Ligand binding and protein dynamics: a fluorescence depolarization study of aspartate transcarbamylase from Escherichia coli.
Ligand binding and protein dynamics: a fluorescence depolarization study of aspartate transcarbamylase from Escherichia coli.
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DOI：
10.1021/bi00394a027
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发表时间：
1987-10
期刊：
影响因子：
2.9
通讯作者：
C. Royer;P. Tauc;G. Hervé;J. Brochon
中科院分区：
文献类型：
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作者：
C. Royer;P. Tauc;G. Hervé;J. Brochon
研究了大肠杆菌天冬氨酸转氨甲酰酶两个色氨酸残基的荧光偏振和实时荧光强度衰减随温度的变化。将蛋白质溶解在80%甘油/缓冲液混合物中，温度在-40至20 ℃之间变化，以将去极化限制在Dahans的局部旋转。两种荧光物质贡献了超过95%的发射。它们的荧光寿命相差约4 ns，这取决于观察到的温度和它们对总强度的贡献。偏振和寿命数据的Y-图分析允许通过它们的临界旋转幅度来区分两种旋转物质，第一种是分量1，第二种是分量2。我们认为，这两个物种对应的两个色氨酸残基的蛋白质。在双底物类似物N-（膦酰基乙酰基）-L-天冬氨酸（PALA）的存在下以及在核苷酸效应分子ATP和CTP的存在下对ATCase进行极化和寿命实验。PALA的结合导致色氨酸1的旋转摩擦阻力的热系数的增加和色氨酸2的旋转摩擦阻力的热系数的减少。ATP结合不影响蛋白质阻碍色氨酸旋转的程度，但确实导致色氨酸2的临界旋转幅度发生变化。在CTP存在下获得的结果与用PALA获得的结果相似。
The polarization of the fluorescence and the real-time fluorescence intensity decay of the two tryptophan residues of aspartate transcarbamylase from Escherichia coli were studied as a function of temperature. The protein was dissolved in an 80% glycerol/buffer mixture, and temperatures were varied between -40 and 20 degrees C in order to limit the depolarization to local rotations of the tryptophans. Two fluorescent species contribute to over 95% of the emission. They differ in their fluorescence lifetimes by approximately 4 ns depending upon the temperature observed and their fractional contributions to the total intensity. The Y-plot analysis of the polarization and lifetime data allows for the distinction of two rotational species by their critical amplitude of rotation, the first being component 1 and the second being component 2. We suggest that these two species correspond to the two tryptophan residues of the protein. The polarization and lifetime experiments were carried out for ATCase in presence of the bisubstrate analogue N-(phosphonoacetyl)-L-aspartate (PALA) and in presence of the nucleotide effector molecules ATP and CTP. The binding of PALA results in an increase in the thermal coefficient of frictional resistance to rotation of tryptophan 1 and a decrease in that of tryptophan 2. ATP binding does not affect the degree to which the protein hinders tryptophan rotation but does result in a change in the critical amplitude of rotation of tryptophan 2. The results obtained in the presence of CTP are similar to those obtained with PALA.