Critical role of tyrosine 79 in the fluorescence resonance energy transfer and terbium(III)-dependent self-assembly of ciliate Euplotes octocarinatus centrin

• Published in: Journal of biological inorganic chemistry 2010-09, Vol.15 (7), p.995-1007
• Main Authors: Duan, Lian, Liu, Wen, Wang, Zhi-Jun, Liang, Ai-Hua, Yang, Bin-Sheng
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Aromatics; Biochemistry; Biomedical and Life Sciences; Calcium - metabolism; Calcium-binding protein; Calcium-Binding Proteins - chemistry; Calcium-Binding Proteins - genetics; Ciliates; EF-hand; Euplotes - chemistry; Euplotes - genetics; Euplotes octocarinatus; fluorescence resonance energy transfer; Fluorescence Resonance Energy Transfer - methods; Fluorescent indicators; Humans; Hydrogen bonding; Hydrophobicity; Ions; Kinetics; Life Sciences; Light scattering; Microbiology; Models, Molecular; Molecular modelling; Molecular Sequence Data; Mutagenesis, Site-Directed; Original Paper; Oxygen; Protein Binding; Protozoan Proteins - chemistry; Protozoan Proteins - genetics; Self-assembly; Sequence Alignment; Site-directed mutagenesis; Terbium - chemistry; Tyrosine; Tyrosine - chemistry; Tyrosine - genetics
• ISSN: 0949-8257; 1432-1327
• DOI: 10.1007/s00775-010-0660-z

Ciliate Euplotes octocarinatus centrin (EoCen) is a member of the EF-hand superfamily of calcium-binding proteins. It has been proven, using Tb 3+ 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 Tb 3+ 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 Tb 3+ 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 Tb 3+ (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 Tb 3+ 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 Tb 3+ -dependent self-assembly of EoCen among the three tyrosines.