Lutetium(III)-dependent self-assembly study of ciliate Euplotes octocarinatus centrin.
Ciliate Euplotes octocarinatus centrin (EoCen) is a member of the EF-hand superfamily of calcium-binding proteins, which often associated with the centrosomes and basal bodies. To explore the possible structural role of EoCen, we initiated a physicochemical study of the self-assembly properties of the purified protein in vitro. The native PAGE results indicate that only the integral protein shows multimers in the presence of Lu(3+). The dependence of Lu(3+) induced self-assembly of EoCen on various chemical and physical factors, including temperature, protein concentration, ionic strength and pH, was characterized using resonance light scattering (RLS). Control experiments with different metal ions suggest that Ca(2+) and Lu(3+) bindings to the N-terminal domain of EoCen are all positive to the self-assembly of the protein, and Lu(3+) exhibits the stronger effect, however, Mg(2+) alone cannot take the same effect. The experiments of 2-ptoluidinylnaphthalene-6-sulfonate (TNS) binding and ionic strength demonstrate that the lutetium(III)-dependent self-assembly is closely related to the exposure of hydrophobic cavity. Control experiment on pH value with EoCen and the fragments of it, N-terminal domain of EoCen (N-EoCen), indicates that the electrostatic effect is of small tendency to be served as the main driving force in the self-assembly of EoCen. The specific oligomerization form of the protein was exhibited by cross-linking experiment.
Duan
Lian
L
Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Molecular Science, Taiyuan 030006, China.
Zhao
Ya-Qin
YQ
Wang
Zhi-Jun
ZJ
Li
Guo-Ting
GT
Liang
Ai-Hua
AH
Yang
Bin-Sheng
BS
D000818:Animals; D002118:Calcium / Q000737:chemistry; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D020832:EF Hand Motifs; D016812:Euplotes / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D008187:Lutetium / Q000737:chemistry*; D009282:Naphthalenesulfonates / Q000737:chemistry; D009994:Osmolar Concentration; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins / Q000737:chemistry* / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence; D013696:Temperature
D002135:Calcium-Binding Proteins; D009282:Naphthalenesulfonates; D015800:Protozoan Proteins; D008187:Lutetium; C009497:2-(4-toluidino)-6-naphthalenesulfonic acid; D002118:Calcium