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Self-Assembly Study Of Ciliate Euplotes Octocarinatus Centrin

Posted on:2011-02-18Degree:DoctorType:Dissertation
Country:ChinaCandidate:L DuanFull Text:PDF
GTID:1100360305995311Subject:Inorganic Chemistry
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Centrin is an acidic, low molecular weight (Mr~20000) protein that belongs to the EF-hand superfamily of calcium binding proteins. This calcium-binding protein (centrin) was first identified as a major component of the fibers that link the nucleus to the flagellar apparatus in flagellated unicells, and later, it was shown to be a ubiquitous component of centrioles, centrosomes, and mitotic spindle poles. Cenrin is required for the cell division, centrosome positioning and orientation, mitotic spindle segregation, as well as microtubule severing, and plays an essential role in contraction of centrin-based fiber systems in eukaryotic cells. A distinguishing feature of centrin-based fiber systems is that they all show calcium-induced contractile behavior, which is closely related to the fact that centrins from yeast (Cdc31p), algae (Scherffelia dubia centrin (SdCen)), or humans (HsCen1, HsCen2) may form multimers in the presence of Ca2+.Ciliate Euplotes octocarinatus centrin (EoCen) is first reported by our laboratory, which is cloned from Euplotes octocarinatus, a unicellular eukaryotic protozoon located in a special phylogenic degree. Using EoCen as a model of metal ion-binding target is significant for elucidating the molecular basis of biological effects of rare earth elements. EoCen is a protein of 168 residues, which shares about 60,62 and 66% sequence identity with human centrin 1, human centrin 2 and human centrin 3, respectively, and shares approximately 50% sequence identity with the well studied EF-hand protein calmodulin (CaM). Recently, the structure of the N-terminal domain of EoCen was obtained by NMR (2joj. pdb). Like CaM, centrin consists of two independent domains tethered by a flexible linker, each domain comprising a pair of EF-hand motifs of helix-loop-helix that can potentially bind two calcium ions. EoCen may form multimers in the presence of Ca2+ as same as centrins from other species, in clear contrast with CaM. This dissertation focuses on the metal ion-dependent self-assembly of EoCen. In the present study, nine mutants (Y46F, Y72F, Y79F, N-Y46F, N-Y72F, N-Y79F, N-Tyr46, N-Tyr72 and N-Tyr79) were first obtained by site-directed mutangenesis. In all cases, the presence of the specific mutation and the lack of random mutations were verified by DNA sequence analysis, which was conducted by a commercial company. Recombinant plasmids were transformed in E.coli BL21 (DE3), then the fusion expression of the mutants were performed by the induction of IPTG Fusion protein was cut by PPase and was purified by GST affinity chromatography. At the same time, the wild-type EoCen and the truncated form of it (EoCen, N-EoCen, C-EoCen,Δ23EoCen, P23, and P123), which are previously constructed by our group, were expressed and purified. The final product is examined by SDS-PAGE, and all of the protein products were of high purity.In order to systematically characterize the metal-ion dependent self-assembly of EoCen, we initiated a physicochemical study of the self-assembly properties of the purified protein (EoCen, N-EoCen, C-EoCen,Δ23EoCen, P23, and P123) in vitro. The critical role of the N-terminal domain of EoCen played in the self-assembly was exhibited by chemical cross-linking experiment. The native PAGE results indicate that only the integral protein shows multimers in the presence of Lu3+. The dependence of Lu3+-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 Ca2+and Lu3+binding to the N-terminal domain of EoCen are all positive to the self-assembly of the protein, and Lu3+ exhibit the stronger effect, however, Mg2+ alone cannot take the same effect. The experiments of 2-ptoluidinylnaphthalene-6-sulfonate (TNS) binding and ionic strength effect 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.Tyrosine is one of the most preferred residues in "hot spots" of protein-protein interactions. In addition, tyrosine is of fluorescence properties. It has been proven that EoCen has four calcium-binding sites by using Tb3+ as fluorescence probe. The sensitized emission arises from a nonradiative energy transfer between the three tyrosine residues (Tyr-46, Tyr-72, and Tyr-79) of N-terminal half and the bound Tb3+ions. In addition, To find the most critical one of the three tyrosine residues in the process of fluorescence resonance energy transfer (FRET) and in the protein self-assembly, nine mutants (Y46F, Y72F, Y79F, N-Y46F, N-Y72F, N-Y79F, N-Tyr46, N-Tyr72 and N-Tyr79) of EoCen, which contain one or two tyrosine residues were obtained by site-directed mutagenesis. The aromatic residue-sensitized Tb3+ fluorescence of N-Y79F was mostly affected, which displayed a 50% reduction compared with wild-type N-EoCen. Tyr-79 is of the nearest mean distance (8.8±0.2A) of protein bound Tb3+(at sitesⅠ/Ⅱ), which is calculated via the Forster mechanism. The steady-state and time-resolved fluorescence parameters of the wild-type N-EoCen and the three double mutants suggest that Tyr-79, which exists in a hydrophobic environment, has the highest quantum yield (3.27×10-2) and a relatively long average lifetime (2.20 ns). The standard deviation of the average lifetime of Tyr-79 (σ=0.271 ns) is the least among the three tyrosine residues. In addition, molecular modeling shows that a critical hydrogen bond is formed between the 4-hydroxyl group of Tyr-79 and the oxygen from the side chains of residue Asn-39. Kinetic experiments of tyrosine and Tb3+fluorescence demonstrate that the tyrosine fluorescence quenching is largely originated from the self-assembly of EoCen, and that the quenching degrees of the mutants differ from one another. Resonance light scattering (RLS) and cross-linking analysis carried out on the full-length single mutants (Y46F, Y72F, and Y79F) showed that Tyr-79 also plays the most important role in the Tb3+-dependent self-assembly of EoCen among the three tyrosines.Monastrol, a cell-permeable inhibitor, considered to specifically inhibit kinesin Eg5, can cause mitotic arrest and monopolar spindle formation, thus exhibit antitumor properties. Centrin, a ubiquitous protein associated with centrosome, plays a critical role in centrosome duplication. Moreover, a correlation between centrosome amplification and cancer has been reported. In this study, it is proposed for the first time that centrin may be another target of the anticancer drug monastrol since monastrol can effectively inhibit not only the growth of the transformed Escherichia coli cells (pGEX-6p-1-EoCen) and the division of the Euplotes octocarinatus cells in vivo, but also the Lu3+-dependent self-assembly of EoCen in vitro. The two closely related compounds (Compound 1 and 2) could not take the same effect. Fluorescence titration experiments suggest that four monastrols per protein is the optimum binding pattern, and the binding constants at different temperatures were obtained. Detailed thermodynamic analysis indicates that hydrophobic force is the main acting force between monastrol and centrin, and the extent of monastrol inhibition of centrin self-assembly is highly dependent upon the hydrophobic region of the protein, which is largely exposed by the binding of metal ions.Melittin, a basic 26-amino acid residue amphiphilic peptide from bee venom, is widely used as a tool for the study of protein-lipid and protein-protein interactions in artificial and biological membranes. In the present study, we proved that the main acting force in the binding of melittin to EoCen is the electrostastic force. Evidence that melittin binding of EoCen have inhibited the Lu3+-induced self-assembly of EoCen is preliminary. The two action mode of centrin may underlie the same molecular mechanism.
Keywords/Search Tags:Centrin Self-Assembly, Metal Ion, Tyrosine, Monastrol, Melittin
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