Date of Completion
pore-forming toxin, proline isomerization, hemolysin, conformational exchange, NMR, protein structure
Field of Study
Structural Biology and Biophysics
Doctor of Philosophy
Proteins are complex macromolecules that play a critical role in all biological processes. Their ability to change shape, or conformation, in response to environmental factors is intimately linked with function. Thus, understanding the mechanisms underlying conformational changes is imperative for complete understanding of protein function(s), and an overall comprehension of biological systems. This work explores the structural and dynamic attributes of conformational exchange in the C-terminal domain of pore-forming toxin Hemolysin II (HlyIIC), and the coiled-coil leucine zipper, GCN4p, using nuclear magnetic resonance (NMR). The HlyIIC domain contains a sole proline at position 405 that leads to equal populations of cis and trans states in thermodynamic equilibrium. NMR structure determination revealed that HlyIIC has a novel topology, consisting of five β-strands and two α-helices. Arranged in a “pseudobarrel fold” held together by a core consisting of three layers of hydrophobic amino acids. Isomerization of the G404-P405 peptide bond induces movement of the proline-containing loop, causing perturbations in adjacent loops, and smaller changes at distant sites transmitted through core hydrophobic side chains. The cis/trans equilibrium is temperature dependent, with the trans state favored entropically, and the cis state favored enthalpically. A trans-stabilized variant of the domain, P405M-HlyIIC, exists in a monomer/dimer equilibrium that shifts in a concentration and temperature dependent manner. Preliminary structural studies by NMR show domain swapping of β5. Further investigation can help to elucidate the structural significance of P405. To understand how changes in electrostatic charge interactions affect protein structure we determined the NMR structures of the GCN4p coiled-coil homodimer at three pH values. The structures show that as the pH went from neutral to acidic, and ion-pair interactions are broken there is an unwinding of the coiled-coil superhelix, yet the helical character of the monomers is retained. Side chain dynamics increased at lower pH, notably for residues that participated in intermolecular ion pairs. NMR provides the unique advantage to study proteins in solution, and alter the solution conditions to probe changes in structure and stability.
Kaplan, Anne R., "Adventures in Structureland: Exploring Protein Conformational Plasticity by NMR" (2019). Doctoral Dissertations. 2244.