Dilution-induced phase separation polypeptide and application thereof

Abstract

The invention discloses a dilution induced phase separation polypeptide and application thereof. The invention accidentally discovers a polypeptide TP presenting abnormal phase change kinetics, studies liquid-liquid phase separation properties and molecular mechanisms induced by TP dilution, and discovers that liquid-liquid phase separation can be induced by diluting a homogeneous TP solution, so as to realize the zoning of biomacromolecules, and the discovery subverts the traditional understanding of liquid-liquid phase separation, so that the method has the advantages of simple operation, low cost and high efficiency. The application also verifies that the TP dilution-induced coagulation effect can realize the compartmentalization of guest molecules, regulate the optical output of the guest, promote the protein in the coagulation to form secondary liquid-liquid phase separation and optimize the enzymatic reaction, thereby proving the practicability of the TP dilution-induced coagulation; a brand new regulation strategy is provided for self-assembly and function regulation of biological macromolecules, and the method has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to dilution-induced phase separation polypeptides and their applications. Background Art

[0002] Compartmentalization is the most fundamental feature of all living organisms. By separating active components into different compartments, the living system can achieve precise biological functions such as material transfer, energy conversion, and information exchange. Analyzing the molecular pathways by which nature constructs complex compartmental structures not only provides a unique perspective for exploring the essence of cell life but also brings important insights into understanding the origin mechanism of life on Earth. However, the efficient concentration of biomolecules within compartments faces thermodynamic challenges - the entropy loss accompanying this process makes it thermodynamically unfavorable. To achieve the compartmentalization of biomacromolecules, cells and their precursor systems have evolved various compartment construction strategies: forming membrane-like compartments through the self-assembly of amphiphilic lipids, polysaccharides, polypeptides, and even inorganic structures. Different from traditional membrane structures or membrane-like structures, liquid–liquid phase separation (LLPS), as a new mechanism for concentrating biomacromolecules into membraneless condensates, has gradually been recognized as an important way to achieve cell compartmentalization in recent years. Polypeptides with phase separation ability can encapsulate other molecules into the phase separation system and play a role. Summary of the Invention

[0003] To make up for the deficiencies of the prior art, the present invention provides a polypeptide and its applications.

[0004] To achieve the above object, the present invention adopts the following technical solutions:

[0005] In a first aspect of the present invention, there is provided a polypeptide, the polypeptide comprising an amino acid sequence selected from the following groups:

[0006] (1) an amino acid sequence obtained by substituting, deleting, adding, or inserting at least one amino acid residue in SEQ ID NO:1;

[0007] (2) an amino acid sequence having at least 90% sequence identity with SEQ ID NO:1.

[0008] Further, the substitution is the substitution of one or more of leucine, glutamic acid, and alanine.

[0009] Further, leucine is substituted with isoleucine.

[0010] Further, glutamic acid is substituted with valine and / or aspartic acid.

[0011] Further, alanine is substituted with aspartic acid.

[0012] Further, the sequence of the polypeptide is as shown in any one of SEQ ID NO: 1-5.

[0013] The second aspect of the present invention provides a polypeptide condensate, which comprises the polypeptide described in the first aspect of the present invention.

[0014] The third aspect of the present invention provides a polynucleotide, which encodes the polypeptide described in the first aspect of the present invention or the polypeptide condensate described in the second aspect of the present invention.

[0015] The fourth aspect of the present invention provides a vector, which contains the polynucleotide described in the third aspect of the present invention.

[0016] In some embodiments, examples of the vector include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, polyomaviruses (such as SV40), λ phage, M13 phage, plasmids. Specific examples of the vector include, but are not limited to, pcDNA3.3, pMD18-T, pOptivec, pCMV, pEGFP, pIRES, pQD-Hyg-GSeu, pALTER, pBAD, pcDNA, pCal, pL, pET, pGEMEX, pGEX, pCI, pEGFT, pSV2, pFUSE, pVITRO, pVIVO, pMAL, pMONO, pSELECT, pUNO, pDUO, Psg5L, pBABE, pWPXL, pBI, p15TV-L, pPro18, pTD, pRS10, pLexA, pACT2.2, pCMV-SCRIPT.RTM., pCDM8, pCDNA1.1 / amp, pcDNA3.1, pRc / RSV, PCR2.1, pEF-1, pFB, pSG5, pXT1, pCDEF3, pSVSPORT, pEF-Bos.

[0017] The fifth aspect of the present invention provides a host cell, which comprises the polynucleotide described in the third aspect of the present invention or the vector described in the fourth aspect of the present invention.

[0018] In some embodiments, the host cell is a cell for receiving, maintaining, replicating, and amplifying the vector. The host cell can also be used to express the polypeptide encoded by the vector. When the host cell divides, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid. In one embodiment, the host cell is a genetic package that can induce the expression of variant polypeptides on its surface. In another embodiment, the host cell is infected with the genetic package.

[0019] In some embodiments, the host cell can actually be any cell available for the expression vector, including prokaryotic cells and eukaryotic cells. The prokaryotic cells include but are not limited to eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae, such as Escherichia, for example, Escherichia coli (DH5α, BL21DE3, BL21DE3pLysS, JM109, TOP10, HB101, SCS110, E. coli JM110); Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella, for example, Salmonella typhimurium; Serratia, for example, Serratia marcescens; and Shigella, as well as Bacilli, such as Bacillus subtilis and Bacillus licheniformis; Pseudomonas, such as Pseudomonas aeruginosa; and Streptomyces.

[0020] Eukaryotic cells include but are not limited to protist cells, animal cells or fungal cells. The animal cells include mammalian cells, avian cells and insect cells. Among them, mammalian cells include but are not limited to CHO cells, F2N cells, CSO cells, BHK cells, Bowes melanoma cells, HeLa cells, 911 cells, AT1080 cells, A549 cells, 293T cells, 293F cells.

[0021] The sixth aspect of the present invention provides the use of the polypeptide described in the first aspect of the present invention, the polypeptide condensate described in the second aspect of the present invention, the polynucleotide described in the third aspect of the present invention, the vector described in the fourth aspect of the present invention, and the host cell described in the fifth aspect of the present invention in liquid-liquid phase separation, enrichment or in the preparation of products for regulating liquid-liquid phase separation and enrichment.

[0022] The seventh aspect of the present invention provides the use of the polypeptide described in the first aspect of the present invention, the polypeptide condensate described in the second aspect of the present invention, the polynucleotide described in the third aspect of the present invention, the vector described in the fourth aspect of the present invention, and the host cell described in the fifth aspect of the present invention in regulating the compartmentalization of guest molecules or in the preparation of products for regulating the compartmentalization of guest molecules.

[0023] The eighth aspect of the present invention provides the application of the polypeptide described in the first aspect of the present invention, the polypeptide condensate described in the second aspect of the present invention, the polynucleotide described in the third aspect of the present invention, the vector described in the fourth aspect of the present invention, and the host cell described in the fifth aspect of the present invention in aggregation-induced luminescence or in the preparation of products with aggregation-induced luminescence.

[0024] Furthermore, the product includes an aggregation-induced luminescence probe.

[0025] The ninth aspect of the present invention provides the application of the polypeptide described in the first aspect of the present invention, the polypeptide condensate described in the second aspect of the present invention, the polynucleotide described in the third aspect of the present invention, the vector described in the fourth aspect of the present invention, and the host cell described in the fifth aspect of the present invention in promoting enzymatic reactions or in the preparation of products for promoting enzymatic reactions.

[0026] The tenth aspect of the present invention provides any one of the following methods:

[0027] (1) A method for regulating liquid-liquid phase separation and enrichment, the method comprising using the polypeptide described in the first aspect of the present invention, the polypeptide condensate described in the second aspect of the present invention, the polynucleotide described in the third aspect of the present invention, the vector described in the fourth aspect of the present invention, and the host cell described in the fifth aspect of the present invention;

[0028] (2) A method for regulating the compartmentalization of guest molecules, the method comprising using the polypeptide described in the first aspect of the present invention, the polypeptide condensate described in the second aspect of the present invention, the polynucleotide described in the third aspect of the present invention, the vector described in the fourth aspect of the present invention, and the host cell described in the fifth aspect of the present invention;

[0029] (3) A method for promoting aggregation-induced luminescence, the method comprising using the polypeptide described in the first aspect of the present invention, the polypeptide condensate described in the second aspect of the present invention, the polynucleotide described in the third aspect of the present invention, the vector described in the fourth aspect of the present invention, and the host cell described in the fifth aspect of the present invention;

[0030] (4) A method for promoting enzymatic reactions, the method comprising using the polypeptide described in the first aspect of the present invention, the polypeptide condensate described in the second aspect of the present invention, the polynucleotide described in the third aspect of the present invention, the vector described in the fourth aspect of the present invention, and the host cell described in the fifth aspect of the present invention.

[0031] Advantages and beneficial effects of the present invention:

[0032] Surprisingly, a polypeptide TP showing anomalous phase transition kinetics was discovered in this application. The liquid-liquid phase separation properties and molecular mechanisms induced by TP dilution were studied. It was found that liquid-liquid phase separation could be induced by diluting a homogeneous TP solution, thereby achieving the compartmentalization of biomacromolecules. This discovery overturned the traditional understanding of liquid-liquid phase separation. This application also verified that the compartmentalization of guest molecules, the regulation of the optical output of guests, the promotion of the formation of secondary liquid-liquid phase separation in condensates, and the optimization of enzymatic reactions could be achieved by using the condensation effect induced by TP dilution. It not only demonstrated the practicality of TP dilution-induced condensates but also provided a new regulatory strategy for the self-assembly and functional regulation of biomacromolecules, with broad application prospects. Description of the Drawings

[0033] Figure 1 It is a diagram of the phase separation effect induced by TP solution dilution. Among them, 1A is a diagram of TP solutions with different concentrations during the dilution process in a system of 20 mM Tricine, pH = 7.4. 1B is a bar chart of the static light scattering intensity corresponding to each concentration in 1A (N = 5). 1C is a diagram of the OD550 signal intensity detected by a microplate reader when TP alternates between two concentrations of 1 mM and 0.75 mM.

