RESUMEN
The binding of aromatic amino acids to the ligand response domain of the tyrosine repressor (TyrR) protein (TyrR(lrd)) of Haemophilus influenzae was investigated using circular dichroism and fluorescence spectroscopy. The induced secondary structural changes were unique for each aromatic amino acid and were further influenced by the presence or absence of ATP. Tyrosine was found to have the highest affinity for TyrR(lrd) in the absence of ATP, whereas the affinity for ATP itself increased in the presence of tyrosine. Binding of tyrosine is therefore the conformational trigger for the activation of TyrR whereas ATP is regarded as a conformational co-activator.
Asunto(s)
Adenosina Trifosfato/farmacología , Proteínas de Escherichia coli , Haemophilus influenzae/química , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Tirosina/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sitios de Unión/efectos de los fármacos , Dicroismo Circular , Secuencia Conservada , Ligandos , Fenilalanina/metabolismo , Unión Proteica/efectos de los fármacos , Estructura Secundaria de Proteína/efectos de los fármacos , Espectrometría de Fluorescencia , Relación Estructura-Actividad , Termodinámica , Triptófano/metabolismo , Tirosina/agonistasRESUMEN
The secondary structure of the ligand response domain of the Haemophilus influenzae tyrosine repressor, TyrR(lrd), was investigated using CD spectroscopy which revealed 42.5% alpha-helix, 17.6% beta-sheet, and 39.9% loops. Quaternary structure analysis by fluorescence anisotropy showed that TyrR(lrd) is monomeric at a concentration of 100 nM to 2 microM but that the protein readily dimerizes in the presence of its natural ligand ATP. Equilibrium unfolding studies of TyrR(lrd) using guanidinium hydrochloride suggested a two-state model with no detectable stable intermediates. The unfolding transition monitored by CD spectroscopy was responsive to tyrosine and ATP resulting in a shift to higher denaturant concentrations in the presence of these ligands. Differential scanning calorimetry yielded melting temperatures, T(m), of 51.15 and 58.07 degrees C for the unliganded and for the ATP-liganded protein, respectively. ATP is thus proposed to be a major structural cofactor for the molecular architecture of TyrR(lrd).