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

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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.

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Abbreviations

CaM:

Calmodulin

EoCen:

Wild-type ciliate Euplotes octocarinatus centrin

FRET:

Fluorescence resonance energy transfer

HEPES:

4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid

IPTG:

Isopropyl-d-thiogalactopyranoside

LB:

Luria–Bertani

Phe:

Phenylalanine

PBS:

Phosphate-buffered saline

RLS:

Resonance light scattering

TNS:

2-pToluidinylnaphthalene-6-sulfonate

N-EoCen:

N-Terminal domain (1–101) of wild-type EoCen

Y46F/Y72F/Y79F:

The three single mutants of full-length EoCen in which one tyrosine (Y) residue has been replaced by phenylalanine (F)

N-Y46F/N-Y72F/N-Y79F:

The three single mutants of N-EoCen in which one tyrosine (Y) residue has been replaced by phenylalanine (F), each of which contains two tyrosine residues

N-Try46/N-Tyr72/N-Tyr79:

The three double mutants of N-EoCen, each of which contains a single tyrosine residue

Tyr:

Tyrosine

TEMED:

N,N,N′,N′-Tetramethylethylenediamine

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Acknowledgments

This work was supported by the National Natural Science Foundation of PR China (No. 20901048 and No. 20771068), and the Natural Science Foundation of Shanxi Province (No. 2007011024). The Ph.D Programs Foundation of Ministry of Education of China (20091401110007).

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Authors and Affiliations

  1. Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Molecular Science, Shanxi University, Taiyuan, 030006, China

    Lian Duan, Wen Liu & Bin-Sheng Yang

  2. Institute of Biotechnology, Shanxi University, Taiyuan, 030006, China

    Ai-Hua Liang

  3. Chemical Department, Changzhi University, Changzhi, 046011, China

    Zhi-Jun Wang

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  1. Lian Duan
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  2. Wen Liu
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  3. Zhi-Jun Wang
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Correspondence to Bin-Sheng Yang.

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Duan, L., Liu, W., Wang, ZJ. et al. Critical role of tyrosine 79 in the fluorescence resonance energy transfer and terbium(III)-dependent self-assembly of ciliate Euplotes octocarinatus centrin. J Biol Inorg Chem 15, 995–1007 (2010). https://doi.org/10.1007/s00775-010-0660-z

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