Human centrin is a 20 kDa calcium sensor protein, belonging to the EF-hand superfamily, which has both structural and regulatory roles. Two highly conserved Homo sapiens isoforms of centrin(centrin 1 and centrin 2) have been included in this study both are associated with the microtubule organizing center. Sfi1 is a 1242 amino acid protein comprised of 23 tandem centrinbinding sites (CBS). These centrin binding sites are not conserved allowing for a systematic study of the interaction. Both centrin and Sfi1 are essential Eukaryotic proteins that have been found to be co-localized to the centrioles, basal bodies, and primary cilium. Structural information is available for the yeast homologue of this complex known as Cdc31-Sfi1p (PDB 2DOQ) associated with the duplication of the half bridge, yet the effect of a naturally occurring mutation with well-defined phenotype known as vfl2 has not been investigated to date. In Homo sapiens the centrin (E105K) variant corresponding to the vfl2 phenotype has been link to no centriole duplication. Our goal is to investigate the structural consequence of the centrin mutation (E105K) and its effect on the key salt-bridge interaction required for complex formation and to determine the possibility of an alternate salt-bridge interaction within the centrin (E105K)-Sfi1p21complex. Crystal screens were performed in collaboration with the HTSlab and Visual data examined for Hs centrin 1 WT-, Hs centrin 1(E105K)- and Hs centrin 2 (E105K)-Sfi1p21complexes. Optical identification of possible complex crystals was achieved with the result of said visual data which was composed of images taken with novel Second Order Non-lineal Imaging of Chiral Crystals (SONICC), UV-T, and light microscopy. Suspected centrin-Sfi1p21 complex crystals were found in similar crystallization conditions, thus increasing our confidence in the identification of such crystals. Crystals that were positive in all three viewing methods were considered to have a high possibility of been the crystallized centrin-Sfi121 complex of interest. These triple positive crystals will be subject to diffraction analysis to ascertain if they are indeed protein crystals and their diffraction pattern collected for high resolution structure determination.

VFL2 and wild type centrin-Sfi1p21 complex crystallization screenings

251st National Meeting (2016)

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