[0034] Figure 2 It is a micrograph of the morphology of TP condensates under differential interference contrast microscopy;

[0035] Figure 3 It is a concentration-correlation phase diagram of the phase separation of Tricine and TP;

[0036] Figure 4 It is a concentration-correlation phase diagram of the phase separation of Tris and TP;

[0037] Figure 5 It is a concentration-correlation phase diagram of the phase separation of TEA and TP;

[0038] Figure 6 It is a concentration-correlation phase diagram of the phase separation of phosphate and TP;

[0039] Figure 7 It is a micrograph sequence showing that TP condensates are prone to wet the glass interface;

[0040] Figure 8 It is a diagram showing the correlation between buffer salt concentration and condensate size revealed by dynamic light scattering;

[0041] Figure 9 It is a fitting diagram of the fusion kinetics curve of TP condensates. Among them, 9A is an example micrograph of the fusion process of TP condensates. 9B is a scatter plot of the correlation between eccentricity and time based on morphological measurements during the fusion process. 9C is a function fitting diagram of the correlation between the relaxation time and the final radius of TP condensate fusion;

[0042] Figure 10 It is a graph for detecting the molecular exchange kinetics by fluorescence recovery after photobleaching experiment;

[0043] Figure 11 It is a graph showing the full Raman spectra inside and outside the TP condensate;

[0044] Figure 12 It is a graph for constructing a standard curve of phenylalanine concentration - signal intensity to calculate the polypeptide concentration inside the TP condensate;

[0045] Figure 13 It is a graph for comparing the positions of key concentration points in the phase diagram during the TP phase separation event;

[0046] Figure 14 It is a graph showing the deconvolution of the secondary structure in the amide I region of the TP condensate;

[0047] Figure 15 It is a graph for comparing the dilution - induced LLPS model with the classical LLPS model;

[0048] Figure 16 It is a graph showing the structural prediction of the TP monomer by AlphaFold3;

[0049] Figure 17 It is a graph showing the disorder analysis of the TP sequence by the IUPred3 website;

[0050] Figure 18 It is a graph showing the prion - like feature analysis of the TP sequence by the PLAAC website;

[0051] Figure 19 It is a graph for analyzing the structure of the TP monomer in the high - concentration homogeneous phase by nuclear magnetic resonance spectroscopy;

[0052] Figure 20 It is a graph showing the conformational transition of TP during dilution revealed by circular dichroism spectroscopy;

[0053] Figure 21 It is a graph showing that the addition of trifluoroethanol can promote the formation of TP condensates in the phase diagram;

[0054] Figure 22 It is a predicted structure of the TP - 3A mutant in AlphaFold3;

[0055] Figure 23 It is a test graph for the dilution - induced phase separation effect of the TP - 3A mutant;

[0056] Figure 24 It is a test graph for the dilution - induced phase separation effect of the TP - 2E mutant;

[0057] Figure 25 It is a graph showing the effect of 1,6 - hexanediol on TP condensates;

[0058] Figure 26 It is a test chart of the dilution-induced phase separation effect of the TP-2A mutant;

[0059] Figure 27 It is a display chart of the research on the correlation between the TP phase separation ability and the pH value;

[0060] Figure 28 It is a summary chart of the concentration correlation phase diagrams of various salts and TP phase separation;

[0061] Figure 29 It is the concentration correlation phase diagram of sodium chloride and TP phase separation under the condition of 10 mM Tirs;

[0062] Figure 30 It is the concentration correlation phase diagram of magnesium chloride and TP phase separation under the condition of 10 mM Tirs;

[0063] Figure 31 It is the concentration correlation phase diagram of sodium sulfate and TP phase separation under the condition of 10 mM Tirs;

[0064] Figure 32 It is a display chart of the mutation positions of the TP homologous peptide;

[0065] Figure 33 It is a comparison chart of the dilution-induced liquid-liquid phase separation ability of the TP homologous peptide;

[0066] Figure 34 It is a display chart of the dilution-induced liquid-liquid phase separation energy funnel of TP;

[0067] Figure 35 It is a chart of recruiting guest molecules by using the dilution-induced enrichment effect of TP;

[0068] Figure 36 It is a structural formula of a fluorescent small molecule and a fluorescence microscope image after enrichment;

[0069] Figure 37 It is a display chart of the guest polypeptide sequence and the corresponding isoelectric point for testing the dilution-induced enrichment ability of TP;

[0070] Figure 38 It is a confocal imaging display and fluorescence distribution analysis chart of the guest polypeptide enriched in the TP condensate;

[0071] Figure 39 It is a bar chart of the partition coefficient of the guest polypeptide and the small molecule;

[0072] Figure 40 It is a trend chart showing that increasing the RNA concentration can promote the TP phase separation;

[0073] Figure 41The condensates formed by TP and RNA with different concentrations of buffer salts have different spatial distribution diagrams;

[0074] Figure 42 It is the pattern diagram of the aggregation-induced emission effect and the display diagram of the dilution-induced emission effect of TP;

[0075] Figure 43 It is the pattern diagram of the formation of multi-compartmentalized distribution of eGFP-Gal3 inside the TP condensate by dilution induction;

[0076] Figure 44 It is the concentration threshold diagram of the phase separation formed by eGFP-Gal3 itself;

[0077] Figure 45 It is the confocal imaging diagram of the formation of multi-compartmentalized distribution of eGFP-Gal3 inside the TP condensate by dilution induction;

[0078] Figure 46 It is the diagram of the fusion event of the eGFP-Gal3 condensate inside the TP condensate;

[0079] Figure 47 It is the 3D reconstruction and fluorescence distribution display diagram of the multi-level phase separation system;

[0080] Figure 48 It is the fluorescence bleaching display diagram of the secondary compartments formed by eGFP-Gal3 inside the TP condensate;

[0081] Figure 49 It is the schematic diagram of dilution-induced TP condensate acting as a microreactor to accelerate the enzymatic reaction;

[0082] Figure 50 It is the diagram of the co-localization of His-Gal-10 and TEV in the condensate shown by DIC and confocal fluorescence microscopy;

[0083] Figure 51 It is the polarized light microscope photo revealing that dilution-induced enrichment enhances the crystal production rate. Detailed implementation mode

[0084] The following provides definitions of some terms used in this specification. Unless otherwise specified, all technical and scientific terms used herein generally have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains.

[0085] The following further elaborates on this invention in combination with specific embodiments. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit this invention. Without departing from the scope of this invention, the main features of this invention can be used in various embodiments.

[0086] Embodiment

[0087] 1 Experimental Materials and Methods

[0088] 1.1 Reagent Materials

[0089] The polypeptides were all purchased from Anhui Guotai Pharmaceutical Co., Ltd. Through high performance liquid chromatography and mass spectrometry analysis, the sample purity exceeded 98%. The suppliers of other experimental related reagents (analytical pure) are as follows: Tris, Tricine, phosphate, and TEA were purchased from Sinopharm Group, 1,6 - hexanediol, guanidine hydrochloride, and hexafluoroisopropanol were purchased from Macklin, and DAPI, ThT, and bovine serum albumin (BSA) were purchased from Solarbio. Total yeast RNA was purchased from Beyotime. The experimental water used was ultrapure water treated by Milli - Q.

[0090] 1.2 Concentration Measurement and Treatment

[0091] 1.21 For polypeptides and proteins containing tryptophan and tyrosine

[0092] First, dissolve the freeze - dried powder of the polypeptide used in the experiment in ultrapure water to prepare a high - concentration stock solution. Take a part and dilute it 1:9 in 6M liquid guanidine hydrochloride. Take 2 μl and use a NanoDrop ultraviolet - visible (UV - Vis) spectrophotometer (ThermoFisher) to measure the molar concentration of the polypeptide. The concentration measurement is based on the NanoDrop user manual. According to the Beer - Lambert equation, the absorbance (A) of the polypeptide at a wavelength of 280 nm is related to the molar extinction coefficient (ε) of the polypeptide, the optical path length (L), and the peptide concentration (c), that is:

[0093] A = εLc (1)

[0094] The calculation of the molar extinction coefficient of the polypeptide is based on the sum of the molar extinction coefficients of tryptophan and tyrosine in its sequence at 280 nm:

[0095] ε=(nW * 5500)+(nY * 1490) (2)

[0096] The values of the molar extinction coefficients of tryptophan and tyrosine are adopted as recommended in the NanoDrop user manual.

[0097] 1.22 For polypeptides labeled with fluorescent probes

[0098] For TMR - labeled TP, the concentration of TMR - TP is measured by the absorption of the TMR fluorescent molecule at 555 nm, and the TMR extinction coefficient is ε = 90,000 M -1 cm -1 .

[0099] For FAM-labeled polypeptides, the concentration of the labeled polypeptides was measured by the absorption of the FAM fluorescent molecule at 493 nm, and the FAM extinction coefficient was ε = 83,000 M -1 cm -1 .

[0100] 1.23 Nucleic acid samples

[0101] For the quantification of total yeast RNA used in the experiment, NanoDrop was used for measurement under the RNA detection module.

[0102] 1.3 Induced liquid-liquid phase separation process

[0103] Polypeptides related to liquid-liquid phase separation were stored in a stock solution dissolved in pure water at a high concentration and their concentration was detected. They were diluted in the target buffer solvent system according to experimental needs. For the system of diluting polypeptides to induce liquid-liquid phase separation, the polypeptides were first diluted in the target buffer at a higher concentration to maintain a stable single-phase solution, and then further diluted with the target buffer to achieve phase separation. For experiments requiring fluorescence imaging, 1 mol% of TMR-TP was added to the system to characterize TP.

[0104] 1.4 Liquid-liquid phase diagram drawing

[0105] To draw the phase diagram of the correlation between polypeptide concentration and salt concentration, the polypeptide solution was added to a 48-well crystal culture plate (BioJane), and the experiment was carried out at room temperature (25 °C). The well plate used was pretreated with 2 mg / mL BSA for 30 minutes and then rinsed three times with deionized water to make its surface insensitive to protein attachment, enhance the resistance to condensate wetting, and facilitate observation under the microscope. To prevent evaporation, buffer was added to the reservoir of the well plate and the wells were covered with a transparent sealing film. Images were taken at each time point using an upright microscope (Nikon E200).

[0106] 1.5 Light scattering measurement

[0107] Static light scattering (SLS) measurement was carried out at room temperature (25 °C) using a Zetasizer Nano ZS90 (Malvern). A sample cell irradiated with a 4 mW, 633 nm laser was used, and the scattering angle was set to 90°. Detection started after each sample was equilibrated for 30 seconds, and at least three measurements were made. The data was processed using Zetasizer software (Malvern).

[0108] For dynamic light scattering (DLS) measurements, the sample preparation is the same as that for SLS measurements. Since the hydrated particle size of the sample is greater than 1 μm under certain conditions, the correlation between the scattered light intensity and the particle diffusion behavior decays due to factors such as gravity, reducing the accuracy of the algorithm for fitting the particle size. Therefore, the autocorrelation functions (ACFs) actually detected in the experiment are used to determine the particle size. The longer the decay time, the larger the particle size.

[0109] 1.6 Detection of the fusion of liquid-liquid phase separation condensates

[0110] The 8-well chamber coverslip (Cellvis) used to observe the condensates was pre-incubated with 2 mg / ml BSA for 30 min and rinsed 3 times with deionized water. After the sample was equilibrated for 5 min, it was placed on a DMI8 inverted microscope (Leica) and observed under a 63× oil immersion objective. To facilitate the observation of more large-sized condensate fusion events before the condensates were sedimented by gravity and wetted the coverslip, 200 mM Tricine was used as the buffer system and the final concentration of the polypeptide was 200 μM. The focal length was aligned with the condensates near the bottom surface of the slide, and continuous shooting was performed for 10 min, with each frame taken at 0.5 s. Data processing was measured using LAS X (Leica) to obtain the major axis L max and minor axis L min values during the fusion process of two condensates. For the eccentricity A at each time point in each fusion event, A = (L max – L min ) / (L max + L min ), and it was normalized. The relaxation time was obtained by fitting using the One phase decay method. The final size of the condensate in the measured fusion event was plotted against its relaxation time as a scatter plot and fitted. The slope was the inverse capillary rate, which could reflect the ratio of the condensate viscosity to the surface tension (η / γ).

[0111] 1.7 Raman spectroscopy acquisition

[0112] Raman spectra were acquired using an Alpha300 R confocal Raman microscope (WITec), and the excitation light parameters for the sample were 532 nm and 60 mW. Raman data were processed using Suite SIX (WITec) and Origin software.

[0113] The method for measuring the polypeptide concentration inside the condensates using Raman spectroscopy refers to previous literature studies. Briefly, the characteristic absorption peak of the phenylalanine side chain at 1005 cm -1 was used as a reference. First, the intensities of the characteristic absorption peaks of free phenylalanine at 1005 cm -1 at different concentrations were measured to make a standard curve. The characteristic absorption peak of the sample condensate at 1005 cm -1The Raman peak intensity at , combined with the standard curve, can be used to calculate the concentration of phenylalanine in the aggregates, and then the concentration of the peptide can be converted. The effectiveness of this method has been verified by BSA protein solutions of different concentrations.

