A method for facilitating isomerization of organic small molecules based on rotation of chemical single bonds

Abstract

The application discloses a method for promoting isomerization of organic small molecules based on chemical single bond rotation, comprising the following steps: irradiating the organic small molecules in vitro by using terahertz pulses, wherein the terahertz pulses are Gaussian pulse electric fields, the pulse standard deviation ranges from 100 to 500 picoseconds, the center frequency ranges from 0.1 to 19 THz, and the maximum electric field intensity ranges from 1.5 to 3.0 V / nm; and the organic small molecules are arginine residues located at the narrowest channel position of water channel protein. When the water channel protein is irradiated by the terahertz pulses, the cis-trans isomerization of the arginine residues can be promoted, and the switching between the on and off states of the water channel protein can be controlled. The method is simple and feasible, non-invasive, and has time and space controllability, and can be used for controlling the closing or opening of the water channel protein on the bacterial cell membrane when a water channel protein-based biomimetic permeable membrane or an industrial fermentation recombinant protein of Escherichia coli is used, and has a wide application prospect in industrial production.

Description

Technical Field

[0001] This invention relates to the field of biophysical technology, and to a method for promoting isomerization of small organic molecules based on chemical single bond rotation. Background Technology

[0002] Aquaporins (AQPs) are a family of transmembrane proteins whose main function is to efficiently and selectively mediate the transmembrane transport of water molecules (some subtypes can also transport small molecules such as glycerol and urea), thereby regulating intracellular and extracellular water balance. AQPs are widely distributed in animals, plants, and microorganisms (bacteria, archaea, fungi, etc.). Different subtypes have specific tissue distributions, such as in the kidneys, skin, lungs, eyes, and brain, maintaining intracellular and extracellular water balance, regulating urine concentration, maintaining the transparency of the eye lens, and promoting cell metabolism and signal transduction. In microorganisms, AQPs play an important role in osmotic pressure regulation, responding to environmental stresses (such as drought, high salinity, and freezing), and maintaining cellular water balance.

[0003] Aquaporin-4 (AQP4) plays a crucial role in the pathophysiology of cerebral edema, especially in in vitro cerebral edema models, where its regulatory mechanisms and potential therapeutic targets have attracted significant attention. AQP4 is a key player in in vitro cerebral edema models because it plays a dual role in the formation and resolution of edema. When culturing astrocytes or neurons in vitro, changing the culture medium to hypertonic or hypotonic fluid immediately causes cells to shrink or swell. However, if AQP4 is knocked out, the change in cell volume under the same stimulus is immediately halved, demonstrating that AQP4 is the "only rate limiter" in the model. Further, by embedding cells into three-dimensional collagen to create "mini-brain slices" and then infusing them with artificial cerebrospinal fluid containing a red fluorescent tracer, vasogenic edema can be simulated, allowing for real-time monitoring of cerebral edema progression: the AQP4-normal group rapidly removes the dye; the AQP4-deficient group retains the dye. Common methods to induce AQP4 expression changes and create cerebral edema-like changes include hypoxia models, stimulation by inflammatory factors, mechanical injury, and lentiviral transfection. Furthermore, pharmaceutical companies use AQP4 as a high-throughput drug screening platform: by dropping small molecules of candidate drugs into culture wells, they can determine within 30 minutes which drug can tighten the AQP4 barrier and thus reduce edema. Therefore, AQP4 serves as both a "pathogenic switch" and a "drug efficacy benchmark" in in vitro cerebral edema models.

[0004] Because aquaporins can efficiently transport water molecules while blocking salt and other impurities, scientists have used biomimetic design to embed them into polymer membranes (such as liposomes or nanofiber membranes). This allows them to maintain their activity and withstand high-pressure environments, thus creating highly efficient seawater desalination membranes to solve the problems of high energy consumption, high cost, and membrane fouling associated with traditional seawater desalination methods (such as reverse osmosis). The Danish company Aquaporin A / S is the world's first to commercialize aquaporin membranes, with its products sold in tonnes and applied in industrial wastewater treatment and seawater desalination. However, natural aquaporins act like "live valves," closing when exposed to acids, calcium ions, or mechanical pressure. Even after embedding them into biomimetic membranes, the channels still close due to stimuli such as sudden pH drops, excessively high salt concentrations, and membrane bending, reducing water flow to a fraction of its original rate. Laboratory studies have shown that the opening probability of bacterial aquaporin AQP-Z drops from 90% to less than 10% when temperatures rise or divalent ions in the solution increase. Therefore, if the switching state of aquaporins can be altered, it is hoped that long-term stable water production and precise water production control can be achieved, and that it can also help clean the permeate membrane to improve membrane durability.

[0005] Furthermore, in the high-density fermentation of E. coli recombinant protein plants, osmotic pressure runaway is a critical risk, exhibiting a delayed manifestation. Feeding glucose and metabolic byproducts can cause a sawtooth-like spike in osmotic pressure. If not properly controlled, the osmotic pressure can exceed 450–600 mOsm, far surpassing the optimal cell window of 300 mOsm ± 20, triggering instantaneous cell dehydration and shrinkage, metabolic imbalance, and lysis cascades, ultimately leading to the scrapping of the entire batch. At the process design level, osmotic pressure control, along with dissolved oxygen, pH, and bottom flow rate, is considered one of the four core control loops, with a risk level classified as "unacceptable," and cumulative economic losses reaching tens to hundreds of millions of yuan. If the switching of aquaporins in cells can be controlled in real-time and reversibly—for example, closing aquaporins when extracellular osmotic pressure surges, preventing intracellular fluid leakage, and allowing cells to quickly escape the effects of osmotic pressure runaway—it's equivalent to equipping the fermenter with a millisecond-level osmotic pressure relief valve, minimizing the risk of a sudden drop in recombinant protein yield due to changes in osmotic pressure.

