This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Centrin is a calcium binding protein which is localized in centrioles in vertebrate cells. The phosphorylation of centrin has been shown to signal the separation of centrosomes at prophase and has been implicated in centriole separation. Recent work by Sanoguet and coworkers has investigated the effect of centrin phosphorylation upon the protein thermal stability and structural dynamics. Using Fourier transform infrared spectroscopy and two-dimensional correlation spectroscopy, they were able to determine the structural changes due to thermal denaturation for both phosphorylated and holo-centrin. The results indicated that phosphorylation affects the unfolding of the loops of the C-terminal domain. Furthermore, there is evidence of a pre-transition involving the aspartate band of the Amide I. In light of these findings, we propose to use the method of Laser Induced Temperature-Jump in conjunction with infrared spectroscopy to monitor the folding dynamics of the C-terminal domain of phosphorylated centrin and holo-centrin. The C-terminal domain of centrin is structurally independent from other domains, and study of this fragment alone will simplify the experiment and its interpretation. The experiments will aim to monitor the aspartate vibrational mode which shows pre-transition absorption changes at low temperatures as well as the vibrations of the loop structures which show high temperature denaturation. A comparison of the phosphorylated and holo-centrin kinetics for these structural changes may yield a greater understanding of the biological function of centrin.
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