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WO2025147594A1 - Peptides et procédé d'utilisation dans la préparation de nanodisques - Google Patents

Peptides et procédé d'utilisation dans la préparation de nanodisques Download PDF

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WO2025147594A1
WO2025147594A1 PCT/US2025/010215 US2025010215W WO2025147594A1 WO 2025147594 A1 WO2025147594 A1 WO 2025147594A1 US 2025010215 W US2025010215 W US 2025010215W WO 2025147594 A1 WO2025147594 A1 WO 2025147594A1
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Prior art keywords
peptide
nanodiscs
native
membrane
amino acid
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Huan BAO
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University of Florida
University of Florida Research Foundation Inc
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University of Florida
University of Florida Research Foundation Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • the disclosure relates to peptides and/or nanodiscs and methods of making and using thereof for extraction of proteins.
  • the present disclosure provides a peptide comprising an amino acid sequence of formula I: DWX1KAFYDKX 2 AEKX 3 KEAX 4 (I) (SEQ ID NO: 41 ) wherein Xi is an amino acid with hydrophobic side chain; X 2 is selected from V or W; X3 is selected from L or W; X 4 is selected from F or W; and wherein the N-terminus of the peptide is modified with a fatty acid, such as hexanoic acid.
  • the peptide the peptide of the present disclosure comprises an amino acid sequence of formula II:
  • the peptide of the present disclosure comprises an amino acid sequence of formula III:
  • the peptide of the disclosure is amidated at the C-terminus.
  • the peptide of the disclosure comprises the amino acid sequence of any one of SEQ ID NOs: 22-40.
  • the disclosure also provides a method of preparing a nanodisc, the method comprising (a) contacting a lipid bilayer comprising a payload with a peptide comprising an amino acid sequence of formula I:
  • the method of the disclosure comprises a peptide that does not comprise SEQ ID NO: 1.
  • the fatty acid is hexanoic acid.
  • the fatty acid comprises a myristoyl moiety, decanoic acid, or palmitoyl moiety.
  • the peptide is amidated at the C-terminus.
  • the peptide comprises the amino acid sequence of any one of SEQ ID NOs: 22-40.
  • the peptide comprises the amino acid sequence of any one of SEQ ID NOs: 7-21.
  • FIGs. 4A-4C illustrate the formation of native nanodiscs from bacterial membranes using DeFrMSPs.
  • FIG. 4A (left panel) depicts an illustration of the reconstitution procedure described herein.
  • FIG. 4A (right panel) shows isolation efficiencies of MalFGKi into nanodiscs with different designs of DeFrMSPs. DDM and buffer were used as positive and negative controls for data normalization.
  • FIG. 4C shows representative gels of extracted MalFGKi.
  • FIGs. 17A-17G depict the optimization and characterization of DeFrMSPs for detergent-free extraction of MalFGKi native NDs from crude membranes.
  • FIG. 17A depicts a representative image of an electrophoresis gel of crude membranes isolated from cells overexpressing MalFGK? that were incubated with the indicated DeFrMSPs or DDM overnight and subjected to pull down experiments. Samples at each step were analyzed by SDS PAGE. In negative control experiments, PBS buffer with the same amount of DMF was used. Red boxes indicated the extracted MalF and MalK.
  • FIG. 17A depicts a representative image of an electrophoresis gel of crude membranes isolated from cells overexpressing MalFGK? that were incubated with the indicated DeFrMSPs or DDM overnight and subjected to pull down experiments. Samples at each step were analyzed by
  • FIG. 17B depicts a graph of evolving DeFrMSP designs based on Hex-18A for converting MalFGK proteoliposomes into NDs. Data were quantified based on the density of the MalFGKo complex band normalized to that of the Hex ISA-extracted sample.
  • FIG. 17C depicts a graph of a SEC profile of native MalFGK2 NDs using a Superdex 200 3.2/300 column.
  • FIGs. 17D-17E depict graphs of diameters of native NDs harboring MalFGF , mGluR7, and HCN1 determined from negative stain EM (FIG. 17D) and DLS (FIG. 17E).
  • FIG. 17F depicts a pie chart of lipids associated with native MalFGK? NDs from LC-MS/MS analysis.
  • FIG. 17G depicts a graph of ATPase activities of MalFGBG WT and mutants in native NDs. Data are shown as mean ⁇ s.d.,
  • FIGs. 18A-18D depict the enhancement and characterization of DeFrMSPs for detergent-free extraction of mGluR7 native NDs from crude membranes.
