| Abstrakt: |
To evaluate the role of zinc in Escherichia colialanyl-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 (s20,w) was 6.3 S and was virtually independent of temperature between 10 and 37°C, similar to the results reported for the native form. However, the s20,wnow decreased significantly as the concentration increased, indicative of a possible change in conformation. The s20,wvalue 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 s20,wof 3.6 S was obtained and with 6.6 M added urea, a peak with a s20,wof 2.7 S was seen. Added Gd-HCl (6 M) gave a single peak with s20,wof 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°C. At 25°C, a diffusion coefficient (D20,w) of 3.3 × 10−7cm2s−1was obtained. The combination of s020,wand D20,wyielded 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°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. |
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