignment was completed and the NMR structure was calculated in reducing, slightly acidic conditions (1mM DTT, pH 5.5). The determined HTH domain shows good similarity with structures predicted by a homology search, while the N-terminal domain has no other homologous structure in the Protein Data Bank (PDB). The structure of the paddle region (27 residues) of the HsapBK(233-260) voltage and Ca+2 activated potassium channel, in DPC micelles, was determined by NMR. It shows a helix-turn-helix loop, which agrees well with the expected structure and could help to verify the proposed models of the voltage gating mechanism. The C-repressor (dimer of 99 residues) of bacteriophage P2 was analyzed by NMR. We assigned the chemical shifts and NMR structure determination is under way.

Livet tas för givet av de flesta. Det finns däremot många som ägnar stora delar av sitt liv för att försöka lösa dess mysterier. En del av lösningen ligger i att förstå hur alla molekyler är sammanlänkade i det gigantiska nätverk som definierar den levande organismen. Under det senaste seklet har en hel del forskning utförts för att kartlägga dessa nätverk. Resultatet av dessa mödor kan vi se i de läkemedel som vi har idag och som har utvecklats för att bota eller åtminstone lindra olika sjukdomar och tillstånd. Dessvärre finns det fortfarande många sjukdomar som är obotliga (t.ex. cancer) och mycket arbete krävs för att förstå dem till fullo och kunna designa framgångsrika behandlingar. Arbetet i denna avhandling beskriver en analytisk plattform som kan användas för att effektivisera kartläggningsprocessen; protein-mikroarrayer. Mikroarrayer är ytor som har mikrometerstora (tusendels millimeter) strukturer i ett regelbundet mönster med möjligheten att studera många interaktioner mellan biologiska molekyler samtidigt. Detta medför snabbare och fler analyser - till en lägre kostnad. Protein-mikroarrayer har funnits i ungefär ett decennium och har följt i fotspåren av de framgångsrika DNA-mikroarrayerna. Man bedömer att protein-mikroarrayerna har en minst lika stor potential som DNA mikroarrayerna då det egentligen är mer relevant att studera proteiner, som är de funktionsreglerande molekylerna i en organism. Vi har i detta arbete tillverkat modellytor för stabil inbindning av proteiner, som lämnar dem intakta, funktionella och korrekt orienterade i ett mikroarray format. Därmed har vi adresserat ett stort problem med protein mikroarrays, nämligen att proteiner är känsliga molekyler och har i många fall svårt att överleva tillverkningsprocessen av mikroarrayerna. Vi har även studerat en metod att tillverka mikroarrayer av proteiner bundna till strukturer, som modellerats att efterlikna cellytor. Detta är särkilt viktigt eftersom många (hälften) av dagens (och säkerligen framtidens) läkemedel är riktade mot att påverka denna typ av proteiner och att studera dessa i sin naturliga miljö är därför väldigt relevant. I ett annat projekt har vi använt protein mikroarrayer för att detektera fyra vanliga droger (heroin, amfetamin, ecstasy och kokain). Detektionen baseras på användandet av antikroppar som lossnar från platser på ytan när de kommer i kontakt med ett narkotikum. Detta koncept kan enkelt utvecklas till att detektera mer än bara fyra droger. Vi har även lyckats att parallellt mäta förekomsten av en annan typ av förening på mikroarray ytan, nämligen det explosiva ämnet trinitrotoluen (TNT). Detta visar på en mångsidig plattform för detektionen av i princip vilken typ av farlig eller olaglig substans som helst - och på en yta! Vi föreställer oss därför att möjliga tillämpningsområden finns inom brottsbekämpning, i kampen mot terrorism och mot narkotikamissbruk etc. Mikroarrayerna har i denna avhandling utforskats med optiska metoder som tillåter studie av omärkta proteiner, vilket resulterar i så naturliga molekyler som möjligt. / Life is a thing taken for granted by most. However, it is the life-long quest of many to unravel the mysteries of it. Understanding and characterizing the incomprehensively complex molecular interaction networks within a biological organism, which defines that organism, is a vital prerequisite to understand life itself. Already, there has been a lot of research conducted and a large knowledge has been obtained about these pathways over, especially, the last century. We have seen the fruits of these labors in e.g. the development of medicines which have been able to cure or at least arrest many diseases and conditions. However, many diseases are still incurable (e.g. cancer) and a lot more work is still needed for understanding them fully and designing successful treatments. This work describes a generic analytical tool platform for aiding in more efficient (bio)molecular interaction mapping analyses; protein microarray chips. Microarray chips are surfaces with micrometer sized features with the possibility of studying the interactions of many (thousands to tens of thousands) (bio)molecules in parallel. This allows for a higher throughput of analyses to be performed at a reduced time and cost. Protein microarrays have been around for approximately a decade, following in the