Structure and behavior of four-helix bundle cavitein systems

This thesis will investigate the sequence to structure relationship of protein folding through the use of template assembled synthetic proteins (TASPs). A cavitand template will be used to bring together four de novo designed peptides to form a four-helix bundled TASP, referred to as a cavitein. The first chapter will detail the rationale behind de novo protein design and use of TASPs to study protein folding. Chapter 1 will also review past and current work relating to the design and structural characterization of synthetic de novo proteins. Chapter 2 will investigate the context dependency of leucine residues within the hydrophobic core of a cavitein. Single leu to ala substitutions resulted in similar destabilization of the cavitein. However, replacing the middle leucine compromised the structure and stability of the cavitein. It was concluded that the middle leucine acted as a linchpin within the peptide sequence. Chapter 3 will examine the first reported crystallization of a TASP. The crystal structure is revealed as a dimer. The cavitein (Q4) was thought to be monomeric. The chapter will compare the solution structures of both the parent and Q4 cavitein. It was found that both parent and Q4 have a weak dimer association at relatively high concentrations. Chapter 4 will discuss glutamine variants used to probe the tolerance of dimer crystallization with respect to sequence variation. The chapter uncovers a key lattice contact for cavitein dimer lattice nucleation and stabilization. Furthermore, in an attempt to control the oligomeric state of caviteins, Chapter 4 will also discuss histidine metal binding caviteins and disulfide caviteins. A cavitein designed to favor a dimer via histidine metal binding was synthesized and crystallized revealing a potential metal binding center. Caviteins designed to favor a monomer conformation were also designed involving either histidine metal binding or disulfide bridges. Both methods proved successful in their monomeric design as shown by solution characterization. Chapter 5 will conclude and summarize the work throughout this thesis. The chapter will also suggest future projects expanding on studies within this thesis as well as suggest other projects pertaining to cavitein research.Science, Faculty ofChemistry, Department ofGraduat