Sood S M, Wu M X, Hill K A, Slattery C W
Department of Biochemistry, Loma Linda University School of Medicine, Loma Linda, California, 92350, USA.
Arch Biochem Biophys. 1999 Aug 15;368(2):380-4. doi: 10.1006/abbi.1999.1314.
To evaluate the role of zinc in Escherichia coli alanyl-tRNA synthetase, hydrodynamic measurements and circular dichroism spectra were obtained for the zinc-depleted protein and compared with those of the native enzyme. At a protein concentration of 5 mg ml(-1), pH 7.5, the sedimentation coefficient (s(20,w)) was 6.3 S and was virtually independent of temperature between 10 and 37 degrees C, similar to the results reported for the native form. However, the s(20,w) now decreased significantly as the concentration increased, indicative of a possible change in conformation. The s(20,w) value did not appear to change as the pH was increased to 9.5. In standard buffer with 3.3 M added urea, a single peak with a s(20,w) of 3.6 S was obtained and with 6.6 M added urea, a peak with a s(20,w) of 2.7 S was seen. Added Gd-HCl (6 M) gave a single peak with s(20,w) of 2. 0 S. Like the native form, laser light scattering studies indicated some heterogeneity and a radius of 6.4 nm which was virtually independent of concentration and temperature in the range of 10-37 degrees C. At 25 degrees C, a diffusion coefficient (D(20,w)) of 3.3 x 10(-7) cm(2) s(-1) was obtained. The combination of s(0)(20,w) and D(20,w) yielded a molecular mass of approximately 179 kDa, which is slightly less than that reported for the native dimeric form (186 kDa). The intrinsic viscosity at 25 degrees C was extrapolated to 5. 3 ml g(-1), a value significantly higher than that reported for the native form, which increased with temperature. These results indicate some conformational and flexibility changes from the native to the zinc-depleted form, which may explain differences in activity. Furthermore, urea denaturation experiments demonstrate the role of zinc in stabilization of AlaRS structure.
为了评估锌在大肠杆菌丙氨酰 - tRNA合成酶中的作用，我们对去除锌的蛋白质进行了流体动力学测量和圆二色光谱分析，并与天然酶的测量结果进行了比较。在蛋白质浓度为5 mg/ml、pH值为7.5的条件下，沉降系数（s(20,w)）为6.3 S，并且在10至37摄氏度之间几乎与温度无关，这与天然形式的报道结果相似。然而，随着浓度的增加，s(20,w)现在显著下降，这表明构象可能发生了变化。当pH值增加到9.5时，s(20,w)值似乎没有变化。在添加了3.3 M尿素的标准缓冲液中，获得了一个s(20,w)为3.6 S的单峰，在添加了6.6 M尿素时，观察到一个s(20,w)为2.7 S的峰。添加6 M的Gd - HCl得到了一个s(20,w)为2.0 S的单峰。与天然形式一样，激光光散射研究表明存在一些异质性，并且半径为6.4 nm，在10至37摄氏度范围内几乎与浓度和温度无关。在25摄氏度时，获得的扩散系数（D(20,w)）为3.3×10^(-7) cm²/s。s(0)(20,w)和D(20,w)的组合得出分子量约为179 kDa，略低于报道的天然二聚体形式（186 kDa）。25摄氏度时的特性粘度外推至5.3 ml/g，该值明显高于天然形式的报道值，且随温度升高而增加。这些结果表明从天然形式到去除锌的形式存在一些构象和柔韧性变化，这可能解释了活性上的差异。此外，尿素变性实验证明了锌在稳定丙氨酰 - tRNA合成酶（AlaRS）结构中的作用。
