OMP29 é uma das principais proteínas de membrana externa de Aggregatibacter actinomycetemcomitans (Aa) e está associada à invasão de célula epitelial gengival (CEG). Os objetivos deste estudo foram: analisar filogeneticamente omp29 e omp29 parálogo (omp29par), em cepas de Aa; determinar a interação de OMP29 com proteínas de matriz extracelular e o efeito da sua interação com CEG, pela avaliação da expressão gênica e produção de mediadores inflamatórios. Variações filogenéticas foram observadas para omp29 e omp29par, bem como para os seus promotores e estas relacionam-se com os sorotipos. A proteína recombinante OMP29his interagiu com fibronectina plasmática e celular (p<0,05), mas não com os domínios F30 e 45 ou com colágenos tipo I, III, IV e V, fibrinogênio, laminina e plasminogênio. A interação das mutantes de Aa deficientes em omp29 e/ou omp29par (obtidas pelo sistema LoxP/Cre) e OMP29his com CEG OBA-09 demonstrou que OMP29 regula positivamente il-18 e negativamente il-6r e il-8 (p<0,05). Os dados sugerem que OMP29 está envolvida na evasão do sistema imune. / OMP29 is one of the major outer membrane proteins of Aggregatibacter actinomycetemcomitans (Aa) and is associated with invasion into gingival epithelial cells (GEC). This study aimed to evaluate phylogenetically omp29 and omp29 paralogue (omp29par) in Aa strains; determine the interaction of OMP29 with extracellular matrix proteins and its effect on GEC by gene expression analysis and production of inflammatory mediators. Phylogenetic variations were observed for omp29 and omp29par as well as their promoters, and they were related to the serotypes. The recombinant protein OMP29his interacted with plasma and cellular fibronectin (p <0.05), but not to F30 and F45 domains, neither to collagens I, III, IV and V, fibrinogen, laminin and plasminogen. The interaction of Aa mutants defective in omp29 and/or omp29par (obtained by LoxP system/Cre) and OMP29his with CEG OBA-09 indicated that OMP29 regulates positively il-18 and negatively il-6r and il-8 (p <0.05). Data suggested that OMP29 is involved in bacterial evasion of the immune system.

O desenvolvimento de uma vacina contra malária causada por P. falciparum é prejudicado pelo alto nível de polimorfismo apresentado pelos antígenos desse parasito. O dimorfismo alélico é um padrão no qual os alelos observados de um gene se encontram divididos em duas famílias. A proteína dimórfica MSP-6 se associa à proteína MSP-1 (também dimórfica) na superfície do merozoíto. Genes de msp-6 de 21 isolados obtidos de pacientes do Brasil, mais 2 isolados da Tanzânia, África, foram seqüenciados para estudo da diversidade nucleotídica e distribuição geográfica dos alelos. As duas famílias possuem distribuição global. Não foi verificada associação entre o dimorfismo de MSP-1 e MSP-6. O gene ortólogo de msp-6 em P. reichenowi, grupo irmão de P. falciparum, foi seqüenciado para estudos evolutivos. Os alelos dimórficos de MSP-6 aparentam ter surgido de uma população ancestral polimórfica, tendo sido mantidos no presente por seleção balanceada. O alto grau de conservação encontrado dentro de cada família alélica torna MSP-6 um potencial alvo de uma vacina contra a malária. / The development of a vaccine against malaria caused by Plasmodium falciparum has been hampered by the high level of antigen polymorphism exhibited by this parasite. Allelic dimorphism is a pattern in which every observed allele of a gene is clearly grouped into one of two families. The dimorphic protein MSP-6 forms a complex with MSP-1 (also dimorphic) on merozoite surface. The msp-6 genes were sequenced in isolates obtained from 21 patients from Brazil, plus 2 isolates from Tanzania, Africa, to study nucleotide diversity and geographic distribution of alleles. Both families are globally distributed. Moreover, no association was observed between the MSP-1 and MSP-6 allelic types. Orthologous gene of msp-6 in P. reichenowi, chimpanzee parasite and sister group of P. falciparum, was sequenced for evolutionary studies. Dimorphic alleles of MSP-6 seem to have originated from an ancestral polymorphic population and are maintained by balancing selection. The high degree of conservation observed within each allelic family makes MSP-6 an promising target for vaccine development.

