Abstract
Membrane transport proteins play a crucial role in development, differentiation and survival of eukaryotic cells. Many human diseases have been linked to dysfunction or modified function of membrane transporters, such as cystic fibrosis and adrenoleukodystrophy. Due to technical difficulties, it is possible to crystallizeand yield of all the three dimensional structure of proteins. For this reason, a first idea for the folding of the various regions of the protein could be obtained by homology modeling, if there is provided a representative crystallographic structure. In recent years a large number crystallize transport proteins, and these are created by homology models related proteins. Such an example is secondary activity transporters. As crystallography yields a static, single, dynamic snapshot protein structure outside of the lipid bilayer, which is the physical topology, requires time and other biological approaches. In order to clarify the factors that determine the selectivity ...
Membrane transport proteins play a crucial role in development, differentiation and survival of eukaryotic cells. Many human diseases have been linked to dysfunction or modified function of membrane transporters, such as cystic fibrosis and adrenoleukodystrophy. Due to technical difficulties, it is possible to crystallizeand yield of all the three dimensional structure of proteins. For this reason, a first idea for the folding of the various regions of the protein could be obtained by homology modeling, if there is provided a representative crystallographic structure. In recent years a large number crystallize transport proteins, and these are created by homology models related proteins. Such an example is secondary activity transporters. As crystallography yields a static, single, dynamic snapshot protein structure outside of the lipid bilayer, which is the physical topology, requires time and other biological approaches. In order to clarify the factors that determine the selectivity of substrate structures with low primary homology but high structural similarity is imperative that the combined use of crystallographic data and systematic mutagenesis. The latter biological approach in conjunction with the construction Homology modeling can bean effective and reliable approach to the study of structure-function relationships of transporters secondary activity. In this PhD thesis were studied by biophysical and genetic / molecular approaches two purine transporters in the model ascomycete Aspergillus nidulans, UapA and FcyB. UapA is a uric acid-xanthine / H+co-transporter of the NAT/NCS2 family, forwhich there is a wealth of structural and functional mutations. In this PhD thesis we constructed homology model, relying on the structure of UraA, the first X-ray crystallography of the family. The model was verified by molecular dynamics (Desmond). Calculations with rigid and flexible tether suggested binding site of the carrier substrate, amino acid residues involved, the fastening device and the path of the substrate from the binding site to the cytoplasm (LMCS, Glide-IFD, QSAR). To verify the calculations made by site-directed mutagenesis, mutations of residuesindicated by the calculations that are involved in binding and transport of the substrate. The strains bearing the mutations were characterized phenotypically completely (with growth assays in purines as sole nitrogen sources) and biochemically (by measuring uptake of radiolabeled xanthine and competition experiments) and also studied and their subcellular topology (by microscopic observation of the protein-conjugated GFP). The biological approach verified the calculations mooring and the estimated model. Finally, two other models were constructed homology family, carrier xanthine SNBT1 (in Rattus novergicus) and carrier L-ascorbate (in Homo sapiens). In models with these calculations suggested the binding of the substrate binding site and the residues were compared with the corresponding part of UapA. Found that the residues involved are common, indicating their evolutionary relationship. FcyB is a purine / H+co-transporter of the NCS1/PRT family, featured on A.nidulans. In this PhD thesis we constructed a homology model, based on the structure of Mhp1 of the same family. This model was confirmed by molecular dynamics calculations (Desmond). Finally, binding calculations suggested that the binding of substrates (adenine, hypoxanthine, guanine, cytosine) to the carrier and the affectedamino acid residues. Based on these results and maintenance of residues in primarysequence alignment proposed changes to verify the calculations tether both FcyB, and other family members in A. nidulans (FurA, FurD).
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