X-ray diffuse scattering by proteins in solution. Consideration of solvent influence

Abstract

Thyroglobulin is a protein involved in the synthesis, storage and secretion of thyroid hormones, reported here are some small-angle X-I ay measurements primarily concet ned with determination of its shape parameters in solution. The protein was fractionated on Sephadex G-200, and the portions corresponding to 'native' protein were collected and re-chromatographed on Sepharose 6-B; the protein samples were examined in phosphate buffer at pH 6"9. A radius of gyration of 64 A, a particle volume of ~ 1"7 x 106 ,~3, and an anisotropy factor of 1.12 were found. Assuming a (reasonable) prolate ellipsoidal shape, one obtains an axial ratio of 2: 1, in good agreement with recent electron micrographic results, and implying a high degree of solvation. Some details and problems connected with the SAXS measurements, in particular of the molecular weight, will be discussed.

Calculation of Small-Angle Scattering Patterns

“…The determination of ϕ k q ! , the scattering amplitude of the homogeneous substance filling the macromolecule and its excluded volume, is not trivial, and several authors proposed solutions for it [21,32,33,35,36].…”

“…Fedorov et al [32][33][34] and Ninio et al [35] proposed the so-called cube method, where the models of macromolecules in solution are surrounded by the solvent (or by the matrix where the particles are immersed), and the cube method permits a correct calculation of the volume inaccessible to the solvent. The theoretical intensity is given by…”

“…is the scattering amplitude of the macromolecule in vacuum and ϕ q ! has the same volume of the molecule but with homogeneous electron density r 0 [32,33]. The calculation of the scattering amplitude of a protein macromolecule with known atomic coordinates can be done with the equation,…”

“…direction. In order to perform random orientation over direction Z, one can take N directions, in reciprocal space, on an sphere of radius q, so the average scattering intensity is given by [32,33] Iq ðÞ¼ 1…”

Experimental and theoretical large‐angle X‐ray diffuse scattering by globins in solution. Sensitivity of the method

“…The collimation correction for the slit height was taken into account only for the internal part of the scattering curves. Calculation of scattering indicatrices was based on a combination of methods described earlier [8], the 'cube method', and the 'sphere method'. Similarly to the 'sphere method', the solvent effect was taken into account by introducing effective atomic factors ~i6u)…”