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

SUMMARY Molecular switches alterable between two stable states by environmental stimuli, such as light and temperature, offer the potential for controlling biological functions. Here, we report a circular photoswitchable protein complex made of multiple protein molecules that can rapidly and reversibly switch with significant conformational changes. The structural and photochromic properties of photoactive yellow protein (PYP) are harnessed to construct circular oligomer PYPs (coPYPs) of desired sizes. Considering the light-induced N-terminal protrusion of monomer PYP, we expected coPYPs would expand upon irradiation, but time-resolved X-ray scattering data reveal that the late intermediate has a pronounced light-induced contraction motion. This work not only provides an approach to engineering a novel protein-based molecular switch based on circular oligomers of well-known protein units but also demonstrates the importance of characterizing the structural dynamics of designed molecular switches.

more than two PYP oligomers by making a cPYP and split intein simultaneously, enabling the production of coPYPs of various sizes, including those that include higherorder PYP units. Since the yield of coPYPs produced by the split intein method was too low, all subsequent experiments were performed using cPYP-produced samples.
In the cPYP-based method, the PYP oligomers were generated based on the cPYP gene in the PQE80L vector. A circular PYP oligomer was constructed by inserting a PYP gene with a linker sequentially into a plasmid expressing a single cPYP gene 1 ( Figure S1). The amino acid sequences of coPYPs are as follows. The dimer PYP was not circular but was in a leaner form.

MALDI/TOF, MALS, and DLS
We implemented several spectroscopic methods, matrix-assisted laser desorption ionization time of flight mass spectroscopy (MALDI/TOF), multiangle light scattering (MALS), and dynamic light scattering (DLS), to determine the molecular weight and size of each protein sample nm.

Transmission electron microscopy
The sample solution (5 μL) was loaded onto an EM-grid with a continuous carbon film, which was rendered hydrophilic by glow discharge. After 60 s of sample adsorption, the grid was washed three times with droplets of deionized water, followed by negative staining with 5 μL of 1% uranyl acetate solution. The excess staining solution was blotted using a piece of filter paper. Images were obtained using a TecnaiTM G2 Spirit TWIN (FEI Co., Hillsboro, OR, USA) transmission electron microscope operated at an acceleration voltage of 120 kV and were recorded using an Ultrascan 4000 CCD camera (Gatan Inc., Pleasanton, CA, USA).

Transient absorption (TA) spectroscopy
The method for TA spectroscopy was similar to those of the previous studies [2][3][4] . Briefly, an optical parametric oscillator (LT-2214-PC; LOTIS), which was pumped by the third harmonic wavelength of an Nd:YAG laser (NL 301G; EKSPLA), produced a 460 nm laser as a pump beam. As a probe source, a continuous wave 250 W Xe-lamp was used. The probe light and the pump laser beam were intersected at the sample position at an acute angle. The TA signal was detected using a combination of a spectrometer (SpectraPro 2300i; Princeton Instruments, Trenton, NJ, USA) and a PMT. The TA signal measured using the PMT was digitized using a digital oscilloscope (TDS 3052B; Tektronix). To improve the signal-to-noise ratio, the average signal from 320 estimates was obtained. The concentrations of the chromophores of PYPs were adjusted to approximately 70 μM, which was calculated using an extinction coefficient of 45.5 mM -1 cm -1 at 446 nm. To avoid photosaturation, the sample solution was flowed using a syringe pump and a flow cell (2 mm path length). The repetition rate of the pump beam was 1 Hz. To obtain measurements after a 1s time delay, the flow was stopped, and the repetition rate of the pump beam was adjusted to 0.5 Hz to reduce photosaturation as much as possible. The static x-ray scattering data were processed and analyzed using the ATSAS package (http://www.embl-hamburg.de/biosaxs/software.html) 5,6 . One-dimensional scattering data I(q) as a function of q were obtained by radial averaging. The scattering intensity from the buffer solution were measured for the background and used to eliminate the contribution of the buffer in the static scattering curve of the protein sample.