abstract: Measuring molecular interaction with membrane proteins is critical for understanding cellular functions, validating biomarkers and screening drugs. Despite the importance, developing such a capability has been a difficult challenge, especially for small molecules binding to membrane proteins in their native cellular environment. The current mainstream practice is to isolate membrane proteins from the cell membranes, which is difficult and often lead to the loss of their native structures and functions. In this thesis, novel detection methods for in situ quantification of molecular interactions with membrane proteins are described. First, a label-free surface plasmon resonance imaging (SPRi) platform is developed for the in situ detection of the molecular interactions between membrane protein drug target and its specific antibody drug molecule on cell surface. With this method, the binding kinetics of the drug-target interaction is quantified for drug evaluation and the receptor density on the cell surface is also determined. Second, a label-free mechanically amplification detection method coupled with a microfluidic device is developed for the detection of both large and small molecules on single cells. Using this method, four major types of transmembrane proteins, including glycoproteins, ion channels, G-protein coupled receptors (GPCRs) and tyrosine kinase receptors on single whole cells are studied with their specific drug molecules. The basic principle of this method is established by developing a thermodynamic model to express the binding-induced nanometer-scale cellular deformation in terms of membrane protein density and cellular mechanical properties. Experiments are carried out to validate the model. Last, by tracking the cell membrane edge deformation, molecular binding induced downstream event – granule exocytosis is measured with a dual-optical imaging system. Using this method, the single granule exocytosis events in single cells are monitored and the temporal-spatial distribution of the granule fusion-induced cell membrane deformation are mapped. Different patterns of granule release are resolved, including multiple release events occurring close in time and position. The label-free cell membrane deformation tracking method was validated with the simultaneous fluorescence recording. And the simultaneous cell membrane deformation detection and fluorescence recording allow the study of the propagation of the granule release-induced membrane deformation along cell surfaces. / Dissertation/Thesis / Doctoral Dissertation Electrical Engineering 2018

Cystic Fibrosis (CF) is an autosomal recessive genetic disease that leads to severe malfunction in many organs, but particularly the lungs. The primary cause of this malfunction is the decrease of the airway surface liquid layer on the lung epithelium. The lack of hydration leads to mucus build up on the epithelial lining, leading to blockage of airways. The underlying cause of CF is the dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR), which results from mutations in the protein. Almost 90% of CF patients are caused by the deletion of the phenylalanine at position 508 of CFTR, which is believed to affect the folding and stability of CFTR. The misfolded ΔF508-CFTR undergoes ER associated degradation (ERAD), causing the failure of ΔF508-CFTR trafficking to the cell surface. Small molecule correctors yield moderate improvements in the trafficking of ΔF508-CFTR to the plasma membrane. It is currently not known if correctors increase trafficking through improved cargo loading of transport vesicles or through direct binding to CFTR. In this dissertation, real-time measurements of trafficking were utilized to identify the mechanistic details of chemical, biochemical, and thermal factors that impact CFTR correction, using the corrector molecule VX-809, a secondary mutation (I539T), and low temperature conditions. Each individually improved trafficking of ΔF508-CFTR to approximately 10% of wild-type levels. The combination of VX-809 with either low temperature or the I539T mutation increased the amount of CFTR on the plasma membrane to nearly 40%, indicating synergistic activity. The number of vesicles reaching the surface was significantly altered; however the amount of channel in each vesicle remained the same. Therefore, a 2 step therapeutic approach might be an ideal treatment for CF. The first step would be composed of a compound that mimics the mechanism of stabilization provided by low temperature or the I539T mutation, while the second step would be VX-809 or a similar corrector compound. These studies suggest that understanding how low temperature and second site suppressors alter ΔF508-CFTR could be key to the development of future therapeutics for the effective treatment of CF. The precise pathophysiology of cystic fibrosis is not well studied. The involvement of another transport protein, epithelial sodium channel (ENaC), makes the situation more complicated. ENaC and CFTR are colocalized on the apical surface of epithelia cells. With our fluorescence microscopy techniques, we explored the effects of CFTR on the residence time of ENaC on the cell membrane. A reliable approach measuring the half-life of protein on the cell membrane is required for this study. We present a new approach to quantify the half-life of membrane proteins on the cell surface, through tagging the protein with the photoconvertible fluorescent protein, Dendra2. Total internal reflection fluorescence microscopy (TIRF) is applied to limit visualization of fluorescence to proteins located on the plasma membrane. Photoconversion of Dendra2 works as a pulse chase experiment by monitoring only the population of protein that has been photoconverted. As the protein is endocytosed the red emission decreases due to the protein leaving the TIRF field of view. The half-life of the protein on the plasma membrane was calculated upon imaging over time and quantifying the change in red fluorescence. Our method provides a unique opportunity to observe real-time protein turnover at the single cell level without addition of protein synthesis inhibitors. This technique will be valuable for the future protein half-life study.

