The spectral studies on the effect of Glu 101 to the metal binding characteristic of Euplotes octocarinatus centrin.
Glu is highly conserved as the first amino acid of E-helix of the EF-hand protein. In this paper, Glu 101, the first amino acid of E-helix of the third EF-hand motif in Euplotes octocarinatus centrin (EoCen) was mutated to be Lys by the method of site direct mutation. Tb3+ and TNS were used as fluorescence probes in the study of the effect of this mutation to the metal binding characteristic of EoCen by fluorescence spectra. Results indicate that compared with EoCen, the mutation protein (E101K) displays a different Tb3+ binding characteristic and an increased hydrophobic exposure surface. Polyacrylamide gels electrophoresis indicated that the electrophoretic mobilities of EoCen and E101K are distinctly different. It can be deduced that the conformation of EoCen has been altered by this mutation. The general conditional binding constant of Tb3+ to the three loops of EF-hand sites I-III in E101K was calculated to be (5.64+/-0.57)x10(5)M(-1) according to the modified equation of the single binding process.
Li
Guoting
G
Institute of Molecular Science, Chemical Biology and Molecular Engineering Laboratory of Education Ministry, Shanxi University, Taiyuan 030006, China.
Wang
Zhijun
Z
Zhao
Yaqin
Y
Ren
Liexiang
L
Liang
Aihua
A
Yang
Binsheng
B
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D016812:Euplotes; D018698:Glutamic Acid / Q000737:chemistry*; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D009282:Naphthalenesulfonates / Q000378:metabolism; D015800:Protozoan Proteins / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013725:Terbium / Q000378:metabolism*; D017186:Titrimetry
D009282:Naphthalenesulfonates; D015800:Protozoan Proteins; D013725:Terbium; D018698:Glutamic Acid; C009497:2-(4-toluidino)-6-naphthalenesulfonic acid; D008239:Lysine