[0006] The "on-off" state changes of aquaporins are essentially conformational changes and reversible rearrangements of key structural elements. These changes are driven by isomerization based on rotatable bonds (especially π bonds, peptide bonds, and σ single bonds), ultimately resulting in a variety of biomolecular conformations. Given the importance of the structure-function relationship, regulating molecular isomerization is a promising strategy; however, it is extremely complex when targeting specific molecules in complex systems with intricate interaction networks. Therefore, achieving a molecularly specific and non-invasive isomer regulation method remains highly challenging. Summary of the Invention

[0007] To address the aforementioned problems, the present invention aims to provide a method for promoting isomerization of small organic molecules based on the rotation of chemical single bonds. This method employs terahertz pulses of a certain frequency, maximum electric field strength, and pulse width to irradiate small organic molecules, which can effectively drive the small organic molecules to undergo isomerization based on the rotation of their chemical single bonds (i.e., cis / trans configuration transition).

[0008] Another object of the present invention is to provide an application of a device for emitting terahertz pulses in promoting isomerization of small organic molecules in a product based on chemical single bond rotation.

[0009] To achieve the above objectives, the present invention provides a method for promoting isomerization of small organic molecules based on chemical single bond rotation, comprising: irradiating the small organic molecule in vitro with a terahertz pulse at room temperature, wherein the terahertz pulse is a Gaussian pulse electric field with a pulse standard deviation ranging from 100 to 500 picoseconds, a center frequency ranging from 0.1 to 19 THz, and a maximum electric field strength ranging from 1.5 to 3.0 V / nm, and the small organic molecule is an amino acid residue with a rotatable single bond located at the narrowest channel position of an aquaporin.

[0010] This invention promotes isomerization of amino acid residues with rotatable single bonds at the narrowest channel position of aquaporins by irradiating them with terahertz pulses, thereby promoting the isomerization of these amino acid residues based on the rotation of their chemical single bonds.

[0011] Furthermore, the amino acid residue is an arginine residue, and the center frequency range of the terahertz pulse is 3–6 THz or 14.5–17.5 THz.

[0012] Within this terahertz pulse parameter range, the terahertz pulse has a high probability of promoting cis-to-trans isomerization of the guanidin group on the arginine residue of the aquaporin based on a single bond, that is, it has a high probability of promoting the aquaporin to switch from the closed to the open state. Outside this frequency range, the probability of successful isomerization and switching of the aquaporin's on / off state decreases sharply.

[0013] Furthermore, the amino acid residue is an arginine residue, and the center frequency range of the terahertz pulse is 10–13 THz.

[0014] Within this range, terahertz pulses have a high probability of promoting single-bond-based isomerization from trans to cis on guanidine residues of aquaporins, meaning a high probability of promoting the switching of aquaporins from open to closed states. Outside this frequency range, the probability of successful isomerization and switching of aquaporin states decreases sharply.

[0015] Furthermore, the aquaporin is one or more of the following aquaporins: AQP1, AQP2, AQP4, AQP5, and AQPZ; the amino acid sequence of AQP1 is shown in Seq ID No. 1; the amino acid sequence of AQP2 is shown in Seq ID No. 2; the amino acid sequence of AQP4 is shown in Seq ID No. 3; the amino acid sequence of AQP5 is shown in Seq ID No. 4; and the amino acid sequence of AQPZ is shown in Seq ID No. 5.

[0016] Furthermore, the amino acid residue is arginine residue number 197 of AQP1, arginine residue number 187 of AQP2, arginine residue number 216 of AQP4, arginine residue number 188 of AQP5, or amino acid residue number 189 of AQPZ.

[0017] The present invention also provides an application of a device for emitting terahertz pulses in promoting the isomerization of small organic molecules in a product based on chemical single bond rotation. The terahertz pulses emitted by the device are used to irradiate aquaporins in vitro. The terahertz pulses are Gaussian pulse electric fields with a pulse standard deviation range of 100 to 500 picoseconds, a center frequency range of 0.1 to 19 THz, and a maximum electric field strength range of 1.5 to 3.0 V / nm.

[0018] Furthermore, the center frequency range of the terahertz pulse is 3–6 THz or 14.5–17.5 THz.

[0019] Furthermore, the center frequency range of the terahertz pulse is 10–13 THz.

[0020] Furthermore, the product is a biomimetic permeable membrane based on aquaporins.

[0021] Furthermore, the product is *E. coli* produced during the industrial fermentation of recombinant protein.