footsteps of the, so far, more successfully used DNA microarrays (developed in the 1990s). Microarrays of proteins are more difficult to produce because of the more complex nature of proteins as compared to DNA. In our work we have constructed model surfaces which allow for the stable, highly oriented, and functional immobilization of proteins in an array format. Our capture molecules are based on multivalent units of the chelator nitrilotriacetic acid (NTA), which is able to bind histidine-tagged proteins. Furthermore, we have explored an approach for studying lipid membrane bound systems, e.g. receptor-ligand interactions, in a parallelized, microarray format. The approach relies on the addressable, DNA-mediated adsorption of tagged lipid vesicles. In an analogous work we have used the protein microarray concept for the detection of four common narcotics (heroin, amphetamine, ecstasy, and cocaine). The detection is based on the displacement of loosely bound antibodies from surface array positions upon injection of a specific target analyte, i.e. a narcotic substance. The proof-of-concept chip can easily be expanded to monitor many more narcotic substances. In addition, we have also been able to simultaneously detect the explosive trinitrotoluene (TNT) along with the narcotics, showing that the chip is a versatile platform for the detection of virtually any type of harmful or illegal compound. This type of biosensor system is potentially envisaged to be used in the fight against crime, terrorism, drug abuse etc. Infrared reflection absorption spectroscopy together with ellipsometry has been used to characterize molecular layers used in the fabrication processes of the microarray features. Imaging surface plasmon resonance operating in the ellipsometric mode is subsequently used for functional evaluation of the microarrays using a well-defined receptor-ligand model system. This approach allows simultaneous and continuous monitoring of binding events taking place in multiple regions of interest on the microarray chip. A common characteristic of all the instrumentation used is that there is no requirement for labeling of the biomolecules to be detected, e.g. with fluorescent or radioactive probes. This feature allows for a flexible assay design and the use of more native proteins, without any time-consuming pretreatments.

This thesis describes the use of Nuclear Magnetic Resonance (NMR) for characterizing two host-pathogen interactions: The behavior of a regulatory RNA of the Hepatitis B virus (HBV) and the attachment of Helicobacter pylori (H. pylori) to the gastric mucosa. NMR is a powerful tool in biomedicine, because molecules ranging from small ligands to biomacromolecules can be studied with atomic resolution. Different NMR experiments are designed to determine structures, or to monitor interactions, folding, stability or motion. Paper I describes the analysis of the motions of a regulatory RNA of HBV. The NMR structure of the RNA had revealed before that several well-conserved nucleotides adopt multiple conformations. Therefore an analysis of possible underlying motions was undertaken using two different NMR techniques, one of which (off-resonance ROESY) was applied to nucleic acids for the first time. The observed motions suggest an explanation why the structurally poorly defined nucleotides are highly conserved. In paper II we improved the ROESY NMR experiment, which is used to measure internuclear distances for structure determination of medium-sized molecules. Using a small protein and an organometallic complex as examples, we demonstrated that the new EASY ROESY experiment yields clean spectra that can directly be integrated to derive interatomic distances. H. pylori, the bacterium involved in peptic ulcer disease and gastric cancer, survives in the harsh acidic environment of the stomach. It possesses many membrane proteins which mediate adherence, raising the question, if their activity is related to membrane composition. In paper III &amp; IV we analyzed therefore the phospholipid composition of H. pylori membranes. In paper III, an advanced method for the analysis of the phospholipid composition of biological membranes was developed. The two-dimensional semi-constant-time 31P,1H-COSY experiment combines information from phosphorus and hydrogen atoms of phospholipids for their unambiguous identification. Furthermore, the high resolution of the two-dimensional experiment allows the quantification of phospholipids where conventional methods fail. In paper IV we applied the new experiment to analyze the lipid composition of whole H. pylori cells, their inner and outer membranes, and of vesicles shed by the bacterium. The goal of this study was to characterize the vesicles which are suggested to play a role in the inflammation process. We established that the outer membrane and the vesicles have similar phospholipid compositions, suggesting that the vesicles are largely derived from the outer membrane. The NMR results presented here elucidate details of molecular systems engaged in pathogenicity, as basis for therapeutic strategies against these pathogens.