Understanding the relationship between sequence and structure: Biophysical studies of beta-bundles and bipartite tetracysteine display in beta sheets

1. Biophysical studies of beta-bundles. Chapter 1 provides an overview of the work on beta-peptide assembly (oligomers of beta3-amino acids) into quaternary structures known as bundles. In particular, the review highlights the work of the Schepartz laboratory, and the progress made thus far towards the rational design of novel non-proteinaceous beta-peptide assemblies with stabilities, and eventually, functionalities analogous to those of natural proteins.1-4.;Chapter 2 details the extensive biophysical characterization (analytical ultracentrifugation (AU), circular dichroism (CD), 1-anilino-8-naphthalenesulfonate (ANS) binding and deuterium exchange NMR (DX NMR)) of an octamer beta-peptide bundle, Acid-1Y. Surprising, we found that Acid-1Y is more stable than its beta-peptide predecessors, Zwit-1F and Acid-1F/Base-1F. Following on this work, the x-ray structural characterization of Zwit-YK, the most stable bundle to date, is described.;Chapter 3 investigates whether beta3-peptide bundle stoichiometry is controlled by the side chain identity within the bundle core. Specifically, we investigated mutations of leucine to valine in the sequences of the octameric bundles. These second generation valine beta-peptides assemble into discrete and stable tetrameric bundles that were extensively characterized by AU, CD, ANS binding and DX NMR.13.;Our interest in modifying the core of beta-bundles was extended in Chapters 4 and 5. These chapters detail the biophysical characterization of novel beta-bundles with alternative core sequences that were predicted using Rosetta (in collaboration with Professor Rhiju Das, Stanford University and Professor David Baker, University of Washington).;Finally, the evolution of bundles with functions that mimic those of natural proteins requires the advancement of screening techniques that will quickly identify beta-peptide sequences that assemble into bundles. Chapter 6 overviews two methods that enable efficient and facile screening of beta-peptide bundle assembly.;2. Bipartite tetracysteine display in beta sheets. The Schepartz laboratory has shown the utility of terminal split-tetracysteine motifs in detecting protein oligomerization and distinguishing between folded and unfolded proteins. Chapter 7 describes the extension of this work in which we engineered intramolecular split tetra-cysteine motifs. We found that this method favors sites with flexible geometries such as loops. This information is critical for directing successful bipartite tetracysteine site designs.