[0114] Raman spectroscopy was used to analyze the secondary structure of the aggregates with reference to the wavelength at 1600 cm -1 -1700cm -1 Amide I region. Suite SIX software was used for baseline subtraction, and origin was used for secondary structure peak separation and fitting. The peak position was fixed at 1615 cm -1 (phenylalanine and tyrosine signals), 1657 cm -1 (α-helix),1670cm -1 (β-sheet),1681cm -1 (disorder).

[0115] 1.8 Circular dichroism spectrum

[0116] A circular dichroism spectroscopy system (Jasco J-1500) was used to characterize the secondary structure of the peptide in solution. The data were collected at room temperature at 25°C, and the light path of the quartz cuvette used was 0.1 cm. The peptide was dissolved in a 20 mM Tricine solution with a pH of 7.4. The spectrum was scanned at a speed of 100 nm / min, with a digital integration time of 1 second and a bandwidth of 2 nm. The spectrum was scanned at least 2 times in the range of 260 nm to 190 nm with a step size of 1 nm. The data were processed using the instrument's own Spectra Manager software and plotted using origin. Ellipticity (θ, deg.cm 2 .dmol -1 ) was used to normalize the signal of the samples based on their concentration.

[0117] 1.9 pH-dependent detection

[0118] Britton–Robinson buffer with a pH range of 2 to 12 was used as a buffer solvent. The buffer was made by mixing 0.04M acetic acid, 0.04M phosphoric acid, and 0.04M boric acid. The pH value was adjusted with 0.2M sodium hydroxide to obtain buffers at different pH values. The buffer was mixed with TP at a volume ratio of 19:1 to make the final concentration of TP 200μM and placed in a 384 flat-bottomed glass plate (Cellvis), 20μL per well. After incubation at room temperature for 30 minutes, each group of photos was taken under a DMI8 inverted microscope, and the absorbance of each group at 550nm was detected using a SynergyH1 microplate reader (Biotek) to evaluate the degree of phase separation.

[0119] 1.10 Fluorescence photobleaching recovery experiment

[0120] For the fluorescence recovery after photobleaching (FRAP) experiment, a Leica Stellaris 5 Confocal Microscope (Leica) equipped with a 63× oil immersion objective and the FRAP module in the Las X (Leica) software were used. Under 200 mM Tricine buffer solution, 5 μL of a 200 μM TP phase separation sample containing 1 mol% of TMR-TP was placed on a BSA-pretreated 8-well chamber coverslip and equilibrated for 5 min. The confocal excitation light wavelength was 561 nm, and the emission light reception range was 570 - 620 nm. Polypeptide condensates with a size of approximately 5 μm near the coverslip surface were searched for FRAP. Three photos were taken as controls before photobleaching. According to the requirements of full-, partial-, and half-FRAP, the photobleaching regions were set as the entire condensate, a 1-μm diameter circular region at the center of the condensate, and a semi-circular region half of the condensate, respectively. The photobleaching region was photobleached at 100% power for 560 ms, and then photos were taken at a rate of one photo every 280 ms for 1 min. In addition, a non-fluorescent region was set as the background, and an unbleached condensate near the photobleached condensate was set as a reference to ensure that the laser used for imaging did not cause additional fluorescence quenching. For full- and partial-FRAP, the region of interest (ROI) for observing the fluorescence signal was the photobleached region. For half-FRAP, the change in the fluorescence intensity of the unbleached half of the condensate also needed to be recorded. The processing of the fluorescence recovery curve was as reported in previous literature. Briefly, it included background subtraction, normalization with the pre-photobleaching fluorescence of the reference ROI, image plotting, and fitting using the One phase decay method to obtain the half-recovery time. At least 4 replicates of the condensate were used.

[0121] 1.11 Guest molecule recruitment experiment

[0122] The recruitment of fluorescent small molecules, polypeptides, and proteins by TP condensates was achieved using the dilution-induced phase separation effect. Briefly, the guest molecule was first mixed with a high concentration of TP in a buffer, and at this time, it was still a homogeneous solution. On this basis, the system was diluted with the buffer to reduce the TP concentration and achieve phase separation.

[0123] The final concentration of DAPI was 0.5 μM, and the excitation light used was 360 nm. The final concentration of ThT was 0.5 μM, and the excitation light used was 450 nm. The final concentration of Yeast RNA (Solarbio) was 200 ng / ml, and SYBR Green II (Thermo Fisher) was used for specific fluorescence imaging tracking, with the excitation light at 488 nm. The final concentration of the FAM-labeled polypeptide was 1 μM, and the excitation light used was 488 nm. The EGFP fusion-expressed protein used an excitation light of 488 nm.

[0124] 1.12 Protein expression and labeling treatment

[0125] The recombinant expression of the protein refers to the previously reported literature. It is obtained through steps such as plasmid transformation, antibiotic screening, expansion culture, sonication, nickel column purification, treatment with an AKTA pure 25 protein purification system (chromatography column: Superdex 75), and protein concentration. Since it only serves as the recruited guest macromolecule in this work, the specific steps are not elaborated.

[0126] The fluorescence labeling of the recombinant protein uses a protein labeling kit (Alexa FluorTM 633 and Alexa FluorTM 488, Thermo Fisher).

[0127] 1.13 Nuclear magnetic resonance spectroscopy

[0128] The 2 mM TP solution is dissolved in 20 mM Tricine buffer (pH 7.4, 10% D2O), and 2,2-dimethyl-2-silapentane-5-sulfonic acid (DSS) is used as the internal standard chemical shift reference. The NMR experiment is carried out at 25 °C using a Bruker Avance 700 MHz nuclear magnetic resonance spectrometer, which is equipped with four radiofrequency channels and a triple resonance cryoprobe with pulsed field gradients. The standard curve is plotted using myoglobin (17.1 kDa), ovalbumin (43.1 kDa), cytochrome C (12.3 kDa), and carbonic anhydrase (29.0 kDa), and the experimental measurements refer to the previously published articles. The DOSY experiment uses the pulse sequence stebpgp1s19, which determines the diffusion coefficient (Dt) by the stimulated echo method. The water peak suppression is achieved by applying a 3-9-19 pulse sequence. The experimental parameters (including the diffusion time and the pulsed gradient field duration) are optimized, and the results are analyzed and fitted using the T1 / T2 module of Bruker TopSpin 3.2 software.

[0129] The chemical shift assignments of the backbone and side-chain atoms are obtained through two-dimensional HCCH-TOCSY (80 ms mixing time) and 1H-1H NOESY (250 ms mixing time) experiments. The 1H-1H NOESY experimental spectrum is used to confirm the NOE effect. The NMR spectrum is processed using NMRPipe and analyzed using NMRView. The parameters for the LBDB structure calculation include the proton distance constraints from nuclear NOE and the dihedral angles calculated from the chemical shifts by TALOS ( ψ). The initial structure was generated using the CANDID module of CYANA. Twenty structures with the lowest energies were selected as models, and the NOE assignments were extended through the SANE program. Further, 200 structures were calculated using CYANA, and the 100 structures with the lowest energies were further refined using AMBER. Finally, 20 conformations with the lowest energies were selected as representative structures and analyzed using MOLMOL and PROCHECK_NMR.

[0130] 2. Experimental Results

[0131] 2.1 Study on the Properties of Dilution-Induced Liquid-Liquid Phase Separation

[0132] 2.11 TP Exhibits Abnormal Dilution-Induced Phase Separation

[0133] In previous experimental studies, this application unexpectedly discovered a polypeptide TP (SYYRPREEEAIPHPLALTHKMGWLQLLGRMF, SEQ ID NO: 1) that exhibits abnormal phase transition kinetics. As Figure 1 shown in A, in a 20 mM Tricine buffer system at pH 7.4, when the concentration of TP is higher than 1 mM, the solution remains clear; but when diluted below the critical concentration (<1 mM), the solution immediately undergoes phase separation, manifested as a significant increase in turbidity.

[0134] Static light scattering experimental data ( Figure 1 B) further verified this phenomenon: when the concentration of TP is 2 mM, the scattering signal intensity is only about 42 kcps, indicating that there are no large-sized molecular assemblies in the solution. Different from common dispersion systems, the scattering signal of TP gradually increases with dilution, reaching ~86 kcps at 1.25 mM; reaching ~318 kcps at the critical 1 mM; when the concentration drops to 0.75 mM, the solution becomes turbid, and the signal intensity surges to ~2470 kcps, indicating the generation of a large number of large-sized assemblies; when it drops to 0.1 mM, it is the maximum signal value of ~6076 kcps, and then the signal decreases with dilution.

[0135] This abnormal phase transition effect is reversible. In the absorbance detection based on the concentration gradient change, the methods of "buffer dilution" and "adding high-concentration TP" were used to make the solution cycle between 1 mM and 0.75 mM, and it was confirmed by the absorbance detection at 550 nm ( Figure 1 C): The phase separation system can be completely redissolved and restored to a clear state when the concentration rises above the critical value, showing an instant response characteristic and complete reversibility.

[0136] Differential interference contrast microscopy (DIC) observations provided direct evidence for this abnormal phenomenon ( Figure 2)。At a TP concentration of 0.5 mM, a large number of spherical droplet-like condensates can be seen in the field of view; while when the concentration rises above the critical value, the condensates completely disappear. This "low-concentration condensation - high-concentration dissolution" phase transition behavior is in sharp contrast to the concentration-dependent phase separation law (low-concentration dispersion, high-concentration aggregation) exhibited by conventional biomacromolecule systems. Through systematic literature research, such inverse concentration phase transition phenomena of single-component have not been recorded in the reported biomacromolecule solution systems. This discovery provides a new perspective for understanding the phase transition regulation mechanism of biomacromolecules.

[0137] 2.12 Construction of the phase diagram of TP dilution-induced phase separation

[0138] Phase diagram analysis is a key means to analyze phase separation behavior. To systematically study the influence of buffer systems on the phase transition of TP, four buffer salts with buffer capacity meeting the pH 7.4 condition were selected: N-tris(hydroxymethyl)methylglycine [N-(Tris(hydroxymethyl)methyl)glycine, Tricine], tris(hydroxymethyl)aminomethane [tris(hydroxymethyl)aminomethane, Tris], triethanolamine [Triethanolamine, TEA], phosphate, and the concentration-phase state correlation phase diagram of them and TP was constructed ( Figures 3 - 6 ). The experimental results show that: First, the phenomenon of dilution-induced phase separation is universal under different buffer systems and different buffer salt concentration conditions, confirming the wide existence of this abnormal phase transition behavior - when the buffer salt concentration is fixed and the TP concentration is gradually increased, the phase states of "homogeneous - phase separation - homogeneous" can be seen to occur in sequence; Second, when the TP concentration is fixed, increasing the buffer salt concentration (such as Tricine increasing from 20 mM to 50 mM) will shift the right critical concentration of phase separation to the right (from 1 mM to 2 mM), that is, the higher the buffer salt concentration, the more favorable it is to promote the phase separation of TP at high concentrations.

[0139] It is worth noting that metastable conditions were generally detected in the boundary regions of the phase diagrams of all buffer systems ( Figures 3 - 6Blue data points). Under these conditions, the condensates formed by diluting the sample disappeared spontaneously within 10 minutes of standing, while slightly further dilution could re-induce phase separation. This kinetic behavior is in an inverted relationship with the classical liquid-liquid phase separation (LLPS) theory and previous related experimental reports: there is a metastable system between the binodal and spinodal, which is characterized by an initial homogeneous phase and will spontaneously phase separate to reach the lowest free energy when activated or left standing for a period of time. However, the observation results of this study show that in the TP system, the metastable state is manifested as the condensates instead relax reversely to return to the homogeneous phase, suggesting that there may be a special energy barrier distribution in this system - under specific boundary conditions, the homogeneous phase is the thermodynamically more stable final state.

