Abstract
Proteins have the potential to serve as nanomachines with well-controlled structural movements, and artificial control of their conformational changes is highly desirable for successful applications exploiting their dynamic structural characteristics. Here, we demonstrate an experimental approach for regulating the degree of conformational change in proteins by incorporating a small-molecule linker into a well-known photosensitive protein, photoactive yellow protein (PYP), which is sensitized by blue light and undergoes a photo-induced N-terminal protrusion coupled with chromophore-isomerization-triggered conformational changes. Specifically, we introduced thiol groups into specific sites of PYP through site-directed mutagenesis and then covalently conjugated a small-molecule linker into these sites, with the expectation that the linker is likely to constrain the structural changes associated with the attached positions. To investigate the structural dynamics of PYP incorporated with the small-molecule linker (SML-PYP), we employed the combination of small-angle X-ray scattering (SAXS), transient absorption (TA) spectroscopy and experiment-restrained rigid-body molecular dynamics (MD) simulation. Our results show that SML-PYP exhibits much reduced structural changes during photo-induced signaling as compared to wild-type PYP. This demonstrates that incorporating an external molecular linker can limit photo-induced structural dynamics of the protein and may be used as a strategy for fine control of protein structural dynamics in nanomachines.
Highlights
Various nanomachines based on small-molecule moieties have been designed and synthesized to control structural changes and mimic numerous mechanical movements at atomic-length scales [1,2]
We chose a protein known to undergo a large conformational change and attached a non-photoactive small-molecule linker to the protein to limit the extensive structural change
The small-angle X-ray scattering (SAXS) data show an increase of Radius of gyration (Rg) value in the ground state of SML-photoactive yellow protein (PYP) by 0.9 Å compared with that of wild-type PYP, which corresponds to an approximate volume increase of 5500 Å3
Summary
Various nanomachines based on small-molecule moieties have been designed and synthesized to control structural changes and mimic numerous mechanical movements at atomic-length scales [1,2]. Controlling the structural changes of proteins and manipulating motion via structural regulation of proteins in molecular nanomachines are challenging, since proteins are complicated macromolecules with many levels of structural organization from the primary structure (a sequence of amino acid residues) to the quaternary structure (interaction of protein subunits) In this regard, one of the important challenges in developing protein-based nanomachines is to achieve fine control of the mechanical motions within proteins. In the case of a protein nanocage [4], an ATP-driven chaperonin was turned into a light-driven cage by binding the azobenzene-based linkers These studies have focused on inducing structural changes of proteins using photo-switchable small molecules. We investigated the possibility and the mechanism of regulating the degree of conformational change by mutations and intramolecular conjugations using a linker molecule
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