Although the basic structure of biological membranes is provided by the lipid bilayer, most of the specific functions are carried out by membrane proteins (MPs) such as channels, ion-pumps and receptors. Additionally, it is known, that mutations in MPs are directly or indirectly involved in many diseases. Thus, structure determination of MPs is of major interest not only in structural biology but also in pharmacology, especially for drug development. Advances in structural biology of membrane proteins (MPs) have been strongly supported by the success of three leading techniques: X-ray crystallography, electron microscopy and solution NMR spectroscopy. However, X-ray crystallography and electron microscopy, require highly diffracting 3D or 2D crystals, respectively. Today, structure determination of non-crystalline solid protein preparations has been made possible through rapid progress of solid-state MAS NMR methodology for biological systems. Castellani et. al. solved and refined the first structure of a microcrystalline protein using only solid-state MAS NMR spectroscopy. These successful application open up perspectives to access systems that are difficult to crystallise or that form large heterogeneous complexes and insoluble aggregates, for example ligands bound to a MP-receptor, protein fibrils and heterogeneous proteins aggregates. Solid-state MAS NMR spectroscopy is in principle well suited to study MP at atomic resolution. In this thesis, different types of MP preparations were tested for their suitability to be studied by solid-state MAS NMR. Proteoliposomes, poorly diffracting 2D crystals and a PEG precipitate of the outer membrane protein G (OmpG) were prepared as a model system for large MPs. Results from this work, combined with data found in the literature, show that highly diffracting crystalline material is not a prerequirement for structural analysis of MPs by solid-state MAS NMR. Instead, it is possible to use non-diffracting 3D crystals, MP precipitates, poorly diffracting 2D crystals and proteoliposomes. For the latter two types of preparations, the MP is reconstituted into a lipid bilayer, which thus allows the structural investigation in a quasi-native environment. In addition, to prepare a MP sample for solid-state MAS NMR it is possible to use screening methods, that are well established for 3D and 2D crystallisation of MPs. Hopefully, these findings will open a fourth method for structural investigation of MP. The prerequisite for structural studies by NMR in general, and the most time consuming step, is always the assignment of resonances to specific nuclei within the protein. Since the last few years an ever-increasing number of assignments from solid-state MAS NMR of uniformly carbon and nitrogen labelled samples is being reported, mostly for small proteins of up to around 150 amino acids in length. However, the complexity of the spectra increases with increasing molecular weight of the protein. Thus the conventional assignment strategies developed for small proteins do not yield a sufficiently high degree of assignment for the large MP OmpG (281 amino acids). Therefore, a new assignment strategy to find starting points for large MPs was devised. The assignment procedure is based on a sample with [2,3-13C, 15N]-labelled Tyr and Phe and uniformly labelled alanine and glycine. This labelling pattern reduces the spectral overlap as well as the number of assignment possibilities. In order to extend the assignment, four other specifically labelled OmpG samples were used. The assignment procedure starts with the identification of the spin systems of each labelled amino acid using 2D 13C-13C and 3D NCACX correlation experiments. In a second step, 2D and 3D NCOCX type experiments are used for the sequential assignment of the observed resonances to specific nuclei in the OmpG amino acid sequence. Additionally, it was shown in this work, that biosynthetically site directed labelled samples, which are normally used to observe long-range correlations, were helpful to confirm the assignment. Another approach to find assignment starting points in large protein systems, is the use of spectroscopic filtering techniques. A filtering block that selects methyl resonances was used to find further assignment starting points for OmpG. Combining all these techniques, it was possible to assign nearly 50 % of the observed signals to the OmpG sequence. Using this information, a prediction of the secondary structure elements of OmpG was possible. Most of the calculated motifs were in good aggreement with the crystal structures of OmpG. The approaches presented here should be applicable to a wide variety of MPs and MP-complexes and should thus open a new avenue for the structural biology of MPs. / Biologische Membranen bestehen hauptsächlich aus Lipiden, ihre Funktion wird jedoch vor allem durch die eingebetteten Membranproteine (z.B. Kanäle, Ionenpumpen und Rezeptoren) bestimmt. Mutationen in dieser Proteinklasse können zum Auftreten verschiedener Krankheitsbilder führen, weshalb die Untersuchung der dreidimensionalen Struktur von Membranproteinen nicht nur von strukturbiologischem, sondern auch von pharmakologischem Interesse ist. In den letzten Jahren wurde eine Methode, die Festkörper NMR Spektroskopie, für Strukturuntersuchungen an Proteinproben im festen Aggregatzustand entwickelt. Diese Arbeit beschäftigt sich mit drei verschiedenen Präparationsarten von Membranproteinen, die eine Aufnahme von hochaufgelösten Festkörper NMR Spektren erlauben. Als Modelsystem wurde das Protein G der äußeren Membrane (outer membrane protein G, OmpG) von Escherichia coli gewählt. Eine wichtige Vorraussetzung zur Berechnung der Proteinstruktur aus den NMR-Spektren, ist die Zuordnung der einzelnen Signale zur jeweiligen Aminosäure in der Proteinsequenz. In dieser Arbeit wurde eine Methode entwickelt, die das Auffinden von Startpunkten für die sequentielle Zuordnung in großen Membranproteinen, wie zum Bsp. OmpG (281 Aminosäuren), erlaubt. Multidimensionale NMR Experimente mit verschieden spezifisch markierten Proben wurden durchgeführt und ermöglichten die Zuordnung von 50 % der NMR Signale der OmpG Proteinsequenz. Zur Überprüfung der gewonnenen Daten wurden diese zur Vorhersage von Sekundärstrukturelementen genutzt. Es konnte gezeigt werden, dass die berechneten Strukturmotive in guter Übereinstimmung zu den bisher veröffentlichten OmpG Strukturen liegen. Die in dieser Arbeit angewendeten Methoden sollten auf eine Vielzahl anderer Membranprotein anwendbar und somit einen neuen Weg zur Strukturbiologischen Untersuchung von Membranproteinen eröffnen.

