| Membrane proteins have important biological functions, such as information transfer across the cell membrane, regulation of membrane fluidity, permeability and structure, and membrane fusion. Therefore, membrane proteins can be used as references for engineering protein-based biosensors or drug delivery machineries. However, the first necessary step is to understand the mechanism of membrane proteins. We have developed mammalian system that can be used as a tool for both studying the mechanism of influenza virus fusion protein and engineering its properties via reverse engineering.; Enveloped viruses contain surface proteins that mediate fusion between the viral and target cell membranes following an activating stimulus. Acidic pH induces the influenza virus fusion protein hemagglutinin (HA) via irreversible refolding of a trimeric conformational state leading to exposure of hydrophobic fusion peptides on each trimer subunit. Using our mammalian system, we have demonstrated product-catalyzed autocatalytic refolding of WT HA and our engineered mutants. Partially activated states do not exist at the scale of the cell, activation of HA leads to aggregation of cell surface trimers, and newly synthesized HA refold spontaneously in the presence of previously activated HA. In order to alter the pH-dependence, we randomly mutated HA, and screened for mutants with altered activation pH's. Sequence analysis showed that mutations in high pH mutants were mostly found at the trimeric interface. One mutation found in a low pH-mutant destabilizes the activated conformation of HA. Further investigation is required to learn the role of mutations in alteration of pH-dependence.; When a high pH activating HA is co-expressed with a low pH activating mutant, the low-pH mutant activation was catalyzed by the activation of the high pH mutant. A product-catalyzed feedback mechanism was also observed in the actual membrane fusion. Thus, it should be possible to create protein switches responding to alternative stimuli that modulate membrane fusion via autocatalysis. |