Time-resolved x-ray solution scattering
Time-resolved x-ray solution scattering (TRXSS) data were collected at the ID14B beamline at Advanced Photon Source (Argonne, IL, USA) following an established experimental protocol [7][8][9] . Before the experiment using coPYP-4, a control experiment was performed using monomer PYP to optimize the experimental conditions. The control experiment effectively

Reversibility of coPYP-4.
A Photoswitchable protein refers to a protein that can control the transition between two stable states, ground and final photoproduct, using light. In order to effectively apply such a photoswitchable protein to bio-engineering, its transition must be reversibly controlled depending on the presence or absence of light. In TRXSS data, the standard deviation is significantly smaller than the amplitude of signal intensity at each q point ( Figure S4). These low standard deviations indicate that the repeatability and reproducibility of the TRXSS experiment was maintained high even though the TRXSS data were obtained from more than 150 repeated pump-probe measurements at the same focal points of the sample capillary. Such high repeatability and reproducibility demonstrate that during the TRXSS experiment, coPYP-4 reversibly regulates the structural changes between the ground and final photoproduct, implying that the reversibility of coPYP-4 was maintained more than 150 cycles.

Elimination of solvent heating contribution from time-resolved difference scattering curves.
Time-resolved difference scattering curves involve both the contribution of the structural changes for the target protein and the contribution from the solvent heating induced by the energy transferred from the excited proteins into the solvent ( Figure S5). Therefore, the contribution of the solvent heating needs to be removed from the time-resolved difference scattering curves to extract only the contributions from the structural change of the protein. To this end, we reconstructed the contribution of solvent heating as a linear combination of temperature change at constant pressure and density change at a constant temperature using a well-established method 8,9 . Subsequently, the contribution of solvent heating is eliminated from the difference scattering curves, and only the contribution for the structural changes of the protein is extracted.

Kinetic analysis of coPYP-4 using SVD and PCA
The kinetics of structural changes in coPYP-4 were determined by applying singular value decomposition (SVD). During the SVD analysis, the data matrix of TRXSS, A, was  (Figures S6A and S6B). Figure S6D shows the significant three rSVs scaled with singular values. By globally fitting the rSVs by two exponential sharing where ∆Stheory(qi, tj) is the theoretical difference scattering curve at given q and t values, ∆SCk(qi ) with velocity-rescale thermostat (τT = 0.1 ps, T = 300 K) and Parrinello-Rahman barostat (τT = 0.5 ps, P = 1 bar). After equilibration, production simulation was conducted with the equilibrated system for 500 ns. The candidate structures were sampled at 10 ps intervals for (ii). And the theoretical x-ray scattering curves of the candidate structures were calculated using CRYSOL 6 . The theoretical scattering curves were calculated using the values for the electron density of the solvent = 0.334 e/Å 3 , the contrast of the hydration shell = 0.03 e/Å 3 , and the average atomic radius = 1.611 Å .
Ensemble optimization method. For the candidate structures from the MD simulations, we implemented EOM analysis using a well-established method 9,11 . Each candidate structure from the MD simulations is considered the potential conformations to be included in the ensembles for the ground and the light states of coPYP-4. The theoretical x-ray scattering curves of the candidate structures were calculated using CRYSOL 6 . Assuming that all conformers exist with the same probability, the experimental data can be expressed by an ensemble containing M different conformers. The average curves for target protein can be obtained from the structures included in the ensemble and compared with the experimental x-ray scattering data: where Savr(q) is the averaged scattering curve for an ensemble, Sn(q) is the theoretical X-ray scattering curve for the n-th conformer, and M is the total number of conformers in an ensemble.
In the general algorithm, 50 ensembles (chromosomes) containing 20 different structures randomly selected from the pool of candidate structures were first generated. For these 50 chromosomes, mutation and cross operations were performed in each generation. During the mutation, 50% of the components of each chromosome were exchanged for unselected structures of the pool or structures present in other chromosomes of the same generation.
During the crossing, 40% of the components of two randomly selected chromosomes of the same generation were exchanged while maintaining the number of components. After mutation and crossing operation, the number of ensembles in a generation tripled from 50 to 150. The averaged theoretical scattering curve, Savr(q), for each chromosome was compared with the experimental scattering curve to yield the parameter χ 2 red: where, Sexp(q) is the experimental scattering curve, Savr(q) is the averaged theoretical scattering curve for each ensemble (chromosome), α is the scaling factor between the theoretical and experimental curves, K is the number of experimental q-points, and σ(q) is the experimental standard deviation. The 50 chromosomes with the smallest χ 2 red were selected from the 150 chromosomes produced by mutation and crossing of the initial chromosomes and propagated to the next generation (evolution of chromosome). The three processes of mutation, crossing, and evolution of chromosomes were repeated over 1000 generations. At the end of evolution, the most suitable chromosome for experimental data was finally collected to extract the optimal structural parameters of Rg and three-dimensional protein conformations. In the case of the analysis process using the genetic algorithm, ground and light-induced states were separately performed. The EOM analysis process was performed five times for the pool of candidate structures in each state, and the resulting Rg distribution was averaged by calculating the standard deviation. Among the protein conformations in the optimized ensemble, the structure observed with the highest frequency was chosen as the representative structure. In the photoproduct, the newly formed populations were observed around the Rg value of 28.5 Å , indicating that the populations involve the protein conformations corresponding to the final product originating from the light-induced activation of coPYP-4 ( Figure 4). Therefore, in the case of photoproduct, the structure observed with the highest frequency was chosen as the representative structure among the protein conformations in the newly formed populations.
After the EOM analysis, for each state, we calculated the weighted R factor (wR) using the following equation to quantify the agreement between the theoretical scattering curve of the optimized ensemble and the experimental scattering curve. are not likely to be highly affected by the limitations. Therefore, the diversity of structural information allows that the conformational parameters and ensemble structures optimized for x-ray scattering through EOM analysis from the candidate structures can provide a reliable solution to the global structural changes of coPYP-4.
We also reconstructed the SAS curves of the third species, so-called the photoproduct, by varying the photoconversion yield from 0.1 to 0.5 and implemented the EOM analysis using these static curves to investigate the sensitivity of the scaling factor to the structural analysis.
As expected, the optimal distributions of Rg show that the relative ratio of the populations around 28.5 Å corresponding to the final photoproduct gradually becomes larger than that of the populations around 32 Å corresponding to the ground as the photoconversion yield increases ( Figure S10)

