RESUMO
We have recently identified the membranotropic regions of the hepatitis C virus proteins E1, E2, core and p7 proteins by observing the effect of protein-derived peptide libraries on model membrane integrity. We have studied in this work the ability of selected sequences of these proteins to modulate the L(beta)-L(alpha) and L(alpha)-H(II) phospholipid phase transitions as well as check the viability of using both DSC and SAXD to screen a protein-derived peptide library. We demonstrate that it is feasible to screen a library of peptides corresponding to one or several proteins by both SAXD and DSC. This methodological combination should allow the identification of essential regions of membrane-interacting proteins which might be implicated in the molecular mechanism of membrane fusion and/or budding.
Assuntos
Biblioteca de Peptídeos , Espalhamento a Baixo Ângulo , Proteínas Virais/metabolismo , Difração de Raios X , Sequência de Aminoácidos , Varredura Diferencial de Calorimetria , Hepacivirus/química , Dados de Sequência Molecular , Peptídeos/química , Análise de Sequência de Proteína , Temperatura , Proteínas Virais/químicaRESUMO
The previously identified membrane-active regions of the hepatitis C virus (HCV) E1 and E2 envelope glycoproteins led us to identify different segments that might be implicated in viral membrane fusion, membrane interaction, and/or protein-protein binding. HCV E2 glycoprotein contains one of the most membranotropic segments, segment 603-634, which has been implicated in CD81 binding, E1/E2 and E2/E2 dimerization, and membrane interaction. Through a series of complementary experiments, we have carried out a study of the binding and interaction with the lipid bilayer of a peptide corresponding to segment 603-634, peptide E2(FP), as well as the structural changes induced by membrane binding that take place in both the peptide and the phospholipid molecules. Here, we demonstrate that peptide E2(FP) binds to and interacts with phospholipid model membranes, modulates the polymorphic phase behavior of membrane phospholipids, is localized in a shallow position in the membrane, and is probably oligomerized in the presence of membranes. These data support the role of E2(FP) in HCV-mediated membrane fusion, and sustain the notion that this segment of the E2 envelope glycoprotein, together with other segments of E2 and E1 glycoproteins, provides the driving force for the merging of the viral and target cell membranes.
