Application of the small‐angle X‐ray scattering technique for the study of equilibrium enzyme‐substrate interactions of phenylalanyl‐tRNA synthetase from E. coli with tRNA^Phe

Abstract

The small-angle X-ray scattering technique (SAXS) is proposed for the investigation of equilibrium macromolecular interactions of the enzyme-substrate type in solution. Experimental procedures and methods of analysing the data obtained from SAXS have been elaborated. The algorithm for the data analysis allows one to determine the stoichiometric, equilibrium and structural parameters of the enzyme-substrate complexes obtained. The thermodynamic characteristics for the formation of complexes of tRNAphe with phenylalanyl-tRNA synthetase have been determined and demonstrate negative cooperativity for binding of the two tRNAPhe molecules. The structural parameters (I$, &, semi-axes) have been determined for free phenylalanyl-tRNA synthetase and tRNAPhe from E. coli MRE-600 and of enzyme complexes possessing one and two tRNAPhe molecules, indicating structural rearrangements of the enzyme in the interaction with tRNAPhe.Small-angle X-ray scattering; Phenylalanyl-tRNA synthetase; Enzyme-substrate interaction; tRNAPhe

Effect of glucocorticoids and their complexes with apolipoprotein A‐I on the secondary structure of eukaryotic DNA

“…The M s values ranged from 20 to 40 ⅐ 10 6 Da, depending on the methods of DNA isolation and purification and the extent of its degradation. *M s ϭ 26 ⅐ 10 6 agrees well with the values obtained by other methods [20]. The difference between *M s and M s can be explained by the fact that the DNA molecule was approximated by a homogeneous sphere.…”

“…where N is the number of PnS complex particles involved in scattering, and M k is the molecular weight of the PnS complexes [20]. On the roentgenograms, the diffraction maximum at angles h Ϸ 0.266 Å Ϫ1 [20] corresponded to the diameter of the DNA double chain (ϳ24 Å) and confirmed the integrity of the DNA secondary structure.…”

“…It is obvious that the spherical approximation is incorrect in this case. As demonstrated in Figure 1(b), the intensity decay for X-ray scattering corresponds to h k ⅐ I(h) Ϸ const, where k ϭ 2 is characteristic of a sphere [20]. The extent of decay k ϭ 2 is likely between k ϭ 1 for the rod (a DNA chain with bends) and k ϭ 3 for simple Gaussian coils with a flexible chain.…”

“…The data for both apolipoprotein A-I and native DNA preparations (solutions) are presented in Figures 1(a) and 1(b), respectively, obtained by the SAXS method. Analysis of these roentgenograms showed that both apoA-I and DNA preparations were fairly monodisperse, as judged by the mean radius of gyration R g of the PANIN, KUNITSYN, AND TUSIKOV scattering particles calculated for both apoA-I and DNA from the slope of the linear regions on the Guinier graphs [20] (Tables I and II). The apoA-I molecule was shown to be slightly extended with the ellipsoid axis ratio b/a ϭ 3.6 ( Table I).…”

“…The M p value (as well as M s ; see below) was determined by measuring the absolute intensities of zero angle scattering I(0), direct beam, and the protein (or DNA) concentration [20]. The apoA-I molecular mass is known to be 28 kDa [21].…”