G protein-coupled receptors (GPCRs) are one of the largest protein families in mammals. GPCRs are instrumental for hormonal and neurotransmitter signalling and are important in all major physiological systems of the body. Paper I describes the repertoire of GPCRs in Branchiostoma floridae, which is one of the species most closely related species to vertebrates. Mining and phylogenetic analysis of the amphioxus genome showed the presence of at least 664 distinct GPCRs distributed among all the main families of GPCRs; Glutamate (18), Rhodopsin (570), Adhesion (37), Frizzled (6) and Secretin (16). Paper II contains studies of the Adhesion, Methuselah and Secretin GPCR families in nine genomes. The Adhesion GPCRs are the most complex gene family among GPCRs with large genomic size, multiple introns and a fascinating flora of functional domains. Phylogenetic analysis showed Adhesion group V (that contains GPR133 and GPR144) to be the closest relative to the Secretin family among the groups in the Adhesion family, which was also supported by splice site setup and conserved motifs. Paper III examines the repertoire of human transmembrane proteins. These form key nodes in mediating the cell’s interaction with the surroundings, which is one of the main reasons why the majority of drug targets are membrane proteins. We identified 6,718 human membrane proteins and classified the majority of them into 234 families of which 151 belong to the three major functional groups; Receptors (63 groups, 1,352 members), Transporters (89 groups, 817 members) or Enzymes (7 groups, 533 members). In addition, 74 Miscellaneous groups were shown to include 697 members. Paper IV clarifies the hierarchy of the main families and evolutionary origin of majority of the metazoan GPCR families. Overall, it suggests common decent of at least 97% of the GPCRs sequences found in humans, including all the main families.

Single particle electron cryomicroscopy (cryo-EM) is a method of structure determination that uses many randomly oriented images of the specimen to construct a three-dimensional density map. In this thesis, single particle cryo-EM has been used to determine the structure of intact adenosine triphosphate (ATP) synthase from bovine heart mitochondria, an approximately 550 kDa membrane protein complex. In respiring organisms, ATP synthase is responsible for synthesizing the majority of ATP, a molecule that serves as an energy source for many cellular reactions. In order to understand the mechanism of ATP synthase, knowledge of the arrangement of subunits in the intact complex is necessary. To obtain maps of intact ATP synthase showing internal density distributions by single particle cryo-EM, methodological improvements to image acquisition, map refinement, and data selection were developed. Further, a novel segmentation algorithm was developed to aid in interpretation of maps. The use of these tools allowed for construction and interpretation of two maps of ATP synthase, solubilized in different membrane mimetics, in which the arrangement of subunits could be identified. These maps revealed interactions within the complex important for its function. In addition, evidence was obtained for curvature of membrane mimetics around ATP synthase, suggesting a role for the complex in maintenance of mitochondrial membrane morphology.