[0140] 2.13 Characterization of the liquid-like properties of TP condensates

[0141] Microscopic observations show that TP condensates generally exhibit a spherical droplet morphology ( Figure 2 ), and are prone to wetting the coverslip substrate ( Figure 7 ), suggesting that they may have liquid characteristics. To systematically analyze their physical properties, this study revealed the following key properties through multi-scale physical property characterization:

[0142] (1) Size regulation mechanism. Based on the positive correlation between the buffer salt concentration and the phase separation trend in the phase diagram study ( Figures 3 - 6 ), dynamic light scattering (DLS) was used to quantitatively study the particle size distribution law. Given the limitations of the traditional Stokes-Einstein model for particles >1 μm (gravity sedimentation interferes with Brownian motion), the relaxation time τ of the autocorrelation function (ACF) was used to indirectly compare the relative particle size. As Figure 8 shown, in the 20 mM Tricine system, the equivalent particle size corresponding to the τ value is <1 μm, while it increases to ~3 μm at 200 mM, confirming that the buffer salt concentration promotes the formation of larger condensates; in addition, when the buffer salt concentration is fixed (taking Tricine and PBS as examples), the change in the TP concentration (0.2 - 0.5 mM) has no significant effect on the τ value, indicating that the condensate size is mainly regulated by the buffer salt concentration. Since large condensates are beneficial to the accuracy and convenience of microscopic observation experiments, a 200 mM Tricine buffer system was selected for the characterization of this part of the physical properties to obtain condensates suitable for observation.

[0143] (2) Fusion kinetics and rheological properties. The fusion event of condensates is the gold standard for judging whether they have liquid properties. By tracking the kinetic process of two adjacent condensates with similar sizes from contact to complete spheroidization ( Figure 9 A - B), a quantitative relationship between the fusion time (t) and the final radius (R) was established (Figure 9 C). The fitted slope reflects the inverse capillary rate η / γ (viscosity / surface tension) of the system. The η / γ of the TP condensates is 68.88 ± 1.84 s / μm, which is 1 - 2 orders of magnitude higher than that of classical liquid condensates such as Pgranules (2.00 s / μm) and GFP-HP1α (0.35 s / μm), revealing its extremely high viscoelasticity.

[0144] (3) Molecular exchange kinetics. Fluorescence recovery after photobleaching (FRAP) experiments can further reveal the intramolecular flow ability of condensates and were performed on a 4 mol% TMR-TP labeled system. Interestingly, partial photobleaching (Part-FRAP) experiments showed that the fluorescence relaxation time τ of the TP condensates was 2.45 ± 0.06 s, and the fluorescence intensity at the plateau recovered to 40% of that before bleaching ( Figure 10 A). This rapid recovery kinetics is significantly contradictory to the high viscoelasticity characterized in the fusion experiments. To analyze this phenomenon, full photobleaching (Full-FRAP) and half photobleaching (Half-FRAP) experiments were designed for further verification. As Figure 10 shown in B, C, both the full and half photobleaching experiments presented kinetic curves similar to those of the partial photobleaching experiments, with relaxation times of 2.75 ± 0.1 s and 2.48 ± 0.06 s respectively, and the recovery degrees were both approximately 40%. Among them, the results of the full photobleaching experiment indicated that the recovery of its fluorescence was entirely from molecular exchange with free TP in the external solution environment, and the proportion of this exchangeable component was approximately 40% of the condensates; for the half photobleaching experiment, since no significant decrease in fluorescence intensity (<5%) was detected in the unbleached region, it indicated that the internal flow of the condensates was restricted, and the fluorescence recovery in the quenched region was also mainly from molecular exchange with the outside.

[0145] Combining the fusion events and fluorescence recovery after photobleaching experiments, the TP condensates exhibited unique kinetic heterogeneity - the coexistence of surface tension-driven liquid-like macroscopic behaviors (such as spheroid fusion) and restricted molecular mobility. This property may stem from a continuous density gradient from the outside to the inside: the loose outer layer (contributing ~40% of the fluorescence signal) enables rapid exchange (τ ~ 2.5 s), while the high-density inner core restricts molecular movement.

[0146] 2.14 Characterization of the properties of TP condensates by Raman spectroscopy

[0147] In this study, high-resolution Raman confocal microscopy was used to perform in-situ detection of TP condensates. First, a Raman broad-spectrum scan in the range of 400–3800 cm -1 was performed on the TP condensates and their surrounding environmental solutions. As Figure 11As shown, there are significant differences in the spectral signal distribution inside and outside the condensate. Especially in the following regions, the signal differences are particularly significant:

[0148] 1002 cm -1 The peak at corresponds to the breathing peak of phenylalanine (Phe) and is related to the C-C stretching vibration of the benzene ring. This signal is not easily affected by the protein structure and is suitable for the quantification of protein concentration;

[0149] 1220 cm -1 –1400 cm -1 The region is the amide III region, which is mainly generated by the combined action of N-H bending vibration and C-N stretching vibration and can be used for the identification of secondary structures;

[0150] 1450 cm -1 The peak is usually related to the bending vibration modes of CH2 and CH3;

[0151] 1600 cm -1 to 1700 cm -1 The region is the amide I region, which is mainly related to the C=O stretching vibration and is suitable for the analysis of secondary structures;

[0152] 2800–3100 cm -1 The region is usually related to the C-H stretching vibration in fatty acid chains and alkyl chains;

[0153] 3100–3700 cm -1 The region is related to N-H and O-H stretching vibrations (hydrogen bonds in amino acids and proteins). Since the O-H stretching vibration signal contributed by water molecules is very strong, water molecules usually contribute the vast majority of the signals in aqueous solution samples.

[0154] By comparing the heights of the signal peaks related to protein characteristics, it can be roughly inferred that the interior of the TP condensate is an extremely concentrated and crowded environment. Further, the difference in the signal area in the 3100–3700 cm -1 region also supports this conclusion: the signal area in this region inside the condensate is less than 50% of that in the external solution environment. Considering that the polypeptides inside the condensate also contribute a part of the signal in this region, it means that the water molecule content inside the condensate is significantly lower than that outside. This phenomenon is significantly different from previous Raman spectroscopic studies of protein condensates, where the signal differences in this region inside and outside the condensate are usually small, revealing that the TP condensate has unique dehydration and densification characteristics.

[0155] Furthermore, by constructing a standard curve between the phenylalanine concentration and the signal intensity of the breathing peak, the quantification of the polypeptide concentration inside the condensate is achieved. First, by measuring phenylalanine solutions of 10 mM, 20 mM, and 36 mM, a standard curve (R 2= 0.999), and verified with 0.3 mM, 0.6 mM, and 1.2 mM BSA solutions to confirm that the phenylalanine signal can accurately reflect the protein concentration( Figure 12 A–C). Then, the signal intensity of the TP condensate (N = 4) at 1002 cm -1 was substituted into the standard curve for calculation. According to the number of phenylalanine residues in TP (n = 1), the polypeptide concentration inside the condensate was as high as 230 ± 30 mM( Figure 12 D), which is more than 20 times the concentration at the right boundary of phase separation under this detection condition (200 mM Tricine, CT concentration of 10 mM) Figure 13 ). This result indicates that the concentration CD of the heavy phase of TP is significantly higher than that of CT, and it is consistent with the extremely high viscoelasticity (η / γ = 68.88 ± 1.84 s / μm) exhibited in the fusion event, suggesting that the phase separation behavior of TP is significantly different from the classical LLPS model.

[0156] In addition, by analyzing the amide I region peak pattern from 1600 cm -1 to 1700 cm -1 , the secondary structure of the polypeptide inside the condensate can be further explored. Through Gaussian fitting of the peak positions at 1615 cm -1 (phenylalanine and tyrosine), 1657 cm -1 (α-helix), 1670 cm -1 (β-sheet), and 1681 cm -1 (random structure), the results show that the α-helix conformation dominates inside the condensate, accounting for approximately 76.5% of the conformational ratio, followed by the random structure, accounting for 18.7%( Figure 14 ). Different from the preference for random conformations of proteins in most condensates, the polypeptides inside the TP condensate tend to exist in the form of α-helices. This phase separation mode dominated by rigid secondary structures may originate from the orientational stacking effect of amphiphilic α-helix conformations in the TP sequence.

[0157] 2.15 Establishment of a Dilution-Induced Liquid-Liquid Phase Separation Model

[0158] Based on the above experimental results, although the phase separation behavior of TP conforms to the macroscopic characteristics of liquid-liquid phase separation (LLPS) (such as spherical shape and deformation dominated by surface tension), its concentration effect is essentially different from the classical LLPS model. Therefore, a phase transition kinetic model of TP is constructed based on the classical LLPS model, called "dilution induced Liquid-Liquid phase separation (DILLPS)".

[0159] As Figure 15As shown, different from the classical LLPS model, the concentration CT at the right boundary of phase separation in the DILLPS model is much lower than the concentration CD of the heavy phase. Beyond this concentration, the two-phase solution will return to the homogeneous phase. Since the polypeptide concentration span from CT to CD is too high to be experimentally observed (the current highest observation is that 30 mM TP is a clear homogeneous phase), there is no evidence to fully prove whether the TP concentration linearly increases in the 3→4 interval, or there is a secondary phase separation state dominated by the heavy phase and supplemented by the light phase. Therefore, it is represented by a dotted square area and finally enters the homogeneous state of the heavy phase.

[0160] 2.2 Research on the Molecular Mechanism of Dilution-Induced Liquid-Liquid Phase Separation

[0161] 2.21 Prediction of the Phase Separation Trend of TP

[0162] This application attempts to use existing computational prediction tools to evaluate the phase separation trend of TP and its key sites. First, AlphaFold3 is used to predict the structure of the TP monomer. The results show that TP has the characteristics of an N-terminal random coil and a C-terminal α-helix, and the overall confidence pIDDT>70% ( Figure 16 ). Since the conformation of biological macromolecules in solution often deviates from the predicted lowest energy state, this prediction can only be used as a reference to reveal the tendency differences of different regions in the sequence between forming stable secondary structures and disordered conformations.

[0163] Furthermore, protein phase separation prediction tools such as IUPred3 and PLAAC are used to analyze the TP sequence. The prediction results of each evaluation method are as follows ( Figure 17 ,18):

[0164] IUPred3 long disorder (long-term disorder)

[0165] Predicts longer disordered regions (>30 amino acids), suitable for identifying proteins with overall disorder or protein regions with long segment disorders, which are often related to LLPS. The prediction results of TP do not show fragments with a score>0.5, indicating a low tendency for LLPS.

[0166] IUPred3 short disorder (short-term disorder)

[0167] Predicts short disordered regions (<30 amino acids), suitable for identifying local disordered regions in overall ordered proteins, such as binding sites or flexible domains. The results show that there are certain flexible regions at the N-terminal and C-terminal of TP, while the middle region is relatively rigid.

[0168] ANCHOR2

[0169] Predict potential binding sites in disordered regions. A score > 0.5 indicates that the region is disordered in the free state but may undergo conformational changes (such as folding into an α - helix or β - sheet) when binding to a target. The TP prediction results show that the scores of the entire sequence are all < 0.5, indicating no obvious binding tendency and a low likelihood of LLPS.

