tailieunhanh - Báo cáo khoa học: Binding of human centrin 2 to the centrosomal protein hSfi1

hSfi1, a human centrosomal protein with homologs in other eukaryotic organisms, includes 23 repeats, each of 23 amino acids, separated by 10 residue linkers. The main molecular partner in the centrosome is a small, calcium-binding EF-hand protein, the human centrin 2. Using isothermal titration calorimetry experiments, we characterized the centrin-binding capacity of three isolated hSfi1 repeats, two exhibiting the general consen-sus motif and the third being the unique Pro-containing human repeat | ễFEBS Journal Binding of human centrin 2 to the centrosomal protein hSfil Juan Martinez-Sanz Ao Yang Yves Blouquit Patricia Duchambon Liliane Assairi and Constantin T. Craescu The Integrative Imaging Unit INSERM U759 Institut Curie-Recherche Centre Universitaire Paris-Sud Orsay Cedex France Keywords centrosome human centrin protein interactions Sfi1 protein Correspondence C. T. Craescu Integrative Imaging Unit INSERM U759 Institut Curie-Recherche Centre Universitaire Paris-Sud Batiment 112 91405 Orsay Cedex France Fax 33 1 69 07 53 27 Tel 33 1 69 86 31 63 E-mail Website http U759 Received 5 June 2006 revised 2 August 2006 accepted 9 August 2006 doi hSfil a human centrosomal protein with homologs in other eukaryotic organisms includes 23 repeats each of 23 amino acids separated by 10 residue linkers. The main molecular partner in the centrosome is a small calcium-binding EF-hand protein the human centrin 2. Using isothermal titration calorimetry experiments we characterized the centrin-binding capacity of three isolated hSfi1 repeats two exhibiting the general consensus motif and the third being the unique Pro-containing human repeat. The two standard peptides bind human centrin 2 and its isolated C-terminal domain with high affinity 107 M-1 by an enthalpy-driven mechanism with a moderate Ca2 dependence. The Pro-containing repeat shows a binding affinity that is two orders of magnitude lower. The target binding site is localized within the C-terminal domain of human centrin 2. Fluorescence titration and NMR spectroscopy show that the well-conserved Trp residue situated in the C-terminus of each repeat is deeply embedded in a protein hydrophobic cavity indicating that the peptide direction is reversed relative to previously studied centrin targets. The present results suggest that almost all of the repeats of the Sfi1 protein may independently bind centrin molecules. On the basis of this

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