Protein Conformations Explored by Difference High-Angle Solution X-ray Scattering:  Oxidation State and Temperature Dependent Changes in Cytochrome C

Abstract

We demonstrate the use of high-angle X-ray scattering to explore protein conformational states in solution by resolving oxidation state- and temperature-dependent changes in the conformation of horse heart cytochrome c. Several detailed models exist for oxidation-dependent changes in mitochondrial class I c cytochromes determined by X-ray crystallography and solution NMR techniques. These models differ in the magnitude and locations of structural change. Our scattering measurements show that high-angle X-ray scattering can discriminate between these models, and that the experimental scattering data for horse cytochrome c can be best reconciled with selected NMR models for the same protein. These results demonstrate the ability to use high-angle X-ray scattering to resolve conformational states of proteins in solution, and to relate these measurements to detailed structural models. Furthermore, temperature-dependent changes are found in the high angle scattering patterns for horse cytochrome c, illustrating the sensitivity of these measurements to dynamic aspects of protein structure. These results demonstrate the ability to use difference high angle scattering as a quantitative monitor of reaction-linked changes in protein conformation and structural dynamics. Synchrotron-based high-angle scattering holds promise as a widely applicable, high throughput technique for exploring conformational states linked to physiological protein function, for resolving configurational differences between protein structures in solution and crystalline states, and for bridging the gap between solution NMR and crystallographic structure techniques.

Accurate small and wide angle x-ray scattering profiles from atomic models of proteins and nucleic acids

“…(12), sometimes improves comparisons between predicted and experimental scattering profiles. 13,54 As pointed out by Moore, 55 the B-factor is only a rough guide to thermal disorder in solution, at least because B-factors are usually obtained from crystallography and are not necessarily comparable to solution scattering. In addition, correlated thermal motions (not modeled by Debye-Waller factors) contribute to scattering in solution.…”

X-ray diffraction “fingerprinting” of DNA structure in solution for quantitative evaluation of molecular dynamics simulation

“…Recently, a number of reports have shown that wide-angle x-ray scattering provides a direct measure of macromolecular conformation in solution (9)(10)(11)(12)(13)(14)(15). Wide-angle x-ray scattering measures rotationally averaged molecular diffraction.…”

“…Solution x-ray diffraction (SXD) patterns provide one-dimensional (1D) ''fingerprints'' of 3D structure that are directly relatable to atomic configuration by Fourier transform (14)(15)(16)(17). In previous work, we demonstrated that SXD patterns have sufficient resolution to characterize protein and DNA conformations in solution and to discriminate between close but distinguishable crystallographic and NMR models (11,15). Observed broadening of the SXD features compared with those calculated from static structures further indicated that experimental SXD patterns include information on configurational landscapes in solution that can be parameterized in terms of a mean configuration and breadth of the dispersion (14,15).…”

Exploring Fine Structures of Photoactive Yellow Protein in Solution Using Wide-Angle X-ray Scattering

“…The method of calculating scattering patterns using atomic coordinates was first developed by Svergun et al, 9 and was extended to produce accurate calculation of scattering patterns in high angle region using numerical precision. 14,15 In this approach, protein scattering is calculated from average of partial scattering amplitudes from the fixed atomic coordinated in the molecules, A a , and the solvent excluded volume, A s…”

“…e-mail: Hyotcherl.Ihee @kaist.ac.kr tron sources are sensitive to protein conformation states, 14 but also that the measured scattering patterns can be compared quantitatively with calculated patterns from detailed structural models. 14,15 If the time-resolved X-ray wide angle scattering is realized in solution, it will have many advantages over the timeresolved X-ray crystallography. First, it does not require highly-diffracting crystals and this will widen its applicability to a variety of systems.…”