[0170] IUPred3 structural domains

[0171] Identify ordered structural domains in proteins (i.e., regions that fold into stable structures). The TP full - sequence score < 0.5 indicates that it may be relatively stable in the monomer state and is less likely to directly drive LLPS.

[0172] PrD - like (Prion Domain - like Score)

[0173] Evaluate whether a protein has characteristics similar to a prion - like domain (PrLD). The higher the value (closer to 1), the more likely it is to form reversible aggregates, thus promoting LLPS. TP does not exhibit prion - like characteristics.

[0174] PLAAC Score

[0175] Based on the results of combining PrD - like and Hidden Markov Model (HMM) calculations, evaluate whether a protein may rely on PrLD for phase separation. A low PLAAC Score (< 0.5) indicates that TP may not rely on the prion - like mechanism and may involve other interactions (such as electrostatic, hydrophobic, π - π stacking) to drive phase separation.

[0176] 4*PAPA Score

[0177] Calculated by PAPA (Prion Aggregation Prediction Algorithm), evaluate the ability of a protein to form reversible aggregates. The higher the value, the stronger the aggregation tendency and the higher the correlation with LLPS. The N - terminus of TP shows a higher aggregation tendency, but the overall score is still lower than that of typical LLPS proteins.

[0178] Fold Index

[0179] Predict whether a protein is more likely to fold into a stable structure or remain disordered. A Fold index > 0 represents that the protein is more likely to fold into a stable three - dimensional conformation, while a Fold index < 0 represents a stronger disorder and a greater tendency to participate in LLPS. The TP prediction results are similar to AlphaFold3, with the C - terminus tending to form a stable structure and the N - terminus being more flexible.

[0180] In summary, TP has both a certain degree of order and disorder, but the computational prediction results do not support the strong LLPS trend of TP, whether based on IDR prediction or analysis of prion-like features. The C-terminal of TP shows higher rigidity, while the N-terminal is more flexible, and there may be potential binding sites. Therefore, its dilution-induced phase separation phenomenon may not completely depend on IDR or PrLD, does not conform to the common protein phase separation driving mechanism, but may involve more complex intermolecular interactions, environmental factors, and dynamic conformational changes.

[0181] 2.22 Nuclear magnetic resonance spectroscopy reveals the conformation of TP in high-concentration homogeneous phase

[0182] This application selects NMR technology to analyze the structure of the high-concentration homogeneous state of TP in order to obtain detailed information on the conformation of TP. In this study, 20 mM Tricine buffer was used, and the concentration of TP was adjusted to 2 mM to ensure that the sample was in a high-concentration homogeneous state, which was conducive to the accurate acquisition of NMR signals.

[0183] This application uses the Diffusion-Ordered Spectroscopy (DOSY) method. This technique infers the apparent molecular weight and conformational state of molecules by measuring their diffusion coefficients, thus providing reliable data support for analyzing the monomer structure of TP. As shown in Table 1, the apparent molecular weight corresponding to the diffusion coefficient of TP measured by DOSY is approximately 5.2 kDa, indicating that under the selected conditions, TP mainly exists in the monomer form. Further, through the analysis of one-dimensional proton nuclear magnetic resonance (^1H NMR) (Table 2), it was found that TP does not have a secondary structure in the homogeneous state and presents an overall compact and coiled globular conformation ( Figure 19 A, PDB ID: 9KVR). More detailed spectral analysis shows that two pairs of electrostatic interaction residue pairs of arginine (Arg)-glutamic acid (Glu) can be observed on the surface of the TP molecule ( Figure 19 B), while most of the hydrophobic side chains tend to concentrate inside the molecule ( Figure 19 C, blue annotation). This characteristic of internal hydrophobicity and surface electrostatic interaction is very likely an important factor in maintaining the compact conformation inside the molecule, preventing the aggregation of TP molecules, and thus inhibiting or reversing LLPS.

[0184] In summary, by using NMR and DOSY techniques, it is not only clear that TP mainly exists in the monomer form in the high-concentration homogeneous phase, but also reveals its unique conformational characteristics and intramolecular interaction patterns. This provides a solid experimental basis for further exploring the molecular mechanism of TP dilution-induced LLPS and lays the foundation for analyzing the details of key interactions and driving forces in subsequent studies.

[0185] Table 1 Display of Diffusion Ordered Spectroscopy Data of TP and Standard Proteins with Different Molecular Weights

[0186]

[0187]

[0188] Table 2 Statistical Data of NMR Structure Calculation of TP Samples

[0189]

[0190] 2.23 The Conformation of TP Transforms into α-Helix with Dilution of Concentration

[0191] To explore the conformational changes of TP at different concentrations and its relationship with phase separation behavior, circular dichroism (CD) spectroscopy was used to measure the conformational changes of TP during the process of gradually diluting from a high-concentration homogeneous phase to the occurrence of phase separation. A total of four concentration gradients were set. Among them, the solutions remained homogeneous at the first three concentrations, while at the lowest concentration (0.5 mM), it was in the LLPS state ( Figure 20 ). The results showed that at 2.0 mM, TP exhibited a weak α-helix trend, manifested as characteristic negative peaks at 208 nm and 222 nm; at the same time, no typical random coil signal (a negative peak at approximately 195 nm formed due to the exposure of the main chain to the solvent and the lack of a fixed hydrogen bond pattern) was observed, which was consistent with the NMR observation results - although TP lacked an obvious secondary structure, it did not belong to a completely loose random coil state; as the concentration decreased, the α-helix conformation signal in the solution gradually increased and reached a peak under the phase separation condition of 0.5 mM (it should be noted that during LLPS, due to the turbidity of the solution and the coexistence of two phases, laser scattering and signal occlusion may lead to partial signal loss). Combining the NMR structure data, the conclusion can be drawn that under high-concentration homogeneous conditions, TP mainly exists in a relatively compact, spherical conformation, and with dilution, the TP conformation gradually transforms into an extended α-helix state.

[0192] To further verify the correlation between the α-helix conformation and the phase separation of TP, a reagent that promotes the formation of α-helix, 2,2,2-Trifluoroethanol (TFE), was added to the solution. The experimental results showed that the addition of TFE significantly enhanced the phase separation tendency of TP: obvious condensates appeared under the induction of 5% TFE in the condition that was originally a high-concentration homogeneous phase ( Figure 21 ). This further proves the key role of the α-helix conformation transformation in the phase separation process of TP.

[0193] 2.24 Study on the Key Sequence Features Maintaining the Compact Coiled State of Monomers of TP at High Concentrations

[0194] Based on the aforementioned structural research results, it was found that TP tends to form a compact coiled monomer that is more conducive to intramolecular interactions under high-concentration conditions, while gradually transforming into an α-helical conformation at low concentrations, enhancing the potential for intermolecular interactions. This concentration-dependent allosteric response effect is undoubtedly achieved through a special sequence arrangement, enabling the two conformations of α-helix and compact coil to exhibit an "antagonistic tug-of-war" with concentration changes, resulting in this abnormal phase transition kinetic effect. This gives rise to three questions: (1) What sequence features are conducive to maintaining the compact coiled monomer state of TP at high concentrations? (2) What are the driving forces for condensate formation? What are the dynamic changes during the allosteric process? (3) Why does the dilution-induced allosteric effect exist? Next, the three questions will be studied and analyzed in subsections in turn.

[0195] Previous studies have pointed out that proline (Pro) usually has a disruptive effect on the formation of α-helices, and the TP sequence contains three Pro. Through nuclear magnetic resonance structure analysis, it can be observed that the positions of these three Pro are exactly accompanied by an obvious twist of the main chain ( Figure 19 C, marked in magenta), which suggests that they may play a key role in maintaining the compact conformation and intramolecular interactions of TP at high concentrations. To verify this hypothesis, the mutant TP-3A was designed and synthesized, in which all three Pro were replaced with alanine (Ala) that is conducive to α-helix formation. The structural prediction results of TP-3A in AlphaFold3 also support the possibility of being more prone to forming an α-helical conformation ( Figure 22 ).

[0196] In the DILLPS effect test, TP-3A exhibited significantly different phase transition kinetics from the prototype TP. Specifically, in 20 mM Tricine buffer, when the concentration exceeded 1 mM, the prototype TP presented a clear homogeneous state; while TP-3A remained turbid up to 6 mM, and the formation of large-sized assemblies could be immediately observed after mixing with the solvent ( Figure 23 A). In addition, in pure water, TP-3A initially showed a clear homogeneous solution, but the solution became turbid after standing at room temperature for 30 minutes, and large-sized assemblies were visible under an optical microscope ( Figure 23 B), indicating that it is thermodynamically more prone to intermolecular assembly. In contrast, the prototype TP does not undergo phase separation in pure water, and a certain concentration of buffer salt is a necessary condition for its phase separation. Generally speaking, TP-3A lost the DILLPS effect and showed a strong tendency for intermolecular aggregation.

[0197] In addition, according to nuclear magnetic resonance structure analysis ( Figure 19B), the two pairs of salt bridges on the surface of TP molecules may be related to the orientation of the main chain conformation, providing stability for maintaining its compact coiled conformation. To verify this speculation, the mutant TP-2E was further designed and synthesized, and the two Args were replaced by Glu. The purpose was to destroy the original electrostatic effect and to change the hydrophilicity and secondary structure tendency of the original residues as little as possible. In the DILLPS effect test, although TP-2E still retained the ability of dilution-induced phase separation, its critical concentration for reaching high-concentration homogeneous phase was significantly increased ( Figure 24 ): In 20mM Tricine buffer, the critical concentration was increased from 1mM of the prototype TP to 4mM. It is worth noting that the aggregates formed by TP-2E present a large and irregular assembly form, with only a few spherical droplets in the field of view, which further reflects that electrostatic interaction plays a key role in regulating the physical properties of TP aggregates.

[0198] In summary, these results indicate that the compact coiled conformation of TP monomers at high concentrations depends on a special sequence arrangement, in which proline plays a key role in maintaining the compact conformation by blocking the formation of α-helices, while the electrostatic interaction residues on the molecular surface help stabilize this conformation. The mutations of Pro→Ala and Arg→Glu significantly changed the phase behavior of TP in solution, providing a solid experimental basis for in-depth analysis of the molecular mechanism of TP dilution-induced LLPS.

[0199] 2.25 Study on the key driving factors of aggregate formation at low TP concentration

[0200] TP relies on a special sequence arrangement to maintain a compact coiled conformation in a high-concentration homogeneous phase. However, as the concentration is diluted, the conformation gradually changes to an α-helix. It is foreseeable that the conformational transition will be accompanied by the gradual exposure of key interaction sites that drive phase separation, causing the original interaction pattern within the molecule to be reorganized, thereby promoting intermolecular interactions and inducing the occurrence of LLPS. Therefore, clarifying which interactions provide the key driving force for the formation of TP aggregates and how these interactions work together is the basis for further understanding the TP dilution-induced phase separation effect.

[0201] Existing literature shows that electrostatic interaction, hydrophobic interaction, and π–π and cation–π interactions play a vital role in the self-assembly of peptides and proteins. Electrostatic attraction and repulsion can regulate the initial aggregation of molecules; while hydrophobic interaction promotes the formation of aggregates by bringing hydrophobic side chains closer together and reducing the free energy of the system; at the same time, π–π stacking and cation–π interactions provide additional stability for the multivalent interaction network, further strengthening the binding between molecules. On this basis, the study will conduct a systematic analysis from three aspects: hydrophobic interaction, π–π / cation–π interaction, and electrostatic interaction.

