A POLAR OCTAPEPTIDE FUSED TO THE N-TERMINAL FUSION PEPTIDE SOLUBILIZES THE INFLUENZA-VIRUS HA(2) SUBUNIT ECTODOMAIN

As a step toward studying membrane fusion with a simplified molecule, the ectodomain, residues 1-185, of the membrane-anchored subunit HA(2) of the influenza virus haemagglutinin (HA) was solubilized by adding the very polar FLAG octapeptide (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) to t he N-terminal HA(2) fusion peptide. The resulting chimeric protein, F1 85, when expressed in bacteria, folded spontaneously into a soluble tr imer, with a high cc-helical content and a high melting temperature, s tructural characteristics of the low-pH-induced conformation of HA(2). Removal of the FLAG octapeptide by proteolysis with enterokinase conv erted the soluble molecule to one that aggregated, bound nonionic dete rgent, and bound to lipid vesicles, properties of the low-pH-induced c onformation of HA. Thermolysin treatment of the aggregated protein rem oved the nonpolar fusion peptide, regenerating soluble trimers of HA(2 ) (residues 24-185), which is analogous to thermolysin treatment of HA in the low-pH-induced conformation. Thermolysin treatment also dissoc iates F185 from the detergent-protein complex by removing the fusion p eptide. These results suggest that highly polar peptides can be fused to the membrane-binding regions of membrane proteins to increase their solubility. They also indicate that ectodomains of HA(2) made in bact eria have membrane-binding properties similar to those of the same ect odomain generated by low-pH treatment of HA isolated from virus.