A Tale of Two Protein Switches: Engineering, Characterizing, and Understanding a Novel and a Natural Switch

Throughout nature, many proteins provide a specific function in response to some input signal (e.g., phosyphorylation, pH, etc.), a process that is oftentimes described as switching a protein “on” or “off.” The advent of protein engineering has allowed for the creation and understanding of chimeric proteins for uses in a number of applications such as therapeutics, biosensors and energy production. Two proteins, hemagglutinin (HA) of fowl plague influenza virus and a chimeric protein comprising a fusion between the LFA-1 I domain and the EF3 and EF4 hands of calmodulin, have been investigated in this dissertation. Both of these proteins undergo structural rearrangements in response to an input signal in a process that could be described as molecular switching.

Hemagglutinin (HA) is a viral fusion protein that undergoes an irreversible conformational change upon acidification to catalyze the fusion of endosomal and viral membranes. Directed evolution was previously used to identify novel mutants with activation pH across a range of 4.8 – 6.0; wild-type HA activates around pH 5.2. Examination of library mutants has enabled us to identify individual amino acids responsible for phenotypic changes and thus provides additional structure-function insight highlighting the importance of HA1 headgroups in pH sensing.

Secondly, we have improved a previously created chimeric protein switch using the integrin LFA-1 I domain and the EF3 and EF4 hands of calmodulin; the original switch protein had a 2.4-fold increase for the I domain ligand, ICAM-1, in the presence of a calmodulin target peptide. Directed evolution has revealed a mutant switch with an additional twenty-fold increase in ICAM-1 binding in the presence of peptide. Additionally, the specificity of the chimeric protein for the original peptide sequence has been engineered, creating a new switch that responds to a different peptide sequence rendering the original peptide nonfunctional. Both proteins, hemagglutinin and the I domain-calmodulin fusion, represent the importance of conformational changes in controlling protein function. The ability to manipulate these changes underscores the power of protein engineering and directed evolution and can lead to the development of complex biomolecules that can be used in a variety of applications.