  • FIG. 18A depicts a graph for screening DeFrMSP designs for extracting mGluR7-eGFP from crude membranes. Solubilized samples were clarified by ultracentrifugation and extracted mGluR7 proteins were quantified by monitoring the fluorescence of eGFP. ND formation efficiencies of m
  • FIG. 18B depicts a representative image of an SDS-PAGE gel of extracted mGluR7 NDs with different DeFrMSP designs that were subjected to pull down experiments. Samples at each step were analyzed by SDS-PAGE and in-gel fluorescence imaging. Abbreviations: 1, Hex-18A; 2, Hex20B; 3, Hex20BlWA; 4, Hex20BF4W.
  • FIG. 18C depicts a graph of the fractionation of mGluR7 native NDs using a Superdex 200 3.2/300 column.
  • FIG. 18D depicts a representative negative stain EM micrograph of mGluR7 native NDs formed by Hex20BlWA. Scale bar, 30 nm.
  • FIGs. 19A-19F depict the optimization and characterization of DeFrMSPs for detergent- free extraction of HCN1 native NDs from crude membranes.
  • FIG. 19A depicts a graph of the screening DeFrMSP designs for extracting HCNl-eGFP from crude membranes. Solubilized samples were clarified by ultracentrifugation and ND formation efficiencies of HCN1 were quantified based on the GFP fluorescence emission normalized to that of DDM- solubilizcd samples. Inset: extracted HCN1 NDs with different DcFrMSP designs and negative control (PBS) were subjected to pull down experiments. Samples at each step were analyzed by SDS-PAGE and in-gel fluorescence imaging.
  • FIG. 19B depicts a graph of the fractionation of HCN1 native NDs using a Superdex 200 3.2/300 column.
  • FIG. 19C depicts a representative negative stain EM micrograph of HCN1 native NDs formed by Hexl8A. Scale bar, 30 nm.
  • FIG. 19D depicts a graph of the extraction of HCN 1 native NDs using the indicated polymers. The majority of the extracted proteins could not be purified through affinity purification.
  • FIG. 21 depicts a schematic and representative images of the extraction of membrane proteins located in intracellular membranes by DeFrNDs.
  • Purified secretory vesicles and ER microsomes were treated with PBS (negative control), DDM (positive control), and DeFrMSPs (Hexl8A or Hex20BlWA). Samples were then clarified by ultracentrifugation and soluble supernatants were analyzed by SDS-PAGE and western blot using antibodies against either VAMP2 or Sec6ip.
  • FIG. 22 depicts a schematic of one-step reconstitution of native NDs using DeFrMSPs.
  • Traditional ND reconstitution requires the purification of the membrane protein of interest in detergent micelles (dash line) and optimization of experimental procedures to assemble with MSPs and lipids upon the removal of detergents.
  • the materials and methods described herein can directly extract membrane proteins into NDs with native lipids in one step, bypassing the need and limitation of detergent-mediated reconstitution.
  • the methods of the present disclosure do not comprise a reconstitution step using detergent.
  • the disclosure provides a peptide comprising an amino acid sequence of formula I: DWX1KAFYDKX2AEKX3KEAX4 (I) (SEQ ID NO: 41 ) wherein:
  • Xi is an amino acid with hydrophobic side chain
  • X2 is selected from V or W;
  • X3 is selected from L or W;
  • the amino acid with hydrophobic side chain (Xi) is selected from A, V, I, L, M, F, Y, or W.
  • Xi is A.
  • Xi is V.
  • Xi is I.
  • Xi is L.
  • Xi is M.
  • Xi is F.
  • Xi is Y.
  • Xi is W.
  • X2 is V.
  • X2 is W.
  • X3 is L.
  • X3 is W.
  • X4 is F.
  • X4 is W.
  • the peptide of the disclosure comprises an amino acid sequence of formula II:
  • DWX1KAFYDKX2AEKX3KEAX4X5W (II) (SEQ ID NO: 42) wherein X5 is selected from D or E, and X1-X4 are described above.
  • X5 is D.
  • X5 is E.
  • the N-terminus of the peptide comprising amino acid sequence of formula II is modified with a fatty acid, and optionally comprises a hexanoic acid.
  • the peptide of the disclosure does not comprise SEQ ID NO: 1.
