Structural studies of the N-terminal region of cyclin B and Pds1, and calcium coordination and backbone dynamics of the metastasis associated protein, Mts1

The Mts1 protein has been strongly linked to metastasis and is thought to act in part by modulating cell motility. Mts1 belongs to the S100 family of proteins, which are composed primarily of two EF-hand structural motifs. Each EF-hand contains two α-helices joined by a calcium-binding loop. Calcium binding is thought to allow Mts1 to associate with a variety of proteins including actin, myosin, and p53. Like most S100 proteins, Mts1 exists primarily as a homo-dimer.; This work presents calcium-binding studies and analysis of the dynamical qualities of Mts1 in both the apo and calcium bound states. Using nuclear magnetic resonance (NMR) techniques to track residue-specific changes during titration with calcium, we find calcium binding to be ordered, with the N-terminal EF-hand binding calcium first. Intriguingly, concurrent with the filling of the N-terminal EF-hand, resonances from much of helix 4 vanish, indicating a change in conformational or chemical exchange in this region upon calcium binding.; The dynamical properties of proteins are very important for function. Dynamical analysis of Mts1 using NMR indicates that in the apo state, helices 1 and 4 are the least flexible, followed in order of increasing flexibility by helices 2 and 3, the calcium binding loops, and the linker region between the two EF-hands. In the holo state, Mts1 shows increased rigidity in the two calcium-binding loops. Again, resonances for helix 4 vanish. The changes in helix 4 could be of critical importance in specifying Mts1 function.; In addition to the Mts1 results, this thesis presents a structural analysis of the N-terminal regions of two cell cycle regulatory proteins, Pds1 and cyclin B. The destruction of Pds1 and cyclin B are essential for entry into anaphase and exit from mitosis, respectively. The destruction of these proteins is accomplished by the ubiquitin-proteasome system, which recognizes the N-terminal regions of these proteins. Using NMR and circular dichroism, we demonstrate that these regions are unfolded under native conditions. We propose that the unfolded state is necessary for function.