[0022] The present invention has the following beneficial effects:

[0023] This invention provides a method for promoting isomerization of small organic molecules based on chemical single bonds. It employs terahertz pulses with specific frequencies, pulse widths, and maximum electric field strengths to irradiate small organic molecules, effectively promoting isomerization based on chemical single bonds. This invention can be used in biomimetic permeable membranes based on aquaporins. Irradiating the membrane with terahertz pulses of specific parameters promotes the opening of aquaporins, maintaining long-term stable water production and precise control of water production, or promoting the closure of aquaporins to clean the membrane, improving membrane durability and reducing fouling. Furthermore, this invention can be applied to E. coli recombinant protein factories. When a sharp increase in extracellular osmotic pressure is detected, timely terahertz pulse irradiation of E. coli can close aquaporins, preventing intracellular fluid leakage and avoiding the effects of osmotic pressure runaway. Attached Figure Description

[0024] Figure 1 This is a schematic diagram illustrating the regulation of the switching state of the aquaporin AQP4 by terahertz pulsed irradiation in this embodiment.

[0025] Figure 2 This is a schematic diagram showing the change in the dihedral angle of the Arg216 guanidine group when a terahertz pulse is applied, as provided in this embodiment.

[0026] Figure 3 This is a schematic diagram illustrating the changes in the water permeability of the water channel protein AQP4 under the application of different terahertz pulses, as provided in this embodiment.

[0027] Figure 4 This is a schematic diagram of the rotational spectra of the four atoms CG, CD, NE, and CZ on Arg216 of the aquaporin AQP4 in the open / closed states, calculated based on the angular momentum method, provided in this embodiment.

[0028] Figure 5 This is a schematic diagram illustrating the influence of different frequency pulses on the switching state of the aquaporin AQP4 provided in this embodiment.

[0029] Figure 6 This is a schematic diagram illustrating the dependence of the switching probability of the aquaporin AQP4 on the maximum electric field strength and pulse width, as provided in this embodiment. Detailed Implementation

[0030] The embodiments of the present invention will now be described in detail and comprehensively so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby providing a clearer and more explicit definition of the scope of protection of the present invention.

[0031] Terahertz fields are traditionally considered to be high-frequency electromagnetic fields ranging from 0.1 to 10 THz. Currently, in interdisciplinary research in biophysics, the upper frequency limit has been extended to 100 THz. A terahertz pulse refers to a terahertz wave packet with a defined duration. In this invention, when terahertz pulses are used to irradiate aquaporins, the pulses resonate with the four atoms (CG, CD, NE, CZ) on the guanidino side chain of the arginine residue, the narrowest channel position of the aquaporin. The energy of the terahertz pulse is absorbed resonantly by the guanidino group on the arginine residue, increasing the rotational kinetic energy. This increases the probability of rotation based on the rotatable single bond CD-NE, thus increasing the probability of dihedral angle changes based on CD-NE, ultimately causing isomerization of the guanidino group on the arginine residue based on the CD-NE bond. Terahertz pulses with different parameters can cause the guanidinium group of arginine residues to undergo conformational changes from cis to trans and vice versa. Since this arginine residue is the amino acid located at the narrowest part of the aquaporin channel, its guanidinium conformational change causes the aquaporin to switch between open and closed states.

[0032] Example 1

[0033] This embodiment provides the dynamic evolution of the aquaporin AQP4 with and without terahertz pulsed irradiation. This process was simulated using the commonly used molecular dynamics software GROMACS. The specific modeling and simulation methods are as follows:

[0034] 1. Construct a membrane-aquaporin dynamics system in equilibrium of temperature and pressure.

[0035] The crystal structure of the aquaporin AQP4 used for modeling was obtained from the RCSB protein library, entry number: 3GD8. The amino acid sequence of AQP4 is shown in Seq ID No. 3: 32 QAFWKAVTAEFLAMLIFVLLSLGSTINWGGTEKPLPVDMVLISLCFGLSIAT MVQCFGHISGGHINPAVTVAMVCTRKISIAKSVFYIAAQCLGAIIGAGILYLVT PPSVVGGLGVTMVHGNLTAGHGLLVELIITFQLVFTIFASCDSKRTDVTGSIAL AIGFSVAIG H 201 LFAINYTGASMNPA R 216 SFGPAVIMGNWENHWIYWVGPIIGA VLAGGLYEYVFCP 254This sequence represents the original experimental data from protein crystallography, numbered from 32 onwards. It should be noted that residues numbered 1-31, which are not listed, are residues that could not be determined experimentally at this stage. AQP4 was embedded in a lipid bilayer using the Charmm-GUI online modeling website, with the lipid bilayer normal to the z-axis, and immersed in a 0.15 mol / L KCl solution. The components of the system were described by Charmm36 force field parameters. First, the energy of the simulated system was minimized using GROMACS-2020.7 software to eliminate poor interatomic contacts. Then, a V-rescale heat bath and a Berendsen followed by a Parrinello-Rahman pressure bath were applied to stabilize the average temperature at 303.15 K and the average pressure at 1 bar to obtain an isothermal and isobaric ensemble.