  • SEQ ID NO: 1 (also referred to herein as “18A”) comprises the sequence DWLKAFYDKVAEKLKEAF, which has neither an N- nor a C-terminal chemical modification.
  • the peptide comprises the sequence DWLKAFYDKVAEKWKEAFDW comprising a hexanoic acid modification at its N-terminus (SEQ ID NO: 34).
  • the peptide of the present disclosure comprises a chemical modification at the N- and/or C- terminus.
  • the peptide is amidated at the C-terminus.
  • the disclosure provides a peptide comprising the amino acid sequence of any one of SEQ ID NOs: 22-40.
  • Exemplary peptides of the disclosure are provided in Table 1 .
  • the disclosure contemplates a peptide comprising the amino acid sequence of any one of SEQ ID NO: 7-40, including a peptide comprising the amino acid sequence of any one of SEQ ID NO: 22-40, as well as peptides comprising one, two, three, or four amino acid substitutions within the amino acid sequences set forth herein.
  • the disclosure further provides a method of preparing a nanodisc, the method comprising (a) contacting a lipid bilayer comprising a payload with a peptide comprising an amino acid sequence of formula I; and (b) purifying a nanodisc comprising the payload. The method does not comprise a reconstitution step using detergent.
  • the disclosure further provides a method of preparing a nanodisc, the method comprising (a) contacting a lipid bilaycr that docs not comprise a payload with a peptide comprising an amino acid sequence of formula I; and (b) purifying a nanodisc that does not comprise the payload. The method does not comprise a reconstitution step using detergent.
  • Cells expressing the payload in membranes will typically be homogenized and subjected to ultracentrifugation to isolate cell membranes. These membrane fractions will be resuspended in PBS buffer and incubated with the design DeFrMSPs on ice for 20 hours with gentle shaking. The resulting samples are then optionally clarified by ultracentrifugation.
  • Nanodiscs harboring the payload are isolated by any suitable method, including, e.g., performing affinity purification using the supernatant from ultracentrifugation and further purifying through size-exclusion chromatography to remove aggregated materials.
  • Proteins were eluted in buffer C (50 mM Tris-HCl (pH 8), 500 mM Imidazole, 400 mM NaCl, 5% glycerol, 2 mM P-mercaptoethanol, 0.02% DDM), desalted in buffer A supplemented with 0.02% DDM using PD MiDiTrap G-25 (GE Healthcare).
  • buffer C 50 mM Tris-HCl (pH 8), 500 mM Imidazole, 400 mM NaCl, 5% glycerol, 2 mM P-mercaptoethanol, 0.02% DDM
  • the HEK293 GnTI- cells were grown at a density of 3.0 x 10 6 per mL and infected with high titer P3 BacMam viruses. 10 mM sodium butyrate was added to the culture to enhance the expression of proteins after 12 h post-infection. The culture was further incubated at 30 °C for 48 hr and harvested by centrifugation.
  • cDNA corresponding to HCN1SM in the modified pEG BacMam vector was extracted from large volumes of bacterial cultures using Endotoxin free Plasmid Purification kits (Qiagen) and transfected into suspension cultures of Freestyle HEK 293 cells (Thermo Fisher Scientific) using Trans-IT Pro Transfection reagent (MIRUS) following manufacturer’s instructions.
  • the Freestyle HEK93 cells were not experimentally authenticated or tested for mycoplasma contamination. Post transfection, cells were grown at 12-14 hr at 37 °C, following which sodium butyrate was added to the cultures to a final concentration of 10 mM and cultures were grown at 30 °C for another 48 hr.
  • Cells were collected by centrifugation at 3,000g for 20 min and washed twice with chilled 150 mM NaCl, 2 OmM Tris, pH 8.0. Cell pellets were subsequently resuspended in lysis buffer (300 mM NaCl, 40 mM Tris, 10 mM DTT, 20% glycerol, 1 mM EDTA, 2 mM cholesterol hemi succinate (CHS), pH 8.0 supplemented with IX Halt protease inhibitor cocktail (Thermo Fisher Scientific)) and stored at -80°C.
