Calcium-dependent self-assembly of human centrin 2.
Human centrin 2 (HsCen2) is a member of the EF-hand superfamily of calcium-binding proteins, often associated with the centrosomes and basal bodies. These organelles exhibit different morphological aspects, including a variety of centrin-containing fibers that connect the two centrioles or other structural elements of the pericentriolar space. The molecular basis of the Ca(2+)-sensitive fibers and their precise role in centrosome duplication are not known. To explore the possible structural role of HsCen2, we initiated a physicochemical study of the self-assembly properties of the purified protein in vitro. Using light scattering experiments, we investigated the temporal evolution of the assembly process and characterized the dependence on various chemical and physical factors, including temperature, di-cation concentration, ionic strength, protein concentration, and pH. The reversible self-assembly revealed many features of a large-size protein polymerization, with nucleation and elongation steps. Kinetic and equilibrium experiments show that a hydrophobic fluorescent probe (ANS) inhibits the polymerization by interfering with the nucleation step, probably through interactions with the apolar exposed sites on the protein surface. A truncated form of HsCen2, lacking the first 25 residues (Delta25HsCen2), shows no detectable self-assembly, pointing to the critical role played by the N-terminal fragment in the supermolecular organization of HsCen2. As revealed by isothermal titration experiments, the isolated N-terminal domains bind with a significant affinity (2 x 10(5) m(-1)) to preformed oligomers of Delta25HsCen2 through an entropy-driven mechanism.
Tourbez
Martine
M
INSERM and Institut Curie-Recherche, Centre Universitaire Paris-Sud, 91405 Orsay Cedex, France.
Firanescu
Claudia
C
Yang
Ao
A
Unipan
Laura
L
Duchambon
Patricia
P
Blouquit
Yves
Y
Craescu
Constantin T
CT
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins; D002151:Calorimetry; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002502:Centrioles / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D013696:Temperature
D000817:Anilino Naphthalenesulfonates; C472752:CETN2 protein, human; D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; D005456:Fluorescent Dyes; C027132:1-anilino-8-naphthalenesulfonate; D002118:Calcium