I. Biophysical studies on PIN WW domains: A model for understanding folding and stability of beta-sheet proteins. II. Towards understanding templated beta-sheet self-assembly.

摘要

This thesis utilizes protein engineering and chemical synthesis to understand the many facets of β-sheet folding and stability. The introductory chapter describes the underlying forces driving protein folding, discusses biophysical methods for studying the proteins and peptides relevant to this thesis, introduces the PIN WW domain, a β-sheet miniprotein that serves as an ideal experimental system to evaluate protein folding and misfolding, and provides background on protein aggregation and amyloidosis.; Chapter 1 demonstrates that cyclization of the PIN1 WW domain, a 34-residue three-stranded β-sheet structure, leads to more stable β-sheets, despite removing a favorable electrostatic interaction between its termini. Optimization of the linker connecting the N- and C-termini using information based on the previously determined structures is important to achieve maximum stability.; Chapter 2 and Appendix A describes the incorporation of β-turn peptidomimetics into a β-sheet miniprotein. Many of the b-turn peptidomimetics are hydrophobic, thus there incorporation into proteins leaves them with less than perfect solubility properties. These studies reveal that a more polar analog of the dibenzofuran-based β-turn mimetic improved solubility and resistance to aggregation without compromising thermodynamic solubility.; Chapters 3 and 4 explore the influence of backbone hydrogen bonding on folding and stability of PIN WW domain. The importance of individual backbone hydrogen bonds was probed by amide-to-ester substitution using α-hydroxy acids that retains the naturally occurring side-chains and stereochemistry of the L-amino acids. The α-hydroxy acids can be synthesized by methods outlined in Appendix B. The removal of hydrogen bond donor has more influence than weakening of the hydrogen bond acceptor (amide to ester carbonyl). The extent of destabilization imparted by amide-to-ester substitutions is strongly context dependent. Backbone hydrogen bonding appears to lower the transition state energy of the WW domain as reflected by that amide-to-ester mutants have lower folding, rate than that of wt PIN.; Finally, Chapter 5 focuses on the development of small, simple peptidomimetics to study amyloid fibril formation. The peptidomimetic is comprised of a template, peptide strands, and end groups that can be varied to probed structural requirements for amyloidogenesis. The template holds the strands at a separation of approximately 10 Å, allowing corresponding hydrophobic side-chains in the strands to pack into a condensed U-shaped core, not stabilized by intramolecular hydrogen bonds.{09}Stacking of the U-shaped peptidomimetics is stabilized by intermolecular hydrogen boding and hydrophobic interactions between the inwardly directed side-chains in the core of the U-shaped peptidomimetic. The charge and composition of the end groups in combination with buffer composition influence higher order pacing of filaments.{09}Relative stability of all the peptidomimetics derived from their thermodynamic solubility is approximately 7 kcal/mol on average. Two forces, amphiphilicity and β-sheet propensity, enable the peptidomimetic to adopt micelle-like structure where hydrophobic core formed by hydrophobic side-chains and template is shielded by hydrophilic patches formed by hydrophilic side-chains and end groups.