Experiment-restrained rigid-body MD simulation
Prior to the MD simulations of coPYP-4 which were used for the EOM analysis, two different starting conformations of coPYP-4 were obtained. The starting conformation for the ground state of coPYP-4 was constructed from the crystal structure of monomer PYP in the ground state (PDB ID: 2PHY) using the experiment-restrained rigid-body (ERRB) MD simulation.
After the construction, each monomer PYP constituting the starting structure for the ground state constructed from the ERRB MD simulation was replaced with the structure of the monomer PYP in the signaling state (PDB ID: 2KX6). The ERRB MD simulation method is nearly identical to that described in a previous study 8,[19][20][21][22][23] . In the ERRB MD simulation, the crystal structure of monomer PYP in the ground state (PDB ID: 2PHY) was used as a starting point. The structures were divided into a number of rigid bodies composed of several amino acids. The rigid bodies were allowed to move under the influence of the chemical and χ 2 force fields. Because the atomic structure within a rigid body was constrained to be the same as that of the crystal structure, the force field within the rigid body was not considered. However, the van der Waals interactions between rigid bodies and the N-C bond-length corrections between rigid bodies were included in the chemical force field. The χ 2 force field was introduced to drive the molecular structure generated by MD simulations toward a structure that yields a difference scattering curve that matches the experimental difference scattering curve 6,24 .
The total potential (U) on the rigid bodies has the van der Waals term (ULJ), the χ 2 term, and the bond correction term (B) as follows: where c1, c2, and c3 are the weighting parameters to scale the magnitude of the three terms appropriately. ULJ and χ 2 , which define the agreement between the experimental data and theoretical value, are as follows (the scaling factor cs and reduced units are used): 12 (7) where, rij is the distance between atom i and atom j, Iexp(q) is the experimental scattering curve of coPYP-4, Ical(q) is the theoretical scattering curve of the model structure generated from the crystal structure of monomer PYP (PDB ID: 2PHY), and σ q is the experimental standard deviation. 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 atoms, N, is as follows: 12 (8) In the L-J potential, σ is defined as the r value where the corresponding potential is zero and rij (= ri − rj) is the relative position vector between atom i and atom j. If the distance between the two atoms is smaller than σ, the repulsion increases steeply. The following two types of σ values were defined 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. M F R= (10) where i i   Ff is the total force on the rigid body defined as the sum of fi acting on the ith atom within the rigid body. The total torque acting on each rigid body with respect to the COM was as follows: The velocity of ′ relative to R was as follows: ii   rr (12) where ω is the angular velocity with respect to the COM of the rigid body. The moment of inertia with respect to the COM was as follows: The rotational equation of motion of a rigid body around R can be expressed as: Eq. (5)