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

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.


Preparation of Photoactive Yellow Protein (PYP)
Samples were purified using a previously published method [1,2]. Cysteine mutations were introduced in the reconstructed apo-PYP at the 7 th and 91 st amino acids positions in the pQE80 vector, which was over-expressed in Escherichia coli BL21 (DE3) by isopropyl β-D-1thiogalactopyranoside (IPTG) induction. After harvesting and sonication of the mutated PYP from E. coli, the resulting apo-PYP was incubated with a large amount of the linker molecule and Tris(2-carboxyethyl)phosphine (TCEP), and then reconstituted with p-coumaric anhydride and purified through Ni affinity and ion-exchange chromatography. We used enterokinase as a protease to remove the tag without any residual amino acids. The purified protein was further diluted in 20 mM Tris buffer (pH 7.0, 20 mM NaCl).
Sinapinic acid was used as a matrix material for measurement. The laser source for MALDI-TOF was the third harmonic of an Nd:YAG laser (355 nm), and the repetition rate was 1 Hz.

Circular Dichroism.
To confirm the secondary structure of the G7C-M91C mutant PYP and SML-PYP, circular dichroism (CD) spectra were measured using a CD spectrometer (Jasco-815, JASCO Inc., Japan) with a 2-mm quartz cuvette at room temperature. The spectral window ranged from 190 nm to 260 nm at 0.2-nm intervals. The baseline was measured with the same buffer in the same cuvette and subtracted.

Experiment-Restrained Rigid-Body molecular dynamics (MD) Simulation. This method was
almost identical to that used in a previous study [3,4]. Here, a crystal structure (2PHY) was used as a starting point. The structures were divided into a number of rigid bodies comprising several amino acids. The rigid bodies were allowed to move under the influence of a chemical and χ 2 force field. Because the atomic structure within a rigid body is constrained to that of the crystal or nuclear magnetic resonance (NMR) structure, the force field within the rigid body is not considered; however, Van der Waals interactions between rigid bodies and N-C bondlength corrections between rigid bodies are included in the chemical force field. The χ 2 force field is introduced to drive the molecular structure generated by MD simulations toward a structure that yields a different scattering curve that matches the experimental difference scattering curve. Thus, the total potential (U) on the rigid bodies has the Van der Waals term ULJ, the χ 2 term, and the bond correction term Bij as follows: where c1, c2, and c3 are weighting parameters used to scale the magnitude of the three terms. ULJ and χ 2 , which define the agreement between the experimental data and theoretical values, are calculated as follows; scaling factor cs and reduced units are used in this calculation.
The force field is the gradient of the total potential; therefore, the total force acting on the i-th particle among the total number of particles N is as follows: In the L-J potential, σ is defined as the r value where the corresponding potential is zero and rij (= ri − rj) is the distance between particle i and particle j. If the distance between the two particles is smaller than σ, repulsion increases steeply. We defined the following two types of σ values for the L-J potential between rigid bodies: for the N-C bond between two rigid bodies consisting of helices, σN-C = 1.28 Å, and for the atoms between two rigid bodies, σa-a = 1.
The velocity of ' i r relative to R is as follows: where ω is the angular velocity with respect to the COM of the rigid body. The moment of inertia with respect to the COM is as follows: The rotational equation of the motion of a rigid body around R can be expressed as 4 Eq. (5) updates R and, combining eqns.     Whereas wild-type PYP and mutated PYP show similar CD spectra, the CD spectrum of SML-PYP indicates that its a-helix content is reduced probably due to unfolded N-term region.

Figure S6. Guinier region of SAXS data for BM(PEG) 2 -incorporated PYP (SML-PYP) in the dark state.
PYP is a highly soluble protein and even after the incorporation of BM(PEG)2 the solubility of the protein was maintained. To avoid any distortion of X-ray scattering curve originated from the aggregated species, the solution was cautiously centrifuged in 10,000 g for 10 min prior to performing the SAXS measurements. The Guinier plot of the original SAXS data shows excellent linearity with an R-square value of 0.9999, confirming the absence of aggregation in the protein solution. Figure S7. UV-vis spectra of PYP with/without continuous LED illumination. The spectrum with continuous LED illumination barely shows the peak corresponding to pG, indicating that pG has been converted to pB under the used illumination condition. The fraction of pG is estimated to be less than 2.5%. When this spectrum was measured, the concentration of the solution was 10 µM and the volume was 2 mL. For the SAXS measurement, the concentration was 0.71 mM, but the volume (1 µL) was much smaller, thus, the total number of proteins in the SAXS measurement was even smaller than that in the UV-vis measurement.
In addition, the same LED source was used with tighter focusing for the SAXS measurement, and thus, the pG fraction under continuous LED illumination is expected be even much smaller than 2.5% in the SAXS measurement.