Application of the small-angle x-ray scattering technique for the study of two-step equilibrium enzyme-substrate interactions

Abstract

The small‐angle x‐ray scattering (SAXS) technique is used for the investigation of two‐stage equilibrium macromolecular interactions of the enzyme‐substrate type in solution. Experimental procedures and methods of analyzing 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 double‐stranded oligonucleotide with Eco dam methyltransferase (MTase) have been determined and demonstrate a high cooperativity of MTase binding when the ternary complex containing the dimeric enzyme is formed. The structural parameters (Rg, Rc, semiaxes) have been determined for free enzyme and polynucleotides and of enzyme‐substrate complexes, indicating structural rearrangements of the enzyme in the interaction with substrates. © 1996 John Wiley & Sons, Inc.

Tetrahydrocortisol–apolipoprotein A-I complex specifically interacts with eukaryotic DNA and GCC elements of genes

“…Previously, the SAXS method was successfully used in our study on interaction of specific tRNA with tRNAsynthetases [18], of oligonucleotides with methyltransferases [19], in evaluating the structural changes in HDL [20], and revealing the fractional composition of lipoproteins in plasma and blood serum [21]. SAXS was proven useful in studying the mechanisms of molecular interaction, in particular, allowing one to analyze stoichiometry and equilibrium constants of macromolecular complexes, structural and weight properties of macromolecules.…”

“…From estimates obtained in our SAXS experiments (Table 4) and from the results of studies reported in [18,19] one can assume that in these interactions the equilibrium is shifted essentially to the formation of complexes, i.e., it is highly cooperative. Then, upon formation of PnS complexes in the mixtures, parameter n in scheme (2) will grow up to some maximum value with increasing protein concentration (C p ) in the mixtures.…”

“…In the absence of THC small interaction of DNA with apoA-I and RNA-PM was observed. The size of PnS complexes (here P stands for RNA-PM; S denotes DNA aggregates with THC-apoA-I; n is the number of P molecules in the complex) within the Table 2 Structural and mass parameters of native DNA from rat liver estimated using SAXS accuracy of measurements corresponded to the size of DNA molecules in a free state (R g = 39.2±0.6 nm), while reliably changed only the values of I(0) related to concentration and molecular mass of PnS complexes [18,19]:…”

“…ApoA-I molecules were shown to possess a slightly oblong shape with the ellipsoid axes ratio b/a = 3.6, radius of gyration R g = 2.35 nm and molecular mass M = 27.4 kDa, which is close to the values we obtained earlier [7]. It is known that macromolecules of short single-chain oligonucleotides in a solution can have a common shape of the particles like ellipsoid of revolution or cylinder [19]. Thus, the values of radius of gyration (R g = 1.2 ± 0.06 nm) and volume (V = 6.308 nm 3 ) we obtained for oligonucleotide 1 correspond to the structure of cylinder with 0.727 nm radius and 3.8 nm height.…”

“…To estimate the maximum stoichiometry of the complex formed as a result of interaction between oligonucleotide 1 (further oligonucleotide) and apoA-I we used the SAXS experimental data from the first four mixtures, the highly cooperative equilibrium scheme, and the methodology we developed earlier [18,19]:…”