We will use crystallography and radiation foot printing to learn how nucleotides and activating peptides influence the conformation of Arp2/3 complex. This structural information will contribute to the group effort to model the entire structural pathway of Arp2/3 complex activation and nucleation of actin filament branches. Project 1. We will determine the structures of activator peptides derived from WASp, N-WASp or Scar/WAVE1 bound to Arp2/3 complex by x-ray crystallography. We will use mutation and biochemical characterization to characterize the contributions of key residues of activator proteins for binding Arp2/3 complex. Using insights from NMR and crystallography we will design and characterize biochemically activator proteins with mutations that might compromise their binding to or activation of the Arp2/3 complex. Project 2. Determine how nucleotides and WASp/Scar proteins influence the equilibrium between the active and inactive conformations of Arp2/3 complex. We will obtain high resolution structures of active conformations of Arp2/3 complex to document the conformational changes required to activate the complex for nucleating actin filaments and synchrotron radiation protection to identify residues in Arp2/3 complex that change their exposure to the solvent upon binding nucleotides and activator peptides. Project 3. Determine how Arp2/3 complex initiates an actin filament branch. We will crystallize and determine the structures of actin with a bound WH2 peptide or p40 insert peptides. Use x-ray crystallography to determine the structure of activated Arp2/3 complex with the first subunits of the nucleated actin filament. We will attempt to crystallize Arp2/3 complex associated with WA and one or two actin monomers. Project 4. Determine the structure of actin filament branches mediated by Arp2/3 complex. We will use synchrotron radiation protection to identify amino acid residues at interface between Arp2/3 complex and the side of actin filaments.
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