title: Critical role of tyrosine 79 in the fluorescence resonance energy transfer and terbium(III)-dependent self-assembly of ciliate Euplotes octocarinatus centrin.
abstract: Ciliate Euplotes octocarinatus centrin (EoCen) is a member of the EF-hand superfamily of calcium-binding proteins. It has been proven, using Tb3+ as a fluorescence probe, that EoCen has four calcium-binding sites. The sensitized emission arises from nonradiative energy transfer between the three tyrosine residues (Tyr46, Tyr72, and Tyr79) of the N-terminal half and the bound Tb3+ ions. To determine the most critical of the three tyrosine residues for the process of fluorescence resonance energy transfer, six mutants of the N-terminal domain of EoCen, which contain one (N-Tyr46/N-Tyr72/N-Tyr79) or two (N-Y46F/N-Y72F/N-Y79F) tyrosine residues, were obtained by site-directed mutagenesis. The aromatic residue-sensitized Tb3+ fluorescence of N-Y79F was most affected, displaying a 50% reduction compared with wild-type N-EoCen. Among the tyrosines, Tyr79 is the shortest mean distance from the protein-bound Tb3+ (at sites I/II), as calculated via the Förster mechanism. The steady-state and time-resolved fluorescence parameters of the wild-type N-EoCen and the three double mutants suggest that Tyr79, which exists in a hydrophobic environment, has the highest quantum yield and a relatively long average lifetime. The decay of Tyr79 is the least heterogeneous among the three tyrosine residues. In addition, molecular modeling shows that a critical hydrogen bond is formed between the 4-hydroxyl group of Tyr79 and the oxygen from the side chains of the residue Asn39. Kinetic experiments on tyrosine and Tb3+ fluorescence demonstrate that tyrosine fluorescence quenching is largely due to the self-assembly of EoCen, and that the quenching degrees of the mutants differ. Resonance light scattering and crosslinking analysis carried out on the full-length single mutants (Y46F, Y72F, and Y79F) showed that Tyr79 also plays the most important role in the Tb3+-dependent self-assembly of EoCen among the three tyrosines.
authors.affiliation: Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Molecular Science, Shanxi University, Taiyuan, 030006, China.
authors.forename: Lian
authors.initials: L
authors.lastname: Duan
authors.forename: Wen
authors.initials: W
authors.lastname: Liu
authors.forename: Zhi-Jun
authors.initials: ZJ
authors.lastname: Wang
authors.forename: Ai-Hua
authors.initials: AH
authors.lastname: Liang
authors.forename: Bin-Sheng
authors.initials: BS
authors.lastname: Yang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D016812:Euplotes / Q000737:chemistry* / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics; D016415:Sequence Alignment; D013725:Terbium / Q000737:chemistry*; D014443:Tyrosine / Q000737:chemistry* / Q000235:genetics
chemical_list: D002135:Calcium-Binding Proteins; D015800:Protozoan Proteins; D013725:Terbium; D014443:Tyrosine; D002118:Calcium