[0036] 2. Perform molecular dynamics simulations

[0037] After the simulated system reached complete equilibrium, molecular dynamics simulations were performed to analyze the conformational changes of key AQP4 residues. For terahertz pulse stimulation, the terahertz pulse is transmitted through an oscillating Gaussian single-pulse electric field E(t) = A·u·exp(-(t-t0)). 2 / (2σ 2 The expression denoted by cos(2πft+θ) represents the pulse intensities, frequencies, and initial phases of the stimulus, respectively. t0 and σ represent the pulse onset time and standard deviation, respectively. The pulse width of a Gaussian pulse is typically represented by its full width at half maximum (FWHM), where FWHM = 2.354σ. u represents the polarization direction of the stimulus, set to (0,0,1), i.e., perpendicular to the membrane surface, and the initial phase θ is set to 0 degrees. In this embodiment, the dependence of the water permeability properties of aquaporins on terahertz pulse parameters was analyzed by changing the values ​​of A, σ, and f. Specifically, during the simulation, the key amino acid Arg216 was first positionally constrained in its channel-open and closed conformations (see description below), and simulated for 100 nanoseconds in an NVT (temperature equilibrium) ensemble to stabilize the surrounding environment. Then, the positional constraint of Arg216 was removed, and the simulation was run in the NVT ensemble for 20 nanoseconds. During this time, it was possible to add terahertz pulse stimulation (if added, the pulse start time t0 was set to 10 nanoseconds). Atomic trajectories were recorded every picosecond for data post-processing, and the probability of different conformations of Arg216 appearing within the 2 nanosecond range spanned by the pulse was statistically analyzed when terahertz pulses were applied.

[0038] 3. Perform molecular rotation spectrum calculations

[0039] To confirm the effective driving force of conformational changes in key residues of aquaporins, the rotational spectrum of the molecules was calculated and analyzed. A common molecular spectroscopy method is to calculate the infrared absorption spectrum (also known as the molecular vibrational spectrum). Infrared absorption spectra are the spectra formed when molecules absorb light of a specific wavelength under infrared illumination, leading to vibrational or rotational energy level transitions. Infrared light absorption only occurs when the dipole moment changes during molecular vibration or rotation. Therefore, the relationship between the infrared absorption spectrum or spectral density I(ω) and frequency ω is usually obtained by performing a Fourier transform on the autocorrelation function of the total dipole moment of the system, i.e.

[0040]

[0041] in,

[0042]

[0043] It is the total dipole moment of the target molecule, where n represents the total number of atoms, and e is the total dipole moment of the target molecule. j It is the charge of the j-th atom, r j (t) is the position vector of the j-th atom at time t. In this embodiment, to more accurately describe the rotational properties of the molecule, the angular momentum J(t) = ∑ i r i (t)×m i v i (t)(m i ,v i (t),r i (t) represents the mass of the i-th atom, its velocity relative to the molecular center of mass at time t, and its distance, respectively. M(t) is used instead of M(t) for molecular spectral analysis. Angular momentum, as a dynamic variable characterizing molecular rotation, can effectively reflect the rotational behavior of a molecule relative to its rotatable bonds. Combined with changes in the dihedral angles of atoms, the dynamic evolution state of the molecule can be further analyzed.

[0044] Figure 1 This illustration shows a schematic diagram of the regulation of the switching state of the aquaporin AQP4 by terahertz pulsed irradiation, as provided in this embodiment. Figure 1 The left image shows the open state of the aquaporin AQP4 (grayish-white helical structure). The black and white spheres in the channel represent water molecules passing through it. The 216th amino acid residue—arginine (Arg216, gray rod-shaped structure)—and the 201st amino acid residue—histidine (His201, gray rod-shaped structure)—form the narrowest region of the aquaporin channel. These two key amino acids have been labeled in the aforementioned amino acid sequence. Previous studies have found that the guanidino side chain rotation of Arg216 plays a crucial role in the switching between the open and closed states of the aquaporin. The two elliptical coils in the image indicate the guanidino group of Arg216. Figure 1The upper part of the right figure shows the structure of Arg216 and His201 in a closed channel, represented by van der Waals spheres. The size of the spheres is linearly related to the atomic radius. The guanidinium group is oriented downwards as indicated by the arrow, exhibiting a cis configuration. It can be seen that there is almost no gap between Arg216 and His201, preventing water molecules from passing through. However, as... Figure 1 The lower part of the right figure shows the structure of Arg216 and His201 in an open channel. The guanidino group is pointing upwards as indicated by the arrow, exhibiting a trans configuration. It can be seen that there is a gap between Arg216 and His201, just large enough to accommodate one water molecule (represented by black and white van der Waals spheres) to pass through. The terahertz pulses provided in this embodiment, which meet specific parameter ranges, can promote the switching of aquaporins between open and closed states.