[0202] First, 1,6 - hexanediol (1,6 - HD) is a commonly used reagent for studying the influence of hydrophobic interactions in LLPS. Its hydrophobic chain segments can insert into the hydrophobic regions of proteins or polypeptides, thereby interfering with and weakening these hydrophobic interactions, leading to the dissolution or disappearance of droplets or condensates. However, it has less impact on LLPS systems that do not rely on hydrophobic interactions. Therefore, it is suitable for identifying whether hydrophobic interaction is the key driving factor for condensate formation. As Figure 25 shown, adding 5% 1,6 - HD can dissolve most of the TP condensates, and when the concentration is increased to 10%, all the condensates disappear in the field of view, indicating that hydrophobic interaction makes an important contribution to the formation of TP condensates. It should be noted that according to the nuclear magnetic resonance structure analysis ( Figure 19 C), in the high - concentration monomer state, most of the hydrophobic residues are located in the core of the globular - like conformation; therefore, the transition to the α - helix conformation will gradually expose these hydrophobic residues, thus transforming the hydrophobic effect that is mainly intra - molecular interaction into a mode that is more conducive to intermolecular interaction.

[0203] Second, in terms of π–π stacking and cation–π interactions, TP contains four aromatic amino acids with potential interaction capabilities. According to the nuclear magnetic resonance conformation ( Figure 19 C) in the high - concentration monomer state, the side chains of two tyrosine (Tyr) are partially exposed to the solvent and are less affected by the conformational change; the side chains of tryptophan (Trp) and phenylalanine are located in the hydrophobic core region and may be more affected by the conformational change. To focus on exploring the influence of conformational change on the potential interaction mode, a mutant TP - 2A was designed, in which the tryptophan and tyrosine located in the hydrophobic core were replaced with the non - aromatic amino acid alanine with similar hydrophobicity. In the DILLPS test, TP - 2A completely lost the ability of phase separation, and no phase - separation response was observed whether in 20 mM Tricine buffer ( Figure 26 ), or under various buffer conditions with different concentrations such as Tris and phosphate, or after adding the α - helix inducer TFE, indicating that π–π stacking or cation–π interaction plays a key driving role in the phase separation of TP; in addition, the exposure of the side chains of Trp and Phe located in the hydrophobic core to the solvent due to conformational change is of great significance for the formation of condensates.

[0204] Finally, in terms of the contribution of electrostatic interaction, the pH - dependence of the phase separation of TP was detected, using Britton–Robinson buffer with a buffering range of 3 - 12 as the background. The results showed that the phase - separation trend of TP was the most obvious in the pH range of 7 - 10 ( Figure 27); at pH 3-5 and 12, the aggregates completely disappeared. Since TP has both acidic and basic amino acids, the theoretical isoelectric point (pI) is about 8.2. The phenomenon that the phase separation tendency increases when the pH is close to pI points to two contradictory explanations:

[0205] (1) Weakening of long-range electrostatic repulsion. When the pH of the solution is close to pI, the net charge of the protein approaches zero, and the long-range electrostatic repulsion between molecules is significantly reduced. This weakening of repulsion makes it easier for molecules to approach each other, and short-range interactions such as hydrophobic, π-π / cation-π interactions between molecules are more likely to form, indicating that electrostatic interactions are not conducive to phase separation.

[0206] (2) Enhancement of local short-range electrostatic attraction. Near the pI, the full ionization of local charges (deprotonation of the carboxyl group of the acidic residue and protonation of the amino group of the basic residue) maximizes the dipole strength and can provide intermolecular salt bridges during the formation of aggregates, suggesting that electrostatic interactions are conducive to phase separation.

[0207] These two explanations are essentially two sides of the same coin of electrostatic action. They do not conflict with each other, reflecting the complexity of electrostatic action in phase separation driving. Which effect is dominant needs to be further explored in combination with the ion effect. It is worth noting that in the pH-phase separation correlation phase diagram, with pH 8 as the dividing line, when the pH is below 8, the coagulation is prone to wetting, while when the pH is above 8, the coagulation is more inclined to maintain a droplet-like morphology - this may be related to the fact that the quartz glass slide is pre-coated with BSA (pI≈4.7) to form a protein film. When the solution pH is greater than 4.7, the protein film is negatively charged. If the solution pH is also lower than the isoelectric point of TP (8.2), TP is positively charged, and the surface of the coagulation formed is positively charged, which is easy to form electrostatic adsorption with the protein film; on the contrary, TP is negatively charged, and the surface of the coagulation formed is electronegative, which produces electrostatic repulsion with the bottom protein film, preventing wetting.

[0208] In summary, during the dilution process, the compact coiled conformation of TP gradually transforms into a relatively extended α-helix, accompanied by the exposure of key hydrophobic residues, π-π stacking / cation-π interaction sites, thereby enhancing the intermolecular interaction and driving the occurrence of LLPS. The electrostatic mechanism is more complicated and will be discussed in detail in the next summary in combination with the ionic effect.

[0209] 2.26 Discussion on the mechanism of dilution-induced conformational transition

[0210] According to literature research, there are relatively few reports on the transformation of protein / peptide conformations with changes in their own concentrations, and there is a lack of systematic theoretical and experimental demonstrations. Most existing studies focus on the process of transformation from monomers to aggregates when the concentration of proteins / peptides increases. This process is often accompanied by mutual induction of intermolecular conformations, from random conformations to ordered secondary structures. For example: as the concentration of peptides increases, bee venom peptides transform from monomers to tetramers with α-helical conformations; after their own concentrations reach a certain threshold, Amyloid-β and α-synuclein spontaneously assemble into β-sheet structures to form amyloid aggregates; most proteins with LLPS effects are exceptions. When the concentration exceeds the phase separation threshold, they will lose their original stable conformations and transform into random conformations, thereby exposing multivalent binding sites and promoting intermolecular assembly to form aggregates. The common logic of these phenomena is that the increase in protein / peptide concentration shortens the distance between molecules, increases the frequency of intermolecular collisions, and makes intermolecular interactions dominant, resulting in the coordinated occurrence of conformational transformation and molecular aggregation. However, for TP, the peptide concentration decreases during the dilution process, and the distance between molecules increases, which is intuitively not conducive to the occurrence of intermolecular interactions, which is obviously contrary to common sense. At present, there is no mature theory that can fully explain this dilution-induced conformational or aggregation effect. Therefore, this section will comprehensively discuss this effect by combining experimental phenomena with existing theories.

[0211] First, from a macroscopic perspective, before and after dilution, the only thing that changes is the concentration of TP itself, while the overall solvent environment (including water structure, ion species and concentration, etc.) remains unchanged. Therefore, it is necessary to consider whether the long-range intermolecular interactions in the solution, especially the electrostatic interactions between polypeptide molecules, play a key role in the conformational transition. Since the isoelectric point of TP is 8.2, in a solution of pH 7.4, the molecule is positively charged as a whole, so there is intermolecular electrostatic repulsion. One possible speculation is that at high concentrations, the intermolecular electrostatic repulsion network restricts the conformational adjustment of TP and makes an auxiliary contribution to the compact coiled monomer conformation; after dilution, due to the increase in the intermolecular distance, it exceeds the electrostatic shielding range, the electrostatic repulsion disappears, and the original compact coiled state cannot be maintained, resulting in a conformational transition.

[0212] If the speculation is true, then the phase separation trend in the experimental phenomenon should be negatively correlated with the electrostatic shielding distance, that is, weakening the Debye length can enhance the phase separation trend. Combined with the previous phase diagrams of different buffer systems for analysis ( Figure 28A), it was found that the monovalent organic buffer salts (Tris, Tricine, TEA) were generally consistent in their influence trends on phase separation, while the divalent inorganic salt (phosphate) was significantly superior to the former three. The Debye distances at various concentrations of the four buffer salts were calculated as follows: The ionic dissociation forms of each buffer salt at pH 7.4 were calculated through the Henderson–Hasselbalch formula:

[0213]

[0214] It was solved that at pH 7.4, the protonated Tris and TEA were 83% and 71% respectively; phosphate existed in the form of 40% NaH2PO4 and 60% Na2HPO4; while Tricine was special. Due to the existence of two pKa values, ~2.3 (corresponding to the carboxyl group), ~8.15 (corresponding to the amino group), 85% of it existed in the zwitterionic form (overall neutral) at pH 7.4, and only 15% was unprotonated and negatively charged.

[0215] Based on the ionization form, the ionic strength corresponding to each solvent was calculated. According to the ionic strength calculation formula:

[0216]

[0217] where c i is the molar concentration (M) of ion i, and z is the charge number of this ion.

[0218] The relationship between the effective ionic strength provided by the four buffer salts and the concentration (C) was calculated as Tris: 0.915C, Tricine: 0.075C, TEA: 0.85C, phosphate: 1.7C. Then, the electrostatic shielding distances in each buffer solution were calculated according to the Debye formula:

[0219]

[0220] where λ D is the Debye distance; I is the ionic strength; ε r is the relative dielectric constant of the solvent; ε0 is the vacuum dielectric constant; k B is the Boltzmann constant; T is the absolute temperature; e is the elementary charge.

[0221] The electrostatic shielding distances of each buffer salt under the conditions of 10, 20, 50, and 200 mM were calculated accordingly, and the results are shown in Table 3. Among them, the phosphate has the highest ionic strength and the most significant electrostatic shielding effect, which is in line with the trend of the phase diagram; Tris is similar to TEA; Tricine is the weakest, and the Debye distance (2.48 nm) only approaches that of 20 mM Tris (2.25 nm) at 200 mM. Since Tricine and Tris are relatively close in structure and buffering capacity, but there are significant differences in the ionic strength provided, they are suitable for comparison as a control. If the conformational transition during dilution is mainly driven by the disappearance of long-range electrostatic repulsion, then the ability of Tricine to promote phase separation should be significantly lower than that of Tris. For example, the TP phase separation trend in a 200 mM Tricine environment should be close to that in a 20 mM Tris environment. Combining with the phase diagram ( Figure 28 A), the overall effects of Tricine and Tris on the TP phase separation trend are similar. Even at 20 mM, the phase separation trend in Tricine (λ D ≈7.84 nm) is slightly stronger than that in Tris (λ D ≈2.25 nm). This result obviously does not support the simple electrostatic repulsion change explanation; at the same time, it also suggests that the key factor for the promotion of phase separation by increasing the buffer salt concentration is not simply the increase in ionic strength, but may be related to other properties of the buffer salt itself. For example, Tricine mainly exists in the zwitterionic form (85%) at pH 7.4. These neutral molecules contribute zero to the ionic strength, but may affect the polypeptide conformation through hydrogen bonding, electrostatics, hydrophobic interactions, or van der Waals forces, and this part of the contribution may be much stronger than the effect brought by the ionic strength effect.

[0222] In a specific experiment, adjusting the pH of the buffer salt inevitably introduces additional neutral salts, causing the ionic strength to deviate from the theoretical value. To further verify the above conclusion, an experiment was designed to specifically explore the effects of ionic strength and ionic type on the TP phase separation trend. 10 mM Tris at pH 7.4 was selected as the background buffer solvent because TP does not undergo phase separation at any concentration under this condition. On this basis, a certain concentration gradient of monovalent neutral salt sodium chloride, divalent cation neutral salt magnesium chloride, and divalent anion neutral salt sodium sulfate were added respectively, and their effects on the TP DILLPS effect were tested and organized into a phase diagram ( Figure 28 A, Figures 29 - 31) The ionic strength and Debye distance corresponding to each neutral salt are shown in Table 4. The results show that the order of the promoting effect of the three salts on the phase separation of TP is sodium sulfate > magnesium chloride > sodium chloride. Among them, TP fails to form a stable aggregate at all concentrations of sodium chloride (even if metastable phase separation occurs), indicating that TP in the solution still thermodynamically tends to be in a homogeneous form, suggesting that the phase separation ability of TP is not sensitive to simple changes in ionic strength. The difference in the effects of magnesium chloride and sodium sulfate further shows that the type of salt ions is more important than the ionic strength in the phase separation of TP. Therefore, the long-range intermolecular electrostatic repulsion network at the macroscopic level is not the key factor driving the conformational change, and it is necessary to explore potential driving forces from more interaction perspectives and in combination with other theoretical models.

