To properly study protein structure requires both (1) a means to induce changes in the conformational state of proteins, i.e., folding or association, and (2) a means to study the conformation of these intermediately-folded or aggregated structures, preferentially at high resolution. To achieve point #2, work in our lab has revealed that small-angle neutron scattering (SANS) is a powerful technique to study the conformation of partially-folded structures, as well as protein n-mers. Unlike X-ray crystallography, SANS is a solution technique and can provide a more accurate representation of the state of the protein in vivo (e.g., deviations from the native state or self-association equilibrium, etc.) free from crystal packing constraints. Furthermore the often restrictive need to crystallize the protein is removed (particularly challenging for partially-folded proteins with exposed hydrophobic domains and membrane proteins that inherently contain large hydrophobic moieties).

To achieve point #1, we have devolved a novel method to control protein conformation with simple light illumination through the interaction of proteins with photoresponsive surfactants. Photoresponsive surfactants can in essence be switched “on” or “off” with exposure to visible or UV light, respectively, through the photoisomerization of an azobenzene group in the surfactant tail. Hence, surfactant binding to the protein and, thus, protein conformation can be tuned with light.

Through this combined strategy of inducing protein conformational changes with light illumination and studying the high-resolution structure of the resulting conformations with SANS, we have been able to study a variety of protein structural events. For example, we recently discovered a new folding intermediate in lysozyme, where SANS data combined with secondary structural information from FT-IR revealed that unfolding occurs primarily in the α domain of lysozyme, while the β domain remains relatively intact. Thus, this “surfactant-unfolded intermediate” of lysozyme was found to be a distinct structure from the well-known α-domain intermediate, which contains a folded α domain and unfolded β domain. In addition, since the swelling of the α domain was found to leave the active site of the enzyme intact (which could be seen from the SANS-determined structures), the light-initiated transition to the surfactant-unfolded intermediate results in an increase in enzyme flexibility and leads to enzyme “superactivy”.

Our combined two-prong approach has also revealed that, in addition to protein folding, light illumination can be used to induce changes in protein self-association. Results with α-chymotrypsin demonstrate that under visible light the protein exists as the monomer with the active site exposed (surfactant switched “on”, thus, protein-protein interactions are replaced with protein-surfactant interactions), while under UV-light the hexamer form is present, thereby preventing access to the active site. Thus, this novel ability to control enzyme form with light (i.e., quaternary structure) can in turn be used to photo-control enzyme function (i.e., activity). Furthermore, SANS data are used to probe for the first time the high-resolution structure of α-chymotrypsin hexamers in solution. It is found that the protein self-associates through the formation of cork-screw type structures. Due to crystal packing constraints, these structures are undetectable with X-ray crystallography.