As one of the mechanisms of antibiotic resistance, bacteria use several families of membrane-embedded α-helical transporters to remove cytotoxic molecules from the cell. The small multidrug resistance protein family (SMR) is one such group of drug transporters that because of their relative small size [ca. 110 residues with four transmembrane (TM) helices] must form at the minimum dimers to efflux drugs. We have used the SMR homologue Hsmr from Halobacterium salinarum to investigate the oligomerization properties of the protein family at TM helix 4. We produced point mutations along the length of the TM4 helix in the full length Hsmr protein and assayed their dimerization and functional properties via SDS-PAGE and bacterial cell growth assays. We found that Hsmr forms functionally dependent dimers via an evolutionarily conserved 90GLxLIxxGV98 small residue heptad repeat. Upon investigation of the large hydrophobic residues in this motif by substituting each large residue to Ile, Leu, Met, Phe, and Val, we determined that Hsmr efflux function relies on an optimal level of dimerization. While some substitutions led to either decreased or increased dimer and substrate-binding strength, several Ile94 and Val98 mutants were equal to wild type dimerization levels but were nonfunctional, leading to the hypothesis of a mechanistic role at TM4 in addition to the locus of dimerization. The functionally sensitive TM4 dimer represents a potential target for SMR inhibition using a synthetic TM4 peptide mimetic. Using exponential decay measurements from a real-time cellular efflux assay, we observed the efflux decay constant was decreased by up to ~60% after treatment with the TM4 peptide inhibitor compared to control peptide treatments. Our results suggest that this approach could conceivably be used to design hydrophobic peptides for disruption of key TM-TM interactions of membrane proteins, and represent a valuable route to the discovery of new therapeutics.

Single particle electron cryomicroscopy (cryo-EM) is a method of structure determination that uses many randomly oriented images of the specimen to construct a three-dimensional density map. In this thesis, single particle cryo-EM has been used to determine the structure of intact adenosine triphosphate (ATP) synthase from bovine heart mitochondria, an approximately 550 kDa membrane protein complex. In respiring organisms, ATP synthase is responsible for synthesizing the majority of ATP, a molecule that serves as an energy source for many cellular reactions. In order to understand the mechanism of ATP synthase, knowledge of the arrangement of subunits in the intact complex is necessary. To obtain maps of intact ATP synthase showing internal density distributions by single particle cryo-EM, methodological improvements to image acquisition, map refinement, and data selection were developed. Further, a novel segmentation algorithm was developed to aid in interpretation of maps. The use of these tools allowed for construction and interpretation of two maps of ATP synthase, solubilized in different membrane mimetics, in which the arrangement of subunits could be identified. These maps revealed interactions within the complex important for its function. In addition, evidence was obtained for curvature of membrane mimetics around ATP synthase, suggesting a role for the complex in maintenance of mitochondrial membrane morphology.

Knowledge about the insertion and stabilization of membrane proteins is a key step towards understanding their function and enabling membrane protein design. Transmembrane helices are normally quite hydrophobic to insert efficiently, but there are many exceptions with unfavorable polar or titratable residues. Since evolutionary conserved these amino acids are likely of paramount functional importance, e.g. the four arginines in the S4 voltage sensor helix of voltage-gated ion channels. This has lead to vivid discussion about their conformation, protonation state and cost of insertion. To address such questions, the main focus of this thesis has been membrane protein solvation in lipid bilayers, evaluated using molecular dynamics simulations methods. A main result is that polar and charged amino acids tend to deform the bilayer by pulling water/head-groups into the hydrophobic core to keep their hydrogen bonds paired, thus demonstrating the adaptiveness of the membrane to allow specific and quite complex solvation. In addition, this retained hydration suggests that the solvation cost is mainly due to entropy, not enthalpy loss. To further quantify solvation properties, free energy profiles were calculated for all amino acids in pure bilayers, with shapes correlating well with experimental in vivo values but with higher magnitudes. Additional profiles were calculated for different protonation states of the titratable amino acids, varying lipid composition and with transmembrane helices present in the bilayer. While the two first both influence solvation properties, the latter seems to be a critical aspect. When the protein fraction in the models resemble biological membranes, the solvation cost drops significantly - even to values compatible with experiment. In conclusion, by using simulation based methods I have been able to provide atomic scale explanations to experimental results, and in particular present a hypothesis for how the solvation of charged groups occurs.