[0045] Figure 2 This embodiment illustrates the change in the dihedral angle of the Arg216 guanidine group when a terahertz pulse is applied. Figure 2 Figure 'a' shows the time-domain waveform of the terahertz pulse, which exhibits a Gaussian distribution. The corresponding maximum electric field intensity A is 3.0 volts per nanometer (V / nm), and the frequency f is 16.8 terahertz (THz, 1 THz = 10⁻⁶). 12 (Hz), pulse standard deviation σ is 300 picoseconds. Figure 2 Figure b shows Arg216 (ball-and-stick structure). The solid circled part is the guanidinium side chain of Arg216. The dark gray spheres and the atoms labeled CG, CD, NE, and CZ represent carbon (C), carbon (C), nitrogen (N), and carbon (C) atoms, respectively. The guanidinium change mainly revolves around the CD-NE CN single bond. The guanidinium rotation will change the CG-CD-NE-CZ dihedral angle (labeled α). Figure 2 Figure c shows the time-varying dihedral angle of Arg216 (CG-CD-NE-CZ) under a 16.8 THz pulse. It can be seen that the dihedral angle changes from approximately 90 degrees to approximately 270 degrees, with a rotation angle of approximately 180 degrees. Furthermore, the time interval during which the dihedral angle changes corresponds precisely to... Figure 2 The region with higher pulse intensity is shown in section a. Due to the change in dihedral angle, as... Figure 2 In the b group, the Arg216 guanidino group flips from facing down to facing up, thus this change represents the aquaporin switching from a closed state to an open state. Figure 2 The graph shown in Figure d illustrates the change in the CG-CD-NE-CZ dihedral angle of Arg216 over time when the irradiation pulse frequency is changed to 11.4 THz. It can be seen that the dihedral angle changes from approximately 270 degrees to approximately 90 degrees, with a rotation angle of approximately 180 degrees. The dihedral angle change corresponds to... Figure 2In the aquaporin, the Arg216 guanidinium group in b flips from facing upwards to facing downwards, indicating that the channel has switched from an open to a closed state. It can be seen that pulses of different frequencies can switch the aquaporin to an open or closed state, and this switching process promotes the rotational isomerization of the Arg216 guanidinium group based on its CN chemical single bond.

[0046] Figure 3 This embodiment illustrates the changes in the water permeability of the aquaporin AQP4 under different terahertz pulses, with gray shading indicating the time intervals of the applied pulses. Figure 3 As shown in Figure a, without the application of a 16.8 THz pulsed irradiation, no water molecules pass through the channel. However, after the pulse is applied, it can be seen that the number of water molecules passing through the channel increases continuously over time, exhibiting a permeation trend. This indicates that the 16.8 THz pulse opens the aquaporin. Figure 3 As shown in Figure b, without the application of an 11.4 THz pulse irradiation, the number of water molecules passing through the channel increases continuously over time, and the channel exhibits a permeation trend. However, after the pulse is applied, the number of water molecules no longer changes, meaning that no new water molecules pass through. This indicates that the 11.4 THz pulse causes the aquaporin to close.

[0047] Figure 4 This embodiment illustrates the rotational spectra of the four atoms CG, CD, NE, and CZ on Arg216 of the aquaporin AQP4 in the open / closed states, calculated using the angular momentum method. Figure 4 Figure a shows the rotation spectra of the four atoms CG, CD, NE, and CZ when the Arg216 guanidino group is facing downwards (as shown in the ball-and-bar structure embedding diagram, corresponding to the closed state of the aquaporin), obtained based on the aforementioned molecular rotation spectrum calculation method. It can be seen that the rotation spectrum intensity is different at different frequencies, representing different rotation modes, and the absorption intensity of the pulse field energy is also different. The aforementioned pulse frequency of 16.8 THz, which effectively drives the aquaporin from the closed to the open state, is selected based on this rotation spectrum, corresponding to the absorption peak position. Figure 4 Figure b shows the rotation spectrum of the four atoms CG, CD, NE, and CZ when the Arg216 guanidino group is facing upwards (as shown in the ball-and-stick structure embedding diagram, which corresponds to the open state of the aquaporin). The aforementioned pulse frequency of 11.4 THz, which effectively drives the aquaporin from the open to the closed state, is selected based on this rotation spectrum, corresponding to the absorption peak position.

[0048] Figure 5 This diagram illustrates the effect of different frequency pulses provided in this embodiment on the switching probability of the aquaporin AQP4. (See diagram for example.) Figure 5As shown, each data point is the statistical data of 50 repetitive simulations under the action of the pulse at this frequency, representing the probability of successfully switching between the "on" and "off" states, that is, the probability of switching from the off state to the open state (the black solid line marked with a square) and from the open state to the off state (the black dashed line marked with a circle). The maximum electric field amplitude of the action pulse is 2.5V / nm, and the standard deviation is 300 picoseconds. Figure 5 It can be seen that at 0 THz, i.e., without the application of a pulse field, the water channel, which was originally closed, has a 20% probability of spontaneously switching to the open state, while the probability of a channel that was originally open spontaneously switching to the closed state is 0. This indicates that it is more difficult for the water channel to spontaneously close than to spontaneously open. Within the range of 0.1–19 THz, the probability of the channel switching from closed to open is greater than in the case of no terahertz pulse (20%), which falls within the effective operating frequency range. Furthermore, this range is also roughly consistent with… Figure 4 The region with a large rotational spectrum amplitude corresponds to the area where pulse fields with frequencies greater than 19 THz cannot drive the channel to switch from closed to open (black solid line). Furthermore, under the action of pulse fields in the range of 1–17 THz, a channel "off -> on" switching probability greater than 50% can be obtained. Similarly, in the range of 0.1–19 THz, the probability of the channel switching from open to closed is greater than in the case without terahertz pulse action (0), which falls within the effective operating frequency range, and this range is also basically consistent with... Figure 4 The region with a larger rotational spectrum amplitude corresponds to pulse fields with frequencies greater than 19 THz, which cannot drive the channel to switch from open to closed (black dashed line). Furthermore, pulse fields in the 2–14 THz range can achieve a channel "on -> off" switching probability greater than 50%. Since the pulse frequencies overlap between the two switching states, 3–6 THz and 14.5–17.5 THz (e.g., [missing information]) are selected. Figure 5 The light gray area (marked within the range) serves as the optimal operating frequency range for the drive channel's "off" to "on" state switching, 10–13 THz (e.g., Figure 5 The dark gray area (marked range) is the optimal frequency range for driving the "on" to "off" state switching. Other frequency ranges cannot or will not effectively drive the "on" to "off" state switching.

