Estudo das interações entre proteína NS1 do hRSV e flavonóides utilizando métodos biofísicos

• Autor(a) principal: Gomes, Deriane Elias [UNESP]
• Data de Publicação: 2014
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: Repositório Institucional da UNESP
• Texto Completo: http://hdl.handle.net/11449/122135
• topic: Biologia molecular; Biofísica; Virologia molecular; Vírus sincicial respiratório humano; Proteínas não estruturais virais; Flavonoides; Biophysics

Human Respiratory Syncytial Virus (hRSV) is the major cause of acute respiratory infections in children, like bronchiolitis and pneumonia. This Paramyxovirus has a single strand RNA with 10 genes that codify 11 proteins. An important factor that contributes for the success hRSV replication is the immune system evasion, process wich Non-Structural Protein 1 (NS1) is involved. This protein can act by inhibiting or neutralizing steps of interferon type I pathway, as well as, by silencing the ribonucleoproteic complex of hRSV. The knowledge of NS1 protein interaction with ligands is important in the search for molecules that could interfere with protein function and hence result in a reduction of hRSV replication among them the flavonoids have been reported to be effective in suppressing viral replication. The aim of this study includes expression and purification of NS1 protein, characterization of its secondary structure and thermal stability through circular dicroism and perform fluorescence spectroscopy interaction study between NS1 and Quercetin. NS1 purification was performed using affinity resin coupled to a liquid cromatograph ÄKTA (GE HEALTHCARE LIFE SCIENCES). Circular dicroism spectra and thermal denaturation were carried out to investigate NS1 secondary structure and stability. Through fluorescence spectroscopy titration results it has been calculated the Stern Volmer constant (Ksv), the binding (Kb) constant and number of ligands per site (n). CD analysis shown that secondary structure composition of NS1 is 75% alpha-helix; 3% beta-sheet; 10% turn and 12% others and, melting temperature of NS1 is around 65°C. Fluorescence results shown that Ksv and Kb were in order of 104 and 105-106M-1, respectively. Ksv dependence with temperature indicates that the interaction between NS1 and quercetin is dynamic. The results suggest that this interaction may potentially contribute to block the xiii protein function and thus quercetin may ...