三氟拉嗪与八肋游仆虫中心蛋白N端半分子的结合及对蛋白功能的影响

doi:10.7503/cjcu20190334

摘要/Abstract

摘要：

采用荧光光谱、圆二色光谱(CD)、等温滴定量热分析(ITC)、电泳及分子对接等分析技术, 研究了三氟拉嗪(TFP)与八肋游仆虫中心蛋白N端半分子(apoN-EoCen)的结合, 考察了TFP对apoN-EoCen性质的影响. 结果表明, 在室温下10 mmol/L Hepes缓冲溶液(pH=7.4)中, TFP与apoN-EoCen以摩尔比1∶1结合于apoN-EoCen的第二个EF-手的E, F螺旋之间, 条件结合常数约为10 ³ L/mol; TFP的结合导致蛋白质二级结构发生改变, α螺旋含量减小, Tb ³⁺敏化荧光强度降低83%, apoN-EoCen切割DNA的类核酸酶活性明显受到抑制; Tb ³⁺仍可占据复合物apoN-EoCen-TFP中蛋白质的2个金属离子结合位置, 条件结合常数约为7.0×10 ⁵ L/mol, TFP的结合不抑制金属离子诱导的蛋白质聚集.

关键词: apoN-EoCen, 三氟拉嗪, Tb ³⁺离子, 光谱分析

Abstract:

Fluorescence spectroscopy, circular dichroism(CD), isothermal titration calorimetry(ITC), electrophoresis, molecular docking and other modern analytical techniques were used to study the combination of the apoN-terminal domain of Euplotes octocarinatus centrin(apoN-EoCen) with Trifluoperazine(TFP) and observed the effect of TFP on the properties of apoN-EoCen. The results showed that TFP could bind to the E and F helix of the second EF hand of apoN-EoCen and could be combined with a molar ratio of 1∶1 stoichio-metry in 10 mmol/L Hepes buffer solution(pH=7.4) at room temperature. The conditional binding constant is about 10 ³ L/mol. The binding of TFP leads to the change of protein secondary structure and the decrease of α-helix content and the decrease of Tb ³⁺sensitized fluorescence by 83%, and the nuclease activity of apoN-EoCen cleaved DNA is obviously inhibited. Tb ³⁺ can still occupy the two metal ion binding sites of the protein in the apoN-EoCen-TFP complex, and the conditional binding constant is about 7.0×10 ⁵ L/mol. The bonding of TFP does not inhibit protein aggregation caused by Tb ³⁺ ions.

Key words: apoN-EoCen, Trifluoperazine, Tb ³⁺, Spectral analysis

中图分类号:

O611
Q501

TrendMD:

叶旭文,张文龙,王志军,赵亚琴,杨斌盛. 三氟拉嗪与八肋游仆虫中心蛋白N端半分子的结合及对蛋白功能的影响. 高等学校化学学报, 2019, 40(11): 2257.

YE Xuwen,ZHANG Wenlong,WANG Zhijun,ZHAO Yaqin,YANG Binsheng. Binding of Trifluoperazine to N-terminal Domain of Euplotes Octocarinatus Centrin and the Influence on Its Function ^†. Chem. J. Chinese Universities, 2019, 40(11): 2257.

图/表 10

Fig.1 Structure of trifluoperazine

Fig.2 Fluorescence spectra produced by the addition of TFP(0.4 mmol/L) to apoN-EoCen(1.0×10-5 mol/L, 1 mL)(A) and titration curve of fluorescent intensity at 305 nm for apoN-EoCen against TFP/apoN-EoCen molar ratio in 10 mmol/L Hepes buffer(pH=7.4) solution(B) (A) Total volumes of TFP from spectrum a to r are 0, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 75, 85, 95, 105 μL, respectively; (B) inset: the fitting curve of lg[(F0-Fi)/(Fi-F∞)] vs. lg[TFP]f.

Fig.3 Calorimetric titration curve of TFP to apoN-EoCen at 25 ℃ The solid lines represent the best fit for one set of sites model.

Table 1 Summary of thermodynamic parameters of TFP and apoN-EoCen binding by ITC and fluorescence spectroscopy at 25 ℃

Method	10^-3K/(L·mol^-1)	n	ΔS/(J·mol^-1)	ΔH/(kJ·mol^-1)	ΔG/(kJ·mol^-1)
ITC	7.60±0.66	1	33.9	-(12.26±0.49)	-22.54
Fluorescence	3.31±0.07	0.7

Fig.4 Far-UV CD spectra of apo-N-EoCen(a) and apo-N-EoCen -TFP(b) in 10 mmol/L Hepes buffer solution(pH=7.4)

Fig.5 Overlap between fluorescence spectrum of apoN-EoCen(a) and absorption spectrum of TFP(b) The concentrations of TFP and apoN-EoCen both are 2.0×10-5 mol/L.

Fig.6 Caroon ribbon model structure of apoN-EoCen-TFP Black dotted line respents the distance between Tyr72(yellow) and TFP(red).

Fig.7 Resonance light scattering spectra of apoN-EoCen with the addition of TFP(A) and apoN-EoCen-TFP with the addition of Tb3+(B) in 10 mmol/L Hepes buffer solution(pH=7.4) Inset: titration curve of intensity at 287 nm against the concentration ratio of Tb3+ to apoN-EoCen-TFP. From spectum a to g the concentration ratios of TFP to apoN-EoCen or Tb3+ to apoN-EoCen-TFP are 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, respectively.

Fig.8 Tb3+ sensitized fluorescence spectra in the absence(A) and presence(C) of TFP, fluorescence intensity of Tb3+ at 545 nm as a function of Tb3+/apoN-EoCen ratio in the absence(a) and presence(b) of TFP(B), Far-UV CD spectra of apoN-EoCen, Tb2-N-EoCen and Tb2-N-EoCen-TFP(D) (A) Using 1 mmol/L Tb3+ solution to titrate the apoN-EoCen. Tb3+/N-EoCen ratio for spectrum a—g was 0, 0.25, 0.50, 1.00, 1.50, 2.00, 2.25, respectively. (B) The data of curve b are taken from the fluorescence intensity at 545 nm of spectra(C) and data of curve a are taken from spectra(A). (C) Using 1 mmol/L Tb3+ solution to titrate the apoN-EoCen-TFP. Tb3+/N-EoCen-TFP was 0, 0.25, 0.50, 0.75, 1.00, 1.25, 1.50, 1.75, 2.00, 2.25, respectively.

Fig.9 Gelatinous map of protein-cleaved pBR322 DNA in the presence of TFP Lane 1: pBR322DNA; lane 2: pBR322DNA+apoN-EoCen; lanes 3 and 4: on the basis of line 2, added 5, 10 times TFP. The concentrations of DNA, apoN-EoCen was 3.5×10-3 μg/μL and 14.8 μmol/L, respectively.

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