Tesis doctoral de Estefania Hurtado Gomez
The phosphoenolpyruvate: sugar phosphotransferase system (pts), only found in bacteria, is responsible for the detection, migration and concurrent metabolic sugar uptake and phosphorylation. The pts catalyses the transfer of an activated phosphoryl group from phosphoenolpyruvate (pep) to the imported carbohydrate. This catalysis occurs through a cascade of five proteins from pep via phosphoiintermediates of the general phosphotransferase enzyme i (ei) and the histidine-containing phosphocarrier (hpr) to substrate-specific enzyme ii permeases (the so-called iiabcsug). The n-terminal of ei binds to hpr in order to transfer the phosphate from histidine to histidine. ei is the first protein in the pts system, which autophosphorylates in the presence of pep and mg2+. Ei is an homodimer with a molecular weight of 60 kda. Each monomer is composed of three domains: the hpr-binding domain and the his domain (containing the phosphorylation active site), which are included in the n-terminal fragment, einsc; and the pep-binding domain, that corresponds to the c-terminal fragment, eicsc, and is responsible for the dimerization. the conformational stability and structure of the ei from streptomyces coelicolor, eisc, were explored in the absence and in the presence of its effectors by using several biophysical probes (namely, fluorescence, far-uv cd, ftir and dsc; as well as computational approaches. The structure of eisc was obtained by homology modelling of the isolated n- and c-terminal fragments of other ei proteins, whose three-dimensional structures were known (protein data bank codes 1zym and 2bg5). The experimental results indicate that, while at physiological ph the dimeric native state of eisc is stable, at low ph the protein populates a partially unfolded state with molten-globule-like features, as suggested by fluorescence, far-uv cd, ftir and ans-binding. The presence of pep and mg2+ did not change substantially the secondary structure of the protein, as indicated by ftir measurements. However, quenching experiments and proteolysis patterns suggest conformational changes in the presence of pep; furthermore, the thermal stability of eisc was modified depending on the effector added. the uniqueness of the pts in bacteria makes this system, and especially the ei, an attractive target for new antibacterial drugs. These drugs can be obtained from peptides or protein fragments capable of interfering the first step of the protein cascade: the phosphorylation of the hpr protein by the enzyme i. To this end, we designed a peptide comprising the active site and the first a-helix of hpr of s. Coelicolor, hprsc, the peptide hpr9-30; we also obtained a larger fragment of hprsc by protein engineering comprising the first forty-eight residues, hpr1-48, and thus, containing the amino acids of the shorter peptide. Both fragments were disordered in aqueous solution, with a similar percentage of helical structure (~ 7 %), and an identical free energy change upon helix formation. In 40 % tfe, however, both fragments acquired native-like helical structure, stabilized by non-native hydrophobic interactions, as shown by the 2d-nmr assignments of the shorter peptide, and the presence of similar noe contacts in both fragments. we have also studied the binding of eisc to hprsc, as well as to the fragments hprl-48 and hpr9-30, to compare the relative affinities, we have characterized the intermolecular recognition processes by spectroscopic (fluorescence, cd and std-nmr) as well as calorimetric methods (itc). The results indicated that eisc showed larger affinity for hprsc than for its fragments, since the association constant for the complex ei-hprsc was three-fold higher than those for the ei-hpr1-48 and ei-hpr9-30. in our search for an inhibitor of the pts system, hampering the binding of eisc to hprsc, we have also used computational methods. We modeled by homology einsc and hprsc, using the solved structures from e. Coli, by swiss-model server and foldx software. Once the complex ei-hprsc was obtained, the fragments were used as templates in positional scanning assays to achieve optimal peptides for binding to einsc. Finally, the optimal peptides were submitted to combinatorial scanning analysis. From these theoretical methodologies, several peptides were proposed, which had a higher affinity for eisc than hprsc itself and, consequently with potential activity as antiobiotics.
Datos académicos de la tesis doctoral «Caracterizacion estructural, plegamiento y reconocimiento intermolecular de ei y fragmentos de hpr del sistema fosfotransferasa en streptomyces coelicolor. busqueda de nuevos antibioticos«
- Título de la tesis: Caracterizacion estructural, plegamiento y reconocimiento intermolecular de ei y fragmentos de hpr del sistema fosfotransferasa en streptomyces coelicolor. busqueda de nuevos antibioticos
- Autor: Estefania Hurtado Gomez
- Universidad: Miguel hernández de elche
- Fecha de lectura de la tesis: 21/06/2007
Dirección y tribunal
- Director de la tesis
- José Luis Neira Faleiro
- Tribunal
- Presidente del tribunal: manuel Cortijo merida
- adrian Velazquez campoy (vocal)
- margarita Menendez fernandez (vocal)
- chris Johnson (vocal)