Regulation of Protein Structural Changes by Incorporation of a Small-Molecule Linker

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.

Reversible molecular motional switch based on circular photoactive protein oligomers exhibits unexpected photo-induced contraction

“… 36 After constructing the SASs, the candidate structures for the EOM analysis were generated by running two sets of MD simulations starting from the two different initial structures obtained from the experiment-restrained rigid-body (ERRB) MD simulation. 24 , 45 , 59 , 66 , 67 , 89 The theoretical X-ray scattering curves were calculated from the candidate structures. Then, the theoretical X-ray scattering curves were compared with the experimental scattering curves of the ground state, first intermediate, second intermediate, and photoproduct using the EOM method to extract the structural parameter and the optimal ensemble structures for each state.…”

“…In addition, applying such molecular switches to the regulation of biological processes requires a thorough understanding of their structural mechanisms. For the functionalization of the molecular switches, various approaches have been developed based on the structural and functional properties of proteins, 19 - 24 as well as protein-protein, 25 , 26 protein-DNA, 27 , 28 and protein-RNA 28 - 30 interactions. These approaches have successfully engineered monomeric proteins to function as molecular switches.…”

Length and Charge of the N-terminus Regulate the Lifetime of the Signaling State of Photoactive Yellow Protein

“…To understand the role of the N-terminus in the photocycle of PYP, N-terminal truncated PYPs (NT-PYPs) have been investigated with various techniques. − ,,,, Studies using small-angle X-ray scattering (SAXS), X-ray crystallography, and nuclear magnetic resonance (NMR) demonstrated that the ground state of NT-PYPs has a similar globular structure to that of wild-type PYP (wt-PYP). In transient absorption (TA) spectroscopy studies, NT-PYPs showed significantly slower dark recovery time from pB 2 to pG than wt-PYP. , It was proposed that the deceleration of the dark recovery observed in NT-PYPs is due to the absence of the interaction between N-terminal amino acids and the β-scaffold surrounding the chromophore-binding domain .…”

“…1,2 To understand the role of the N-terminus in the photocycle of PYP, N-terminal truncated PYPs (NT-PYPs) have been investigated with various techniques. [3][4][5][6][7]24,25,44,45 Studies using small-angle X-ray scattering (SAXS), 24 X-ray crystallography, 25 and nuclear magnetic resonance (NMR) 6 demonstrated that the ground state of NT-PYPs has a similar globular structure to that of wild-type PYP (wt-PYP). In transient absorption (TA) spectroscopy studies, NT-PYPs showed significantly slower dark recovery time from pB 2 to pG than wt-PYP.…”