Vincent D. Lee

The Scripps Research Institute, لا هویا, California, United States

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Publications (3)6.68 Total impact

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    ABSTRACT: Caltractin (centrin) is a member of the calmodulin subfamily of EF-hand Ca2+-binding proteins that is an essential component of microtubule-organizing centers in many organisms ranging from yeast and algae to humans. The protein contains two homologous EF-hand Ca2+-binding domains linked by a flexible tether; each domain is capable of binding two Ca2+ ions. In an effort to search for domain-specific functional properties of caltractin, the two isolated domains were subcloned and expressed in Escherichia coli. Ca2+ binding affinities and the Ca2+ dependence of biophysical properties of the isolated domains were monitored by UV, CD, and NMR spectroscopy. Comparisons to the corresponding results for the intact protein showed that the two domains function independently of each other in these assays. Titration of a peptide fragment from the yeast Kar1p protein to the isolated domains and intact caltractin shows that the two domains interact in a Ca2+-dependent manner, with the C-terminal domain binding much more strongly than the N-terminal domain. Measurements of the macroscopic Ca2+ binding constants show that only the N-terminal domain has sufficient apparent Ca2+ affinity in vitro (1-10 microm) to be classified as a traditional calcium sensor in signal transduction pathways. However, investigation of the microscopic Ca2+ binding events in the C-terminal domain by NMR spectroscopy revealed that the observed macroscopic binding constant likely results from binding to two sites with very different affinities, one in the micromolar range and the other in the millimolar range. Thus, the C-terminal domain appears to also be capable of sensing Ca2+ signals but is activated by the binding of a single ion.
    Journal of Biological Chemistry 09/2002; 277(32):28564-71. DOI:10.1074/jbc.M112232200 · 4.60 Impact Factor
  • Vincent D. Lee, Stacey L. Finstad, Bessie Huang
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    ABSTRACT: The genomic sequence of an actin-related gene in Chlamydomonas reinhardtii has been determined. The deduced amino acid sequence of this gene shares a 63.9% identity with that of a recently reported conventional actin-encoding gene in C. reinhardtii. Phylogenetic analysis shows that the product of this actin-related gene does not fit into conventional actin or any major actin-related protein categories. The actin-related gene in C. reinhardtii contains seven introns in the coding region and, as described for the conventional actin gene, it contains several sequences similar to the 'tub box' sequence motif in its 5'-upstream region. Southern blot analysis of the gene shows a hybridization pattern different from that of the conventional actin gene, indicating that these genes are distinct from one another. Northern blot analysis of poly(A)+RNA shows the messages of the two genes to be very similar in size, yet the message level of the actin-related gene is significantly lower than that of the conventional actin gene.
    Gene 10/1997; 197(1-2):153-9. DOI:10.1016/S0378-1119(97)00254-0 · 2.08 Impact Factor
  • Vincent D. Lee, Bessie Huang