CURRENT PROJECTS IN PROTEIN ENGINEERING AND DIRECTED EVOLUTION
Membrane Fusogens
We are trying to engineer novel proteins capable of inducing triggered membrane fusion. The model system for our work is influenza hemagglutinin (HA), which mediates membrane fusion related to delivery of the viral genetic material to the infected cell. HA is activated by low pH and undergoes a dramatic conformational refolding leading to membrane fusion. Several years ago we demonstrated that the conformational change is autocatalytic and requires the HA fusion peptide. Currently, we are extending these studies to probe the ability to reengineer HA so as to be activated in response alternative stimuli, as well as to test HA's ability to communicate its activation state to other, similar membrane fusogen proteins.
We are trying to engineer novel proteins capable of inducing triggered membrane fusion. The model system for our work is influenza hemagglutinin (HA), which mediates membrane fusion related to delivery of the viral genetic material to the infected cell. HA is activated by low pH and undergoes a dramatic conformational refolding leading to membrane fusion. Several years ago we demonstrated that the conformational change is autocatalytic and requires the HA fusion peptide. Currently, we are extending these studies to probe the ability to reengineer HA so as to be activated in response alternative stimuli, as well as to test HA's ability to communicate its activation state to other, similar membrane fusogen proteins.
Adhesive Protein Switches
The "I domain" of certain integrins binds to protein ligands to mediate cell adhesion, but only in situations in which the integrin has been activated. I domain affinity for its ligand (ICAM-1) is determined by its conformation, which is regulated by a complex structural mechanism in response to cell signaling in leukocytes. We seek to develop a novel protein architecture incorporating I domain such that its conformation and binding activity can be directly regulated by its environment, such as the presence of a second ligand binding to an alternative site and allosterically inducing adhesion. Such proteins could prove to be useful biosensors or cell targeting agents. Directed evolution and yeast surface display are key methods in this work.
The "I domain" of certain integrins binds to protein ligands to mediate cell adhesion, but only in situations in which the integrin has been activated. I domain affinity for its ligand (ICAM-1) is determined by its conformation, which is regulated by a complex structural mechanism in response to cell signaling in leukocytes. We seek to develop a novel protein architecture incorporating I domain such that its conformation and binding activity can be directly regulated by its environment, such as the presence of a second ligand binding to an alternative site and allosterically inducing adhesion. Such proteins could prove to be useful biosensors or cell targeting agents. Directed evolution and yeast surface display are key methods in this work.
Protein Ligation by Sortase A
The transpeptidase enzyme Sortase A (SrtA) catalyzes the ligation of proteins bearing a five amino acid tag sequence to appropriate nucleophiles, notably the N-terminus of another peptide or protein beginning with glycine. We are exploring applications of SrtA to attach proteins to surfaces and to ligate proteins into oligomeric structures. We are also investigating directed evolution of SrtA to alter its substrate specificity.
The transpeptidase enzyme Sortase A (SrtA) catalyzes the ligation of proteins bearing a five amino acid tag sequence to appropriate nucleophiles, notably the N-terminus of another peptide or protein beginning with glycine. We are exploring applications of SrtA to attach proteins to surfaces and to ligate proteins into oligomeric structures. We are also investigating directed evolution of SrtA to alter its substrate specificity.