[0049] It should be noted that, due to the specificity of the pulse frequency and the unique position of the Arg216 residue, the terahertz pulses provided in this embodiment primarily alter the opening and closing states of aquaporins by influencing Arg216. Specifically, different amino acids have different absorption spectra, meaning that the pulse frequencies at which energy can be effectively absorbed by amino acids differ. Furthermore, even for the same amino acid, its absorption spectrum will change depending on its surrounding environment (i.e., the other amino acids it interacts with). Therefore, the pulses within the frequency range described in this embodiment mainly promote the isomerization of the Arg216 arginine residue at the narrowest point of the channel. Moreover, the rotation of the arginine residue under pulse-driven action is only its side chain and does not affect the molecular backbone. For aquaporins, due to the unique position of Arg216, the rotation of the Arg216 side chain under pulse-driven action precisely affects the opening and closing of the channel. Even if other arginine residues are affected by rotation, the entire protein backbone remains stationary, thus not affecting the channel's opening and closing.

[0050] Figure 6 This diagram illustrates the dependence of the switching probability of the aquaporin AQP4 provided in this embodiment on the maximum electric field strength and pulse width. Figure 6 As shown, each data point represents the statistical data from 50 repetitive simulations under the action of the parameter pulse, indicating the probability of a successful "on-off" state switch. The gray shaded area marks the range of parameters that effectively induce the "on-off" state switch, and the pentagram indicates the optimal parameter within that range. Specifically, as... Figure 6 As shown in Figure a, when the maximum electric field strength is greater than 1.5 V / nm, the pulse energy effectively promotes channel opening. Furthermore, as the maximum electric field strength increases, the probability of channel opening gradually increases, but the rate of increase continuously decreases. Considering the need to conserve pulse energy, the optimal maximum electric field strength is 2.5 V / nm. Additionally, as... Figure 6 As shown in Figure b, when the pulse standard deviation is greater than 100 picoseconds, the pulse can effectively increase the probability of successful switching, and the probability continues to increase as the pulse standard deviation increases. However, due to the brief period of pulse application, additional water molecules (such as...) are introduced. Figure 3 (The shaded area shows an increase in water molecules), therefore the pulse width should be as small as possible. Considering the probability of achieving a pulse width close to the optimal maximum electric field strength at 500 picoseconds, the optimal pulse standard deviation is chosen to be 500 picoseconds. Figure 6 As shown in Figure c, under the action of a pulse with a frequency of 11.4 THz, a maximum electric field strength greater than 1.5 V / nm can effectively promote channel closure. Furthermore, as the field strength increases, the probability of successful switching from "on" to "off" increases continuously. Considering that an excessively high field strength may cause significant disturbance to other parts of the system, the optimal maximum electric field strength is chosen to be 3.0 V / nm. Figure 6As shown in Figure d, when the pulse standard deviation is greater than 100 picoseconds, the pulse can effectively increase the probability of successful switching. As the pulse standard deviation increases, the probability gradually increases, but the rate of increase continuously decreases. The optimal pulse standard deviation is 400 picoseconds.

[0051] Example 2

[0052] Using amphiphilic block copolymers as a substrate, nanoscale water channels were formed through directional self-assembly, and AQPs (such as AQP1, AQP2, AQP4, AQP5, or AQPZ) were embedded into the membrane to construct a biomimetic membrane of aquaporins. The amino acid sequence of AQP1 (RCSB protein library entry number: 1J4N) is shown in Seq ID No. 1: 1 MASEFKKKLFWRAVVAEFLAMILFIFSIGSALGFHYPIKSNQTTGAVQDNVK VSLAFGLSIATLAQSVGHISGAHLNPAVTLGLLLSCQISVLRAIMYIIAQCVGAI VATAILSGITSSLPDNSLGLNALAPGVNSGQGLGIEIIGTLQLVLCVLATTDRRR RDLGGSGPLAIGFSVALG H 182 LLAIDYTGCGINPA R 197 SFGSSVITHNFQDHWIF WVGPFIGAALAVLIYDFILAPRSSDLTDRVKVWTSGQVEEYDLDADDINSRVE MKPK 271 Among them, the histidine residue at position 182 (abbreviated as His182) and the arginine residue at position 197 (abbreviated as Arg197) are located at the narrowest position of the aquaporin AQP1, which control the opening and closing of the aquaporin.