[0223] In addition, the above work clarifies that the long-range intermolecular electrostatic interaction has no significant effect on the phase separation of TP. Therefore, the phenomenon of "the stronger the phase separation trend near the pI value" in the previous subsection is more likely to point to the explanation of "enhanced local short-range electrostatic attraction", that is, the participation of electrostatic interaction is beneficial to the formation and stability of aggregates.

[0224] On the other hand, considering from the microscopic level, the ratio of polypeptide molecules to salt ions in the solution changes before and after dilution: when the concentration decreases, each polypeptide molecule has the potential to be "equipped" with more salt ions. Referring to the Counterion Atmosphere Model (CAM), ions are not evenly distributed in the solution but are concentrated around charged molecules, thus playing a greater role in local charge screening. The core mechanisms of this model include: (1) Electrostatic shielding: Counterions neutralize the surface charges of polypeptides, reducing the intensity of electrostatic repulsion or attraction. (2) Ion concentration gradient: Counterions form a non-uniform distribution around charged molecules (following the Boltzmann distribution), affecting the local dielectric environment and intermolecular forces. The counterion atmosphere model has been widely used in the field of nucleic acids, especially in describing how nucleic acid molecules interact with ions in the solution and how ions affect the structure and stability of nucleic acids. However, its research methods are relatively limited, generally small-angle X-ray scattering (SAXS) and molecular dynamics (MD) simulations. For proteins / polypeptides, due to their relatively low charge density, it is difficult to verify, but some reports have shown that counterions can indeed affect the secondary structure of polypeptides.

[0225] In summary, based on the derivation of the counterion atmosphere model: during the dilution-induced phase separation process, the increase in counterions enriched around TP molecules (for example, under the condition of 20 mM Tricine, when diluted from 2 mM to 1 mM, the molar ratio of TP to Tricine increases from 1:10 to 1:20) may cause key interaction sites to be fully occupied, resulting in the following changes:

[0226] (1) Dynamic balance of the local charge environment: The number of counterions around each TP molecule increases, neutralizing the surface positive charges through direct binding and weakening the repulsive force between like charges among molecules.

[0227] (2) Entropy effect of hydration competition: The hydration ability of counterions enables them to compete with TP polar groups (such as hydroxyl groups and amino groups) for binding water molecules, disrupting the hydration layer structure on the TP surface and affecting the polypeptide conformation. The released bound water molecules lead to an increase in the entropy of the system, providing a thermodynamic driving force for phase separation.

[0228] (3) Conformational flexibility and aggregation cooperativity: Local electrical neutrality and the disruption of the hydration layer prompt the TP conformation to transform from a compact coil to an extended state, exposing hidden hydrophobic or polar interaction sites (such as the α-helix hydrogen bond network), thereby promoting molecular aggregation and phase separation.

[0229] Among them, considering that TP exhibits the property of being insensitive to ionic strength (for example, the Debye distance is about 0.66 nm under the condition of 200 mM sodium chloride, and phase separation still cannot occur stably), it implies that the weakening of intermolecular electrostatic repulsion by local electrical neutralization is not the main reason for conformational transformation. However, this counterion salt bridge can assist the salt ions to reside on the polypeptide surface, and then realize the regulation of conformation at the hydration level.

[0230] Combined with experimental data, it is observed in the phase diagram that the promotion ability of various salts for TP LLPS is ranked as: phosphate ≈ sulfate > Tris ≈ Tricine ≈ TEA > magnesium chloride > sodium chloride.

[0231] Based on the counterion atmosphere model, by integrating the ion residence ability, hydration perturbation effect, and conformational cooperative transformation, it can provide a self-consistent theoretical framework for explaining the "compact coil-α-helix" conformational transformation and phase separation phenomenon of TP at low concentrations.

[0232] Table 3 Ion strength and Debye distance corresponding to four buffer solutions at different concentrations

[0233]

[0234]

[0235] Table 4 Ion strength and Debye distance corresponding to three neutral salts at different concentrations

[0236]

[0237] 2.27 Detection of the DILLPS effect of TP homologous peptides

[0238] Homologous alignment revealed that compared with other mammals, there is a specific mutation, the L11I mutation, in the TP sequence of the human species (Homo sapiens). After comprehensive comparison, representative species with homologous sequences such as rhesus macaque (Macaca mulatta), house mouse (Mus musculus), wild boar (Sus scrofa), and Egyptian fruit bat (Rousettus aegyptiacus) were selected, and TP homologous peptides were synthesized and their respective DILLPS abilities were tested ( Figure 32 ). According to the characteristics, the homologous peptides were named TP-L (L11I reverse mutation, sequence: SYYRPREEEALPHPLALTHKMGWLQLLGRMF, SEQ ID NO:2), TP-VL (L11I, E8V, G22S, sequence: SYYRPREVEALPHPLALTHKMGWLQLLGRMF, SEQ ID NO:3), and TP-KL (L11I, E8K, sequence: SYYRPREKEALPHPLALTHKMGWLQLLGRMF, SEQ ID NO:4), and TP-M (derived from house mouse, sequence: SYYRPREDEDLPHPLALTHKMSWLQLLGRMF, SEQ ID NO:5).

[0239] The detection of the DILLPS effect showed ( Figure 33 ):

[0240] TP-L: Retained the dilution-induced phase separation ability comparable to that of wild-type TP, confirming that the L11I mutation has no significant effect on the function;

[0241] TP-VL: The mutation of glutamate to hydrophobic valine (E8V) led to a small number of condensates, but retained the DILLPS effect, suggesting that acidic residues promote phase separation;

[0242] TP-KL: The mutation of glutamate to lysine (E8K) caused a charge reversal and completely lost the phase separation ability. Combining the results of TP-L and TP-VL, it is suggested that Glu8 may provide a key electrostatic adsorption effect for the ability of TP to form condensates;

[0243] TP-M: The mutation of glutamate to aspartic acid (E8D) is a mutation of the same type of amino acid, and the mutation of alanine to aspartic acid (A10D) provides an additional acidic residue. It was observed that the condensate size was relatively larger than that of TP-L, indicating that the additional acidic residue can enhance the phase separation tendency, which may also be related to the pI value being closer to 7.4.

[0244] The universality of the DILLPS effect was confirmed by homologous alignment and the comparison of the ability to phase-separate homologous peptides. In addition, it was found that the electrostatic adsorption effect contributed by Glu8 might be one of the key factors determining the liquid–liquid phase separation ability of TP solution.

[0245] 2.28 Summary of the molecular mechanism of dilution-induced liquid–liquid phase separation of TP

[0246] In summary, the molecular mechanism of dilution-induced phase separation is like an energy funnel Figure 34 as shown: TP exists in the form of a dynamic conformational ensemble, containing two main conformational subtypes: the compact coil state and the α-helical state. At high concentrations, the monomeric form of the compact coil is energetically favored due to the special sequence arrangement, making the intramolecular interactions of TP dominant; as dilution progresses, the change in the solution microenvironment reshapes the energy landscape of polypeptide folding—the local enrichment of counterions (mainly buffer salts that tend to bind to polypeptides) neutralizes the charge and anchors hydrogen bonds on the polypeptide surface through charge neutralization, weakening the intermolecular repulsion. At the same time, it disrupts the hydration layer structure on the TP surface by its hydration ability, releasing the bound water molecules (driven by entropy increase). This process tilts the energy balance towards the α-helical state, and the conformational change exposes the hydrophobic residues and π–π stacking / cation–π interaction sites that are beneficial for intermolecular interactions to the solvent. Meanwhile, with the rearrangement of electrostatic interactions, intermolecular interactions become dominant, promoting polypeptide assembly and aggregation through multivalent binding sites, forming a phase separation event. This mechanism reveals the key role of solvation effects and ion-specific binding in regulating biomolecular phase separation.

[0247] 2.3 Compartmentalization study of dilution-induced liquid–liquid phase separation

[0248] 2.31 TP exhibits dilution-induced compartmentalized enrichment effect

[0249] Phase-separation-mediated biological compartmentalization is one of the core mechanisms for cells to achieve precise spatiotemporal regulation. Membraneless organelles (such as nucleoli, stress granules, P bodies, etc.) formed through liquid–liquid phase separation provide highly dynamic functional microcompartments for cells. Such biological condensates can locally concentrate specific biomacromolecules, significantly increasing the local concentration of target substances, enabling biochemical reactions that are difficult to occur at low concentrations to proceed rapidly in the microenvironment, thereby improving the reaction efficiency. In addition, condensates provide a controlled microenvironment for molecules, contributing to the regulation of signal transduction, gene expression regulation, and stress response. Through isolation and enrichment, biological condensates not only ensure the spatiotemporally ordered distribution of intracellular substances but also can rapidly adjust the local reaction dynamics in response to environmental changes, ensuring cell homeostasis. Therefore, in-depth study of the phase separation mechanism has important scientific value for revealing the basic principles of life activities and developing new therapeutic strategies.

[0250] The dilution-induced phase separation effect of TP provides a brand-new paradigm for condensate to enrich guest molecules: by adjusting the concentration of TP, the recruitment and release of guest molecules can be effectively controlled. Specifically, when TP is in a high-concentration homogeneous phase, guest molecules are uniformly distributed in the solution; when the solution is diluted, although the global concentration of guest molecules will decrease, due to the occurrence of TP phase separation, guest molecules are recruited by the condensate, and their local concentration can be significantly increased, thus realizing the dilution-induced compartmental enrichment effect ( Figure 35 ); correspondingly, increasing the concentration of TP can effectively release guest molecules.

[0251] First, in terms of recruiting small molecules, TP shows a dilution-induced enrichment effect on aromatic fluorescent molecular probes 4’,6-diamidino-2-phenylindole (DAPI) and thioflavin-T (ThT): after diluting the homogeneous solution containing TP and the fluorescent probe, the fluorescent probe will be enriched in the TP condensate, and their partition coefficients (K = C heavy phase / C light phase) reach 7 and 12 respectively, indicating a high enrichment effect ( Figure 36 , Figure 39 ).

[0252] Secondly, in order to detect whether there is selectivity in the charge properties of guest molecules by TP condensates, a series of polypeptides labeled with 5-carboxyfluorescein (FAM) with a pI range of 3-13 were selected as guest molecules for verification ( Figure 37 ). Through dilution, it was found that these polypeptides could all be enriched in the TP condensate, and the partition coefficient range was from 10 to more than 255 (the confocal resolution limit), all in the high enrichment category ( Figures 38 - 39 ), revealing that TP condensates can recruit guest molecules with different charge properties in a broad spectrum.

