Conformational detection of p53's oligomeric state by FlAsH Fluorescence.

The p53 tumor suppressor protein is a critical checkpoint in prevention of tumor formation, and the function of p53 is dependent on proper formation of the active tetramer. In vitro studies have shown that p53 binds DNA most efficiently as a tetramer, though inactive p53 is predicted to be monomeric in vivo. We demonstrate that FlAsH binding can be used to distinguish between oligomeric states of p53, providing a potential tool to explore p53 oligomerization in vivo. The FlAsH tetra-cysteine binding motif has been incorporated along the dimer and tetramer interfaces in the p53 tetramerization domain to create reporters for the dimeric and tetrameric states of p53, though the geometry of the four cysteines is critical for efficient FlAsH binding. Furthermore, we demonstrate that FlAsH binding can be used to monitor tetramer formation in real-time. These results demonstrate the potential for using FlAsH fluorescence to monitor protein-protein interactions in vivo.

The C-terminus of the P22 tailspike protein acts as an independent oligomerization domain for monomeric proteins.

TSP (P22 tailspike protein) is a well-established model system for studying the folding and assembly of oligomeric proteins, and previous studies have documented both in vivo and in vitro folding intermediates using this protein. Especially important is the C-terminus of TSP, which plays a critical role in the assembly and maturation of the protrimer intermediate to its final trimeric form. In the present study, we show that by grafting the C-terminus of TSP on to the monomeric MBP (maltose-binding protein), the resulting chimaera (MBP-537) is a trimeric protein. Moreover, Western blot studies (using an anti-TSP antibody) indicate that the TSP C-terminus in the MBP-537 chimaera has the same conformation as the native TSP. The oligomerization of the MBP-537 chimaera appears to involve hydrophobic interactions and a refolding sequence, both of which are analogous to the native TSP. These results underscore the importance of the TSP C-terminus in the assembly of the mature trimer and demonstrate its potential utility as a model to study the folding and assembly of the TSP C-terminus in isolation.