[0053] The amino acid sequence of AQP2 (RCSB protein library entry number: 4NEF) is shown in Seq ID No. 2: 1 GSELRSIAFSRAVFAEFLATLLFVFFGLGSALNWPQALPSVLQIAMAFGLGIGT LVQALGHISGAHINPAVTVACLVGCHVSVLRAAFYVAAQLLGAVAGAALLH EITPADIRGDLAVNALSNSTTAGQAVTVELFLTLQLVLCIFASTDERRGENPGTPALSIGFSVALG H 172 LLGIHYTGCSMNPA R 187SLAPAVVTGKFDDHWVFWIGPL VGAILGSLLYNYVLFPPAKSLSERLAVLKGLEP 242 Among them, the histidine residue at position 172 (abbreviated as His172) and the arginine residue at position 187 (abbreviated as Arg187) are located at the narrowest position of the aquaporin AQP2, which control the opening and closing of the aquaporin.

[0054] The amino acid sequence of AQP5 (RCSB protein library entry number: 3D9S) is shown in Seq ID No. 4: 1 SKKEVCSVAFLKAVFAEFLATLIFVFFGLGSALKWPSALPTILQIALAFGLAIG TLAQALGPVSGGHINPAITLALLVGNQISLLRAFFYVAAQLVGAIAGAGILYG VAPLNARGNLAVNALNNNTTQGQAMVVELILTFQLALCIFASTDSRRTSPVGSPALSIGLSVTLG H 173 LVGIYFTGCSMNPA R 188 SFGPAVVMNRFSPAHWVFWVG PIVGAVLAAILYFYLLFPNSLSLSERVAIIKGTYEPDEDWEEQREERKKTMELT TR 265 Among them, the histidine residue at position 173 (abbreviated as His173) and the arginine residue at position 188 (abbreviated as Arg188) are located at the narrowest position of the aquaporin AQP5, which control the opening and closing of the aquaporin.

[0055] The amino acid sequence of AQPZ (RCSB protein library entry number: 3NK5) is shown in Seq ID No. 5: 1 MFRKLAAESFGTFWLVFGGSGSAVLAAGFPELGIGFAGVALAFGLTVLTMAF AVGHISGGHFNPAVTIGLWAGGRFPAKEVVGYVIAQVVGGIVAAALLYLIAS GKTGFDAAASGFASNGYGEHSPGGYSMLSALVVELVLSAGFLLVIHGATDKF APAGFAPIAIGLALTLI H 174 LISIPVTNTSVNPA R 189 STAVAIFQGGWALEQLWFFWVVPIVGGIIGGLIYRTLLEKRD231 Among them, the histidine residue at position 174 (abbreviated as His174) and the arginine residue at position 189 (abbreviated as Arg189) are located at the narrowest position of the aquaporin AQPZ, which control the opening and closing of the aquaporin.

[0056] Similar to AQP4 in Example 1, AQP1, AQP2, AQP5, and AQPZ also have Arg and His forming the narrowest part of the channel. The difference is that these two amino acid residues have different numbers in each aquaporin. For example, in AQP1, the narrowest part of the channel is formed by Arg197 and His182, in AQP2 by Arg187 and His172, in AQP5 by Arg188 and His173, and in AQPZ by Arg189 and His174.

[0057] In seawater desalination plants, terahertz pulse emission devices (such as femtosecond lasers from Spectrophysics paired with TOPAS frequency conversion modules or Daylight tunable pulse lasers) are used to directionally irradiate biomimetic membrane modules (such as Aquaporin membranes from Denmark). By monitoring the osmotic pressure, salt concentration, and pH signals across the membrane in real time, pulse parameters are dynamically adjusted to maintain stable water production and on-demand water production even under conditions of pH fluctuations and high salinity, while significantly reducing energy consumption compared to traditional reverse osmosis membranes. Specifically, the application of terahertz pulse-controlled biomimetic membranes can be divided into the following four aspects:

[0058] (1) Controlling channel opening: When the biomimetic membrane causes aquaporins to close due to increased salt concentration or decreased pH, it emits a pulse with a frequency of 16.8 THz, a maximum electric field strength of 2.5 V / nm, and a pulse standard deviation of 500 picoseconds. This pulse energy causes conformational isomerization of the arginine residues at the narrowest point of the channel, changing the guanidine group from cis to trans, thus releasing the channel from its closed state. The aforementioned simulations show that a single pulse can restart the closed aquaporins within a nanosecond range, thereby restoring water flux.

[0059] (2) Controlling channel closure: In the case of membrane fouling or flow restriction, a pulse with a frequency of 11.4 THz, a maximum electric field strength of 3.0 V / nm, and a pulse standard deviation of 400 picoseconds is applied to induce conformational isomerization of arginine residues at the narrowest part of the channel, and the guanidine group changes from trans to cis, thereby promoting channel closure and limiting water flux.

[0060] (3) Precise water production control: By adjusting the maximum electric field strength and pulse width, graded control can be achieved: pulses with low maximum electric field strength and low pulse width can maintain 30% channel openness to meet low flow water production needs; pulses with high maximum electric field strength and high pulse width can increase the open rate to 60%, which is suitable for peak water use scenarios.

[0061] (4) Membrane antifouling and cleaning: After long-term operation, pollutants will block the inlet of the aquaporin in the biomimetic membrane. At this time, applying a pulse with a frequency of 11.4 THz, a maximum electric field strength of 3.0 V / nm, and a pulse standard deviation of 400 picoseconds can effectively clean and peel off the pollutants attached to the channel opening, reduce pollution, and extend the service life of the biomimetic membrane.

