Structure-Based Design of Circularly Permutated and Frame-Shifted Photoactive Yellow Proteins

Abstract

Photoswitchable proteins are powerful tools for external manipulation and probing of complex biochemical processes. In an effort to create new families of photoswitchable proteins that undergo novel types of conformational changes, we designed a circularly permutated PYP (c-PYP) and frame-shifted PYP (fs-c-PYP) variants of photoactive yellow protein (wt-PYP), a relatively small blue-light sensitive protein from Halorhodospira halophila that contains a photoactive 4-hydroxycinnamic acid chromophore. The c-PYP was created by connecting the N- and C-terminal of wt-PYP with a defined linker polypeptides and introducing new N- and C-termini at G115 and S114 respectively. A fs-c-PYP was created by duplication of a beta-sheet segment in c-PYP so that it can form an alternate H-bond registry with an existing beta-sheet. The designed proteins c-PYP and fs-c-PYP are highly soluble and well folded when overexpressed in E. coli, each undergoes PYP-like photocycle upon exposure to blue light. The thermal relaxation of the protein to the dark-adapted state after removal of the blue light source varies considerably between the designed constructs. UV-Vis absorption data indicate that fs-c-PYP recovers much more slowly than wt-PYP whereas c-PYP recovers more quickly than wt-PYP. Fluorescence-monitored GdnMHCl denaturation experiments show fs-c-PYP is the least stable and wt-PYP the most stable dark-state structure, while folding/unfolding of both c-PYP and fs-c-PYP is more cooperative than wt-PYP. This work shows that new classes of photo-controlled conformational change can be created by altering the backbone topology of known photoswitchable proteins.