Thursday, November 12, 2015: 9:30 AM
Canyon A (Hilton Salt Lake City Center)
From a specific combination of amino acids, each with various degrees of affinity or aversion for water, proteins (including most enzymes) will adopt a native state conformation giving rise to a functional protein. However, enzymes are not static entities, as suggested by the textbook lock-and-key mechanism, and instead regularly undergo conformational changes to intermediately-folded states during the course of activity. In fact, for many enzymes the bond formation steps are sufficiently fast so as to render these conformational changes rate limiting. Various hypotheses have been proposed with this in mind, all with the underlying theme that significant conformational flexibility is required during the course of the reaction in order to surpass the activation-energy barrier. Notably this flexibility is not necessarily proximal to the active site. This has been demonstrated through various mutation studies where replacement of amino acids far removed from the active site has led to increases in protein flexibility and reaction rates. These results seem to suggest a general procedure by which enzymatic activity could be increased through enhancements in enzyme flexibility, provided that these enhancements are not achieved at the expense of denaturing the active site. We have developed a novel method that allows protein conformation to be locally and reversibly controlled with simple light illumination. This method relies on the interaction of proteins with photoresponsive surfactants that can be switched from a photo-active to a photo-passive state with exposure to visible or UV light, respectively. In the active form, the surfactant binds to and unfolds proteins, similar to (although in a more specific manner than) traditional surfactants such as sodium dodecyl sulfate. Herein we demonstrate the use this phenomenon as a means to control enzyme activity with simple light illumination. Examples include (1) photoreversible partial and localized unfolding to enhance enzyme flexibility and give rise to activity greater than the native state (i.e., “superactivity”), (2) photoreversible denaturation and hence deactivation of enzymes (i.e., a light-triggered on-off switch for the enzymatic reaction), and (3) photoreversible dissociation of enzyme oligomers or enzyme-inhibitor complexes to provide access to the active site (i.e., enzyme reactivation). To thoroughly understand enzyme function during these processes we use small-angle neutron scattering to study the in vitro structure and hence regions of localized unfolding of non-native enzyme conformations that form in response to photosurfactant and light. Neutron spin echo measurements and molecular dynamics simulations further allow examination of the dynamics of conformational flexibility in partially-folded versus native conformations. Of specific interest is superactivity observed for β-glucosidase, and important enzyme shown to often be rate limiting during biofuel (cellulose) conversion to ethanol, which could lead to a sustainable replacement for gasoline.