Probing protein structures in solution via concentration- and temperature-dependent small- and wide-angle x-ray scattering

Understanding how biomolecules execute their functions requires knowledge not only of their static ground-state structures but also of the conformational dynamics, solvation, and intermolecular interactions that they sample under physiological conditions. High-resolution crystallography, the dominant source of atomic-resolution structures, captures the molecule within a rigid lattice that can favor particular conformations and is largely silent on the structural dynamics present in solution. Here, we develop temperature-dependent small- and wide-angle x-ray scattering as a probe of biomolecular structure, dynamics, and interactions in the solution phase and apply it to two well-characterized model proteins: the Villin headpiece subdomain and the GB3 domain. Concentration-dependent measurements over temperatures spanning supercooled (−16 °C) to fully unfolded (120 °C), extrapolated to infinite dilution, allow us to recover the forward-scattering intensity I0(T), the radius of gyration Rg(T), and the real-space pair-distribution function p(r,T). Both Villin and GB3 exhibit at least two structurally distinct folded conformations and, when unfolded at high concentration (∼20 mg/ml), form transient dimers due to entanglement of the disordered peptide chains. Villin exhibits concentration-dependent dimerization of its folded structure and an anomalous loss of scattering power upon unfolding that we hypothesize arises from release of bound chlorides. Subtracting the fully disordered, high-temperature ensemble represented in p(r,Tmax) from all other curves generates Δp(r,T) with sufficient spatial resolution to track the temperature dependence of secondary and tertiary structural features. These real-space fingerprints of the folds place stringent constraints on candidate atomic structures and may provide a solution-phase route to atomic-level understanding of biomolecular structure and dynamics under the physiological conditions in which they function.