Browsing by Subject "rakennebiologia"

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  • Leo, Jack (Helsingin yliopisto, 2009)
    Trimeric autotransporters are a family of secreted outer membrane proteins in Gram-negative bacteria. These obligate homotrimeric proteins share a conserved C-terminal region, termed the translocation unit. This domain consists of an integral membrane β-barrel anchor and associated α-helices which pass through the pore of the barrel. The α-helices link to the extracellular portion of the protein, the passenger domain. Autotransportation refers to the way in which the passenger domain is secreted into the extracellular space. It appears that the translocation unit mediates the transport of the passenger domain across the outer membrane, and no external factors, such as ATP, ion gradients nor other proteins, are required. The passenger domain of autotransporters contains the specific activities of each protein. These are usually related to virulence. In trimeric autotransporters, the main function of the proteins is to act as adhesins. One such protein is the Yersinia adhesin YadA, found in enteropathogenic species of Yersinia. The main activity of YadA from Y. enterocolitica is to bind collagen, and it also mediates adhesion to other molecules of the extracellular matrix. In addition, YadA is involved in serum resistance, phagocytosis resistance, binding to epithelial cells and autoagglutination. YadA is an essential virulence factor of Y. enterocolitica, and removal of this protein from the bacteria leads to avirulence. In this study, I investigated the YadA-collagen interaction by studying the binding of YadA to collagen-mimicking peptides by several biochemical and biophysical methods. YadA bound as tightly to the triple-helical model peptide (Pro-Hyp-Gly)10 as to native collagen type I. However, YadA failed to bind a similar peptide that does not form a collagenous triple helix. As (Pro-Hyp-Gly)10 does not contain a specific sequence, we concluded that a triple-helical conformation is necessary for YadA binding, but no specific sequence is required. To further investigate binding determinants for YadA in collagens, I examined the binding of YadA to a library of collagen-mimicking peptides that span the entire triple-helical sequences of human collagens type II and type III. YadA bound promiscuously to many but not all peptides, indicating that a triple-helical conformation alone is not sufficient for binding. The high-binding peptides did not share a clear binding motif, but these peptides were rich in hydroxyproline residues and contained a low number of charged residues. YadA thus binds collagens without sequence specificity. This strategy of promiscuous binding may be advantageous for pathogenic bacteria. The Eib proteins from Escherichia coli are immunoglobulin (Ig)-binding homologues of YadA. I showed conclusively that recombinant EibA, EibC, EibD and EibF bind to IgG Fc. I crystallised a fragment of the passenger domain of EibD, which binds IgA in addition to IgG. The structure has a YadA-like head domain and an extended coiled-coil stalk. The top half of the coiled-coil is right-handed with hendecad periodicity, whereas the lower half is a canonical left-handed coiled-coil. At the transition from right- to left-handedness, a small β-sheet protrudes from each monomer. I was able to map the binding regions for IgG and IgA using truncations and site-directed mutagenesis to the coiled-coil stalk and identified residues critical for Ig binding.
  • Hellman, Maarit (Helsingin yliopisto, 2007)
    Structural biology is a branch of science that concentrates on the relationship between the structure and function of biological macromolecules. The prevalence of a large number of three dimensional structures offers effective tools for bio-scientists to understand the living world. Actin is the most abundant cellular protein and one of its main functions is to produce movement in living cells. Actin forms filaments that are dynamic and which are regulated by a number of different proteins. A class of these regulatory proteins contains actin depolymerizing factor homology (ADF-H) domains. These directly interact with actin through their ADF-H domains. Although ADF-H domains possess very similar three dimensional structures to one another, they vary in their functional properties. One example of this is the ability to bind to actin monomers or filaments. During the work for this thesis two structures of ADF-H domains were solved by nuclear magnetic resonance spectroscopy (NMR). The elucidated structures help us understand the binding specificities of the ADF-H family members.
  • Repo, Heidi (Helsingin yliopisto, 2014)
    Lysosomes are membrane enclosed acidic cell organelles found ubiquitously in higher eukaryotes. The lysosomal lumen contains more than 60 soluble lysosomal hydrolases, which degrade and recycle cellular macromolecules. Mutations in genes encoding lysosomal or lysosome related proteins result in over 50 different lysosomal storage disorders (LSDs) affecting 1 out of every 7700 newborn children. For instance, the first described LSD, Pompe disease, is caused by a mutation that impairs the function of lysosomal α-glucosidase (GAA) and that results in lysosomal accumulation of glycogen. In this study, several lysosomal proteins were studied via a variety of techniques to increase the knowledge of lysosomal function and correspondingly, the lysosome associated diseases. In order to better understand its function, the previously not well characterised phospholipase B-like protein 1 (PLBD1) was purified from bovine kidneys. It was crystallised and the structure solved by X-ray crystallography to a 1.9 Å resolution. The structure showed that PLBD1 is a member of the N-terminal nucleophile aminohydrolases superfamily. This would imply that PLBD1 is not an esterase as the name suggests, but an amidase. The finding that the hydrophobic tail of the potential phospholipid substrate does not fit into the acyl binding cavity also argues against phosphoesterase function. As a first step in the protein transport pathway to lysosomes mannose-6- phosphate-tag is added to lysosomal proteins. This is initiated by N- acetylglucosamine-1-phosphotransferase (GlcNAc phosphotransferase), which requires a recognition signal on the folded surface of the lysosomal proteins. In this study, the conservation of the signal in four lysosomal proteins was analysed. The phosphorylated N-glycosylation sites and the lysine residues on the GlcNAc phosphotransferase recognition site are well conserved at the sequence level in orthologous proteins, but not necessarily in the protein family. Based on surface analysis of PLBD1 and comparison to the paralogous PLBD2, the most likely recognition site for the GlcNAc phosphotransferase for the PLBD1 could be suggested. LSD associated mutations affect protein function through several mechanism. Several disease-associated missense mutations disturb the protein fold. A general analysis of four enzymes associated with LSD showed that the disease-associated missense mutations are not equally distributed among the 20 amino acids. Glycine, arginine and proline are clearly over-represented among the mutations comparedto their abundance in protein sequences. The hydrophobic amino acids tend to be under-represented among disease-associated mutations. The amino acids where mutation frequently involves a disease have unique properties that contribute to the protein structure in a way that cannot be compensated by other amino acids. Enzyme enhancement therapy with chemical chaperones is a novel treatment for LSDs and has shown potential also for Pompe disease. In this study, the stabilisation capacity of potential chemical chaperones for GAA were tested. Most of the compounds stabilised rhGAA against thermal unfolding and some stabilised even better than would be expected from their binding affinity. In addition, the compounds were modelled to the active site of a GAA structural model and based on this three factors to be considered in chemical chaperone design were defined. Firstly, the ligand size can vary, but the four OH-groups in the ligand are critical in orienting the molecule and making the binding specific. Last but most importantly, a positive charge and its location determine the strength of binding to GAA. This thesis with its structural studies of lysosomal proteins provides molecular understanding of lysosomal protein biology, which is critical for full understanding lysosome function and its involvement in diseases.