The Effect Of Glu101 To The Conformation And The Biological Functions Of Euplotes Octocarinatus Centrins

Centrin is a protein of 20 kDa, which belongs to the highly conserved EF-hand CaM superfamily of Ca²⁺-binding proteins. Centrin has four Ca²⁺-binding sites (EF-hand) and each site can bind with one Ca²⁺. The localization of centrin is not limited to centrosomes but is related to other cell compartments like the nucleus or the perimembrane space. Centrins play a fundamental role in centriole duplication and contraction of center basal body fibrous. The methods of the mutation of amino acids are widely used in the study of the effect of certain amino acids to the conformation and the biological function of proteins. The advantage of UV mutagenesis is more mutated proteins can be gotten and the disadvantage is that the result of this mutation is not sure and series of filtration work must be done. The method of site direct mutation is better used than UV mutagenesis just because of its clearly mutated site and its simplex mutation result.The canonical sequence for the E-helix, based on the known amino acid sequences of more than 150 members of the CaM superfamily, can be represented as En$$nn$$n-with E being glutamate, n a hydrophobic amino acid, and $ any amino acid. Thus, the glutamate (Glu) at the beginning of the E-helix would be expected to be important to the proper conformation and function of centrin. In this study, we have expressed and purified the Euplotes octocarinatus centrin (EoCen) by biological engineering and the mutanted protein (E101K) with Glu 101, the first amino acid of the E-helix of the third EF-hand in EoCen, changing to Lys was gotten by the method of site direct mutation. Then the effect of Glu101 to the conformation and the biological function of EoCen were studied as follows:Firstly, the effect of the mutation to the basic spectra of centrin was studied by fluorescence and UV spectra. The results indicate that the excitation, emission and absorption spectra of EoCen were not affected by the mutation of Glu101. Additionally, using Tb³⁺ as fluorescence probe, the effect of the mutation to the metal binding characteristic of EoCen was studied by fluorescence spectra. Results indicate that the fluorescence sensitization of Tb³⁺ binding with the third EF-hand site of EoCen was greatly enhanced by the mutation of Glu101. It could be conjectured that the conformation of Centrin was changed by the mutation of Glu101. It is sure that Glu101 was crucial to keep the normal metal binding characteristic of EoCen.Secondly, the effect of the mutation of Glu101 to the interaction of Centrin and rare earth ions was investigated. Results indicate that the degree of the conformation change of centrins which was induced by the interaction of centrins and rare earth ions was larger than the interaction of centrins and Ca²⁺. Moreover, the RLS intensities which were induced by the interaction of E101K and rare earth ions were obviously stronger than interaction of EoCen and rare earth ions. It was deduced that Glu101 was crucial to keep the normal conformation change which was induced by the interaction of EoCen and rare earth ions.Lastly, the effect of the mutation of Glu101 to the interaction of EoCen and melittin was studied. Results indicate that the 1:1 compound was formed by the interaction of EoCen and melittin and the interaction of Centrin and melittin was not been affected by the mutation of Glu101.