[0062] Example 3

[0063] When using *E. coli* as a recombinant protein factory, high-density fermentation often leads to a sudden increase in extracellular osmotic pressure (such as a sudden rise in NaCl or sucrose concentration) due to nutrient consumption or accumulation of metabolic byproducts. At this time, water rapidly flows out through aquaporins (such as AQPZ, the main aquaporin in *E. coli*), causing cell dehydration, growth arrest, or even lysis. Traditional methods (such as adding osmotic protectants) are costly and negatively impact protein expression efficiency.

[0064] In high-density fermentation of recombinant E. coli, a freezing point osmotic pressure module (such as the OsmoTECH full series of osmoremeters from Adawa) and an automation platform (such as Emerson's DeltaV distributed control system) are integrated in the bypass of the fermenter to monitor changes in extracellular osmotic pressure in real time (accuracy ±10 mOsm / L). Simultaneously, using a metal waveguide, tunable bellows, and antenna, pulses are losslessly extracted from a tunable laser (operating frequency band 10–13 THz) and guided to the inner wall of the fermenter. A beam expander (which can be embedded in the fermenter wall) expands the free-space beam to a meter-scale irradiation area, and the pulses are directed towards the fermenter under the trigger of an osmotic pressure feedback signal. When an osmotic pressure increase of ≥100mOsm / L is detected, the antenna immediately emits a terahertz pulse, which causes conformational isomerization of the arginine residue at the narrowest point of the aquaporin AQPZ in E. coli. The guanidino group changes from trans to cis, thereby closing the channel, blocking the leakage of water molecules from the cell, avoiding cell lysis, and keeping the production of recombinant protein stable under osmotic pressure fluctuations.

[0065] As can be seen from the above embodiments, the method for promoting the isomerization of small organic molecules based on chemical single bonds provided by the present invention uses terahertz pulses of a certain frequency, pulse width, and maximum electric field strength to irradiate small organic molecules, which can effectively promote the isomerization of molecules based on chemical single bonds. The present invention can be used in biomimetic permeable membranes based on aquaporins, irradiating the permeable membrane with terahertz pulses of specific parameters to promote the opening of aquaporins, maintaining long-term stable water production, finely controlling water production, or promoting the closure of aquaporins to clean the permeable membrane, improving membrane durability and reducing fouling. In addition, the present invention can also be applied to E. coli recombinant protein factories. When a sharp increase in extracellular osmotic pressure is detected, timely terahertz pulse irradiation of E. coli can be used to close aquaporins, preventing intracellular fluid from leaking out and avoiding the effects of osmotic pressure runaway.

[0066] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for promoting isomerization of small organic molecules based on chemical single bond rotation, characterized in that, include: At room temperature, terahertz pulses were used to irradiate small organic molecules in vitro. The terahertz pulses were Gaussian pulsed electric fields with a pulse standard deviation ranging from 100 to 500 picoseconds, a center frequency ranging from 0.1 to 19 THz, and a maximum electric field strength ranging from 1.5 to 3.0 V / nm. The small organic molecules were amino acid residues with rotatable single bonds located at the narrowest channel position of an aquaporin. The aquaporin was AQP4, and the amino acid sequence of AQP4 was shown in Seq ID No.

3. The amino acid residue was arginine residue number 216 of AQP4.

2. The method for promoting isomerization of small organic molecules based on chemical single bond rotation as described in claim 1, characterized in that, The amino acid residue is an arginine residue, and the center frequency range of the terahertz pulse is 3~6 THz or 14.5~17.5 THz.

3. The method for promoting isomerization of small organic molecules based on chemical single bond rotation as described in claim 1, characterized in that, The amino acid residue is an arginine residue, and the center frequency range of the terahertz pulse is 10–13 THz.

4. The application of a device for emitting terahertz pulses in promoting the isomerization of small organic molecules in a product based on chemical single bond rotation, characterized in that, The device emits terahertz pulses to irradiate aquaporins. The terahertz pulses are Gaussian pulse electric fields with a pulse standard deviation ranging from 100 to 500 picoseconds, a center frequency ranging from 0.1 to 19 THz, and a maximum electric field strength ranging from 1.5 to 3.0 V / nm. The organic small molecule is an amino acid residue with a rotatable single bond located at the narrowest channel position of the aquaporin. The aquaporin is AQP4, and the amino acid sequence of AQP4 is shown in Seq ID No.

3. The amino acid residue is arginine residue number 216 of AQP4.

5. The application of the terahertz pulse emitting device as described in claim 4 in promoting the isomerization of small organic molecules in a product based on chemical single bond rotation, characterized in that, The center frequency range of the terahertz pulse is 3~6 THz or 14.5~17.5 THz.

6. The application of the terahertz pulse emitting device as described in claim 4 in promoting the isomerization of small organic molecules in a product based on chemical single bond rotation, characterized in that, The center frequency range of the terahertz pulse is 10–13 THz.

7. The application of the terahertz pulse emitting device as described in any one of claims 4 to 6 in promoting the isomerization of small organic molecules in a product based on chemical single bond rotation, characterized in that, The product is a biomimetic permeable membrane based on aquaporins.

8. The application of the terahertz pulse emitting device as described in any one of claims 4 to 6 in promoting the isomerization of small organic molecules in a product based on chemical single bond rotation, characterized in that, The product is Escherichia coli produced during the industrial fermentation of recombinant protein.

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