McDonald, Iain2024-08-302024-08-302024-08-302024-08-27https://hdl.handle.net/10012/20937Myristoyl-switching in proteins is an essential form of functional regulation that controls fundamental biological processes such as signal transduction, protein-membrane interactions, and viral infection. In this form of functional regulation, the reversible switching of a saturated C14 fatty-acyl chain covalently attached to the N-terminus of a protein switches between two states: 1) a sequestered state where the myristoyl group is buried in a hydrophobic environment and 2) a state with increased solvent accessibility where the myristoyl group is available for interaction with binding partners. Myristoyl-switching controls protein function by modulating affinity for membrane and protein binding partners, depending on the accessibility of the hydrophobic myristoyl group. Hisactophilin is a membrane binding protein found in Dictyostelium discoideum responsible for binding and bundling actin in a pH-dependent manner, largely driven by the reversible exposure of its myristoyl group. This protein’s myristoyl switch is controlled by an intramolecular network of electrostatic-hydrophobic interactions; at low pH ~1.5 protons are bound by some of the many ionizable groups, resulting in a conformational shift where the sequestered myristoyl group is made more accessible for insertion into cellular membranes. Through a combination of implicit solvent molecular dynamics simulations and experimental methods, residues D57, H89 and H91 were hypothesized to be the residues controlling myristoyl-switching in hisactophilin. Mutation of these residues indicates that the proposed mechanism of pH-switching in hisactophilin is not fully correct. Design and experimental characterization of follow-up mutants indicates that pH-switching may be controlled through an alternative mechanism. Further investigating the molecular mechanisms of myristoyl-switching in this protein will provide valuable insight into how hydrophobic-electrostatic networks regulate function in allosteric proteins.enproteinMaster Thesis