Structural analysis of an equilibrium folding intermediate in the apoflavodoxin native ensemble by small-angle X-ray scattering.

Intermediate conformations are crucial to our understanding of how proteins fold into their native structures and become functional. Conventional spectroscopic measurements of thermal denaturation transitions allow the detection of equilibrium intermediates but often provide little structural detail; thus, application of more informative techniques is required. Here we used small-angle X-ray scattering (SAXS) to study the thermal denaturation of four variants of Anabaena PCC 7119 flavodoxin, including the wild-type apo and holo forms, and two mutants, E20K/E72K and F98N. Denaturation was monitored from changes in SAXS descriptors. Although the starting and final points of the denaturation were similar for the flavodoxin variants tested, substantial differences in the unfolding pathway were apparent between them. In agreement with calorimetric data, analysis of the SAXS data sets indicated a three-state unfolding equilibrium for wild-type apoflavodoxin, a two-state equilibrium for the F98N mutant, and increased thermostability of the E20K/E72K mutant and holoflavodoxin. Although the apoflavodoxin intermediate consistently appeared mixed with significant amounts of either native or unfolded conformations, its SAXS profile was derived from the deconvolution of the temperature-dependent SAXS data set. The apoflavodoxin thermal intermediate was structurally close to the native state but less compact, thereby indicating incipient unfolding. The residues that foster denaturation were explored by an ensemble of equilibrium ϕ-value restrained molecular dynamics. These simulations pointed to residues located in the cofactor and partner-protein recognition regions as the initial sites of denaturation and suggest a conformational adaptation as the mechanism of action in apoflavodoxin.