[0253] 2.32 The spatial distribution of RNA in TP condensates is regulated by the buffer salt concentration

[0254] Since RNA itself has strong electronegativity and can strongly bind to arginine and lysine in TP. Therefore, an experiment was designed to explore whether RNA has a regulatory effect on the dilution-induced phase separation of TP. Yeast total RNA was used as the guest molecule, and 10 mM Tris (pH 7.4) was used as the buffer salt. When TP was at a high concentration (2 mM), different from the completely clear solution without RNA participation, the addition of RNA caused sparse condensates to exist in the solution ( Figure 40 A), and the phase separation trend increased with the increase of RNA concentration ( Figure 40 B), which may come from the direct interaction between RNA and TP; after dilution (0.5 mM), the phase separation ability of TP increased, and RNA was thus further enriched in the newly formed TP condensates in large quantities ( Figure 41A). Additionally, by changing the buffer salt concentration, the spatial distribution of RNA within the TP condensates can be regulated: under the condition of 10 mM Tris buffer, RNA is mainly enriched in the center of the TP condensates, with less distribution at the edges ( Figure 41 A); while increasing the buffer salt concentration to 50 mM can cause the RNA localization to gradually shift from the inside to the outer layer and the edges: the signal intensity ratio of the edge to the center is approximately 4:1. Figure 41 B). This phenomenon may stem from the competition between RNA and buffer salts for different phase separation driving mechanisms of TP: under low-concentration buffer salt conditions, the degree of α-helix formation of TP is insufficient, and the interaction between polypeptides is weak. At this time, the scaffolding effect of RNA becomes dominant, mediating the phase separation of TP through electrostatic interaction with itself as the core, and the formed condensates are also smaller (~1 nm); while under high-concentration buffer salt conditions, the interaction between TP molecules is strong, and it is more inclined to form condensates by self-aggregation (condensates of ~5 nm can be seen in the field of view). On this basis, RNA is recruited. Since the size of total yeast RNA is relatively large and it is not easy to penetrate into the high-density TP condensates, it shows a "shell-like" distribution.

[0255] 2.33 Dilution-induced luminescence effect with the participation of TP

[0256] The dilution-induced enrichment effect of TP can provide a new and economical way to achieve Aggregation-Induced Emission (AIE). Aggregation-induced emission is a phenomenon in which the luminescence intensity of an organic compound increases in the aggregated state. Different from the "Aggregation-Caused Quenching" (ACQ) effect that traditional fluorescent materials are prone to at high concentrations, AIE materials hardly emit light in dilute solutions, but the luminescence intensity increases significantly in the aggregated state. Taking the triphenylethylene molecule (TVP) as an example ( Figure 42 ), in the solution state, its benzene rings can rotate freely, resulting in the dissipation of energy in a non-radiative manner and low luminescence efficiency. However, in the aggregated state, these rotations are restricted, non-radiative dissipation is reduced, and the luminescence efficiency is significantly improved. Figure 42 The schematic diagram and confocal microscopy imaging show that in a homogeneous mixed solution containing 2 mM TP and 500 nM TVP, since TVP is in a free and unaggregated state, almost no fluorescence can be detected in the solution; however, when the solution is diluted 10 times, the TVP molecules emit bright red fluorescence at 620 nm, and its intensity is increased by more than 20 times compared with the initial state.

[0257] This phenomenon provides a theoretical basis and experimental support for the development of a low-cost and highly efficient detection platform based on dilution-induced phase separation to achieve the AIE effect. This strategy of regulating luminescence by molecular aggregation states has broad application prospects in the fields of bioimaging, sensing, and drug delivery, etc.

[0258] 2.34TP forms a multi-level phase separation pattern through dilution-induced enrichment

[0259] Previous work has demonstrated that TP can widely enrich guest molecules by the dilution-induced enrichment effect, achieving effective compartmentalization regulation phenomena. Here, in this study, a protein that undergoes LLPS at high concentrations - galectin-3 (Gal-3) - is used as an example. As a guest protein, it participates in the TP dilution process, simulating the multi-level phase separation hierarchical organization mechanism of the nucleolus. Through dilution-induced enrichment, the concentration of the guest protein is locally increased in the reverse direction, forming secondary phase separation events, providing a new reference model for understanding the formation of complex biological compartments ( Figure 43 ).

[0260] Gal-3 is a multifunctional protein that has been reported to participate in various cellular processes through LLPS, such as cell adhesion, apoptosis, and immune response, etc. The phase separation concentration threshold of eGFP-Gal3 obtained by recombinant expression is relatively high. In the absence of the crowding agent PEG, there are still no phase separation events at 18 mg / ml ( Figure 44 ). In the experiment, 2 mg / ml of eGFP-Gal3 and high-concentration TP together form a homogeneous solution. At this time, there is no signal in each fluorescence channel; as the solution is diluted by half with buffer, TP forms primary condensates as a scaffold, accompanied by the enrichment of eGFP-Gal3, triggering its phase separation inside the TP condensates, forming multi-compartment secondary condensates ( Figure 45 ). As a control, the eGFP protein that does not have the ability of phase separation is evenly distributed in the TP condensates instead of forming secondary compartments ( Figure 46 A). There are also fusion events between these secondary compartments, but kinetically, it is necessary to overcome the hindrance of TP to exclude TP to form its own compartments ( Figure 46 B). By performing 3D reconstruction on the multi-level phase separation system and exploring the fluorescence distribution, it is found that eGFP-Gal3 has three-level components in the system: its signal in the external solution is almost undetectable, while inside the TP condensates, the ratio of the signal intensity inside and outside the secondary compartments is about 10:1; for TP, almost no signal can be detected inside the secondary compartments, suggesting that it is fully excluded to form a "cavity" ( Figure 47 ). Further, through the Part-FRAP experiment, it is revealed that eGFP-Gal3 has extremely strong fluidity inside the secondary compartments, and material exchange is completed during the quenching process, resulting in the decrease of the global signal ( Figure 48)。

[0261] In summary, through the local enrichment effect, the TP condensate can significantly reduce the phase separation threshold of the guest protein (from a high concentration to a low concentration in terms of the global concentration), demonstrating the core role of microenvironment remodeling in regulating biomolecular behavior. This "concentration amplifier" mechanism provides a new perspective for understanding signal transduction and stress response, and also provides a theoretical basis and application inspiration for the design of new biomaterials and drug delivery systems.

[0262] 2.35 Enzymatic catalysis achieved by dilution-induced enrichment of TP

[0263] The excellent ability of TP to enrich guest molecules endows it with the potential to become a special concentration-responsive microreactor. By locally enriching the substrate and the enzyme, the reaction rate of enzymatic catalysis is increased, making the dilution-induced phase separation not only a physical separation but also a switch of functional activity.

[0264] Taking the homologous protein of Gal-3, galectin-10 (Gal-10), as the guest molecule for the enzymatic catalysis reaction as an example. Gal-10 is a protein that is prone to self-assemble into crystals in vivo and in vitro and is related to immune and inflammatory diseases. Previous studies have shown that fusing His-tag to the N-terminus of Gal-10 can effectively inhibit its aggregation tendency, facilitating protein expression, and by digesting the ligation site of His-tag with tobacco etch virus (TEV) protease, Gal-10 without His-tag will regain the ability to self-assemble and form crystals. Based on the properties of this enzyme reaction system, an experiment was designed to compare the crystal formation rates before and after dilution-induced phase separation and evaluate the utility of TP as a microreactor ( Figure 49 ). His-Gal-10 and TEV protease were labeled with Alexa 488 and Alexa 633 respectively, and a ternary mixture solution of His-Gal-10 / TEV protease / TP was established. After dilution, His-Gal-10 and TEV protease were enriched in the TP condensate and showed spatial co-localization ( Figure 50 ). The reaction systems under various conditions were incubated on a shaker at 4°C. Observed through a polarized light microscope, it can be seen that for the diluted low-concentration group, Gal-10 crystals began to appear at 40 minutes and became obvious at 80 minutes ( Figure 51 A), while no observable crystals were present in the high-concentration group within the field of view at 80 minutes ( Figure 51 B). As a reference, in the absence of any TP, it took 180 minutes to observe crystal formation in this catalytic reaction ( Figure 51 C).

[0265] In summary, the experimental results show that the compartmentalized microreactors formed by TP have efficient biocatalytic capabilities and special concentration regulation mechanisms. This system not only fully mimics the dynamic regulation principle of "molecular enrichment-functional emergence" of membrane-free organelles, but also expands the application boundary of phase separation in synthetic biology - by designing the coupling of responsive scaffolds (such as TP) and modular enzyme systems (such as His-TEV protease-Gal-10), in-situ catalytic enhancement and product self-assembly can be programmably achieved, providing innovative tools for intelligent drug factories, dynamic biomaterials and in vitro metabolic pathway reconstruction.

[0266] The description of the above embodiments is only for understanding the method and its core idea of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A polypeptide, characterized in that, The polypeptide comprises an amino acid sequence selected from the following group: (1) An amino acid sequence obtained by substituting, deleting, adding, or inserting at least one amino acid residue in SEQ ID NO: 1; (2) An amino acid sequence having at least 90% sequence identity with SEQ ID NO: 1; Preferably, the substitution is one or more of leucine, glutamic acid, and alanine being substituted; Preferably, leucine is substituted with isoleucine; Preferably, glutamic acid is substituted with valine and / or aspartic acid; Preferably, alanine is substituted with aspartic acid; Preferably, the sequence of the polypeptide is as shown in any one of SEQ ID NO: 1-5.

2. A polypeptide condensate, characterized in that, The polypeptide condensate comprises the polypeptide according to claim 1.

3. A polynucleotide, characterized in that, The polynucleotide encodes the polypeptide according to claim 1 or the polypeptide condensate according to claim 2.

4. A carrier, characterized in that, The vector contains the polynucleotide according to claim 3.

5. A host cell, characterized in that, The host cell comprises the polynucleotide according to claim 3 or the vector according to claim 4.

6. Use of the polypeptide according to claim 1, the polypeptide condensate according to claim 2, the polynucleotide according to claim 3, the vector according to claim 4, and the host cell according to claim 5 in liquid-liquid phase separation, enrichment, or in the preparation of products for regulating liquid-liquid phase separation and enrichment.

7. Use of the polypeptide according to claim 1, the polypeptide condensate according to claim 2, the polynucleotide according to claim 3, the vector according to claim 4, and the host cell according to claim 5 in regulating the compartmentalization of guest molecules or in the preparation of products for regulating the compartmentalization of guest molecules.

8. Use of the polypeptide according to claim 1, the polypeptide condensate according to claim 2, the polynucleotide according to claim 3, the vector according to claim 4, and the host cell according to claim 5 in aggregation-induced emission or in the preparation of products for aggregation-induced emission; Preferably, the product comprises an aggregation-induced emission probe.

9. Use of the polypeptide according to claim 1, the polypeptide condensate according to claim 2, the polynucleotide according to claim 3, the vector according to claim 4, and the host cell according to claim 5 in promoting enzymatic reactions or in the preparation of products for promoting enzymatic reactions.

10. Any one of the following methods: (1) A method for regulating liquid-liquid phase separation and enrichment, characterized in that, The method comprises using the polypeptide according to claim 1, the polypeptide condensate according to claim 2, the polynucleotide according to claim 3, the vector according to claim 4, and the host cell according to claim 5; (2) A method for regulating the compartmentalization of guest molecules, characterized in that the method comprises using the polypeptide according to claim 1, the polypeptide condensate according to claim 2, the polynucleotide according to claim 3, the vector according to claim 4, and the host cell according to claim 5; (3) A method for promoting aggregation-induced emission, characterized in that the method comprises using the polypeptide according to claim 1, the polypeptide condensate according to claim 2, the polynucleotide according to claim 3, the vector according to claim 4, and the host cell according to claim 5; (4) A method for promoting an enzymatic reaction, characterized in that the method comprises using the polypeptide according to claim 1, the polypeptide condensate according to claim 2, the polynucleotide according to claim 3, the vector according to claim 4, and the host cell according to claim 5.

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