UC Berkeley

Domain swapping, the process in which a structural unit is exchanged between monomers to create a dimer containing two versions of the monomeric fold, is believed to be an important mechanism for oligomerization and the formation of amyloid fibrils. Very little is known, however, about the structural determinants and mechanisms of domain swapping. The goal of the work in this thesis is to elucidate these features in the C-terminal domain swapping of bovine pancreatic ribonuclease A (RNase A).

RNase A is a well studied protein that domain swaps under extreme conditions, such as lyophilization from acetic acid. The major domain-swapped dimer form of RNase A exchanges a β-strand at its C-terminus to form a C-terminal domain-swapped dimer. The hinge region between the exchanged beta-strand and the rest of the protein contains an amide bond between N113 and P114 that adopts a cis conformation in the monomer, and a trans conformation in the domain-swapped dimer. In this thesis, we show that this proline acts as a conformational gatekeeper to control domain swapping. Substitution of P114 with an amino acid that favors the trans conformation, such as glycine or alanine, results in significant population of the domain-swapped dimer under physiological conditions. This variant then allowed us to investigate the mechanism of domain swapping under experimentally accessible conditions.

NMR and hydrogen-deuterium exchange demonstrated that compared to variants that do not readily domain swap, P114G shows decreased hydrogen-exchange protection near the C-terminal hinge region, indicating increased local protein flexibility. These results suggest that local conformational fluctuations play a role in the mechanism of C-terminal domain swapping.

To further investigate this potential pathway for C-terminal domain swapping, we used a fragment approach - studying fragments of the two regions, or `domains', in isolation and in combination. A fragment of RNase A containing residues 1-115 represents the large region of the protein that remains after the swapped arm has exchanged. In isolation, this fragment lacks significant secondary and tertiary structure, suggesting that significant unfolding must take place during the swapping event. Upon noncovalent association with a peptide containing residues 112-124, native structure and function can be restored. These data are consistent with a mechanism in which the protein adopts a partially unfolded structure with the domain -swapped arm undocked and exposed.