CONFORMATIONS OF LIGHT-SENSITIVE PROTEINS

Illinois Institute Of Technology, Chicago IL

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Natural protein photosensors are promising tools for engineering optical control into biological systems. We are just beginning to understand how these genetically encoded light detectors can be easily coupled to arbitrary effectors. We have reported a designed LOV2 photoswitch that selectively binds DNA when illuminated. We demonstrated that signal transduction between the two domains is mediated by the local unfolding of a shared, linking helix. SAXS measurements on the protein in the light and dark yield unexpected results. The overall shape barely changes even though the shared helix unfolds in the light. Modeling mandates that the domains associate due to weak interactions on the hydrophobic face in both the light and dark states. Manuscript is in preparation. Although most folding intermediates escape detection, their characterization is crucial to the elucidation of folding mechanisms. Here we outline a powerful strategy to populate partially unfolded intermediates: A buried aliphatic residue is substituted with a charged residue (e.g., Leu[unreadable]Glu-) to destabilize and unfold a specific region of the protein. We apply this strategy to Ubiquitin (Ub), reversibly trapping a folding intermediate in which the [unreadable]5 strand is unfolded (N-[unreadable]5). The intermediate refolds to a native-like structure upon charge neutralization under mildly acidic conditions. We compare the global dimensions of wild-type Ub to the intermediate using SAXS. At pH 7.8, the measured radius of gyration (Rg) is 13.2 [unreadable] 0.2 and 13.8 [unreadable] 0.1 [unreadable] for the two proteins, respectively. This difference matches the 0.5 [unreadable] difference between Rg's of the native state and the model for the N-[unreadable]5 intermediate. This general strategy may be combined with other methods and have broad applications in the study of protein folding and other reactions that require trapping of high-energy states.