  • lysis buffer 300 mM NaCl, 40 mM Tris, 10 mM DTT, 20% glycerol, 1 mM EDTA, 2 mM cholesterol hemi succinate (CHS), pH 8.0 supplemented with IX Halt protease inhibitor cocktail (Thermo Fisher Scientific)
  • PC 12 cells were cultured in Dulbecco’s modified Eagle’s medium, high glucose
  • Cells were pelleted by centrifugation at 1000 x g for 5 min, resuspended, and washed once by repeating the centrifugation step in homogenization media (0.26 M sucrose, 5 mM MOPS, and 0.2 mM EDTA). Cell pellet was resuspended in 3 mL of homogenization medium containing protease inhibitor (Roche Diagnostics), the cells were then physically lysed by passing cells through a ball bearing homogenizer 10 times, which contained a 0.6367 cm bore and a 0.6340 cm diameter ball. Nuclei and large debris were removed by centrifugation in a fixed angle microcentrifuge at 1000 x g for 10 min at 4 °C.
  • the post-nuclear supernatant was collected, and mitochondria were removed by centrifugation at 8,000 x g for 15 min at 4 °C.
  • the post mitochondria supernatant was then collected and adjusted to 5 mM EDTA and incubated on ice for 10 min to assist with removing ribosomes from the endoplasmic reticulum to reduce contamination in the dense core vesicle fraction.
  • a working solution of 50% OptiPrep iodixanol was made by mixing five volumes of 60% OptiPrep with one volume 0.26 M sucrose, 30 mM MOPS, and 1 mM EDTA.
  • This working solution was mixed with homogenization media to prepare solutions of 30% iodixanol and 14.5% iodixanol for a discontinuous gradient.
  • a SW55 tube was prepared by laying 3.8 mL of 14.5% iodixanol on top of 0.5 mL of 30% iodixanol. Then 1.2 mL of the post mitochondrial supernatant was layered on top of this iodixanol gradient. The sample was then centrifuged at 190,000 x g for 5 hours. A clear white band at the interface between the 30% iodianol and 14.5% iodixanol was collected as the dense core vesicle sample.
  • the dense core vesicles sample was then extensively dialyzed in a cassette with 10,000-kDa molecular weight cutoff (24 h with 3 buffer changes of 5 L each) into 120 mM potassium glutamate, 20 mM potassium acetate, and 20 mM HEPES, pH 7.4. After dialysis, sucrose was spiked in to make a final concentration of 10% sucrose and then the sample was flash frozen and stored at -80 C.
  • Lipids were dried under a gentle stream of nitrogen and further with vacuum for 2 h followed by rehydration in reconstitution buffer (25 mM Tris-HCl, pH 7.5, 100 mM NaCl, ImM DTT). Samples were then extruded through a 200 nm filter for liposome preparation. To monitor the binding of sytl to protein-free NDs via fluorescence spectrometry, protein-free liposomes were made using 70% PC and 30% PS. To study the interaction of SecA with lipids, protein-free liposomes were made using 100% PC, 100% PG, or 50% PC with 50% PG.
  • asymmetric liposomes with PC/PG lipids was induced by generating a transmembrane pH gradient as described previously 57 .
  • purified proteins MalFGBG and SNAREs
  • lipids were incubated at a ratio of 1:1000 in reconstitution buffer plus 1% OG on ice for 30 mins.
  • MalFGK proteoliposomes were prepared with 100% PC lipids.
  • donor v-SNARE and protein-free vesicles were prepared with 12% PE, 40% PS, 45% PC, 1.5% NBD-PE and 1.5% Rho-PE, whereas t-SNARE and protein free vesicles were prepared with 15% PE, 25% PS and 60% PC.
  • Detergents were removed by addition of Bio-beads (1/3 volume) and gentle shaking (O/N). Finally, proteoliposomes were purified by flotation in an Accudenze step gradient as described previously 49 .
  • DeFrMSPs The peptides of the present disclosure are referenced herein as “DeFrMSPs.”
  • candidate DeFrMSPs candidate peptides
  • membranes 4 °C overnight with gentle shaking.
  • the resulting materials were clarified by ultracentrifugation. Samples were then loaded onto affinity and size exclusion column to isolate the nanodiscs.
  • Candidate DeFrMSPs (100 mg/ml in DMF) were first diluted to 25 mg/ml in PBS and added to proteoliposomes at 1 mg/ml or crude membranes at 4 mg/ml. Samples were incubated at 4 °C overnight and clarified by centrifugation at 50,000 rpm for 45 mins. Nanodiscs were purified by affinity purification as described above, and further fractionated by size-exclusion chromatography (SEC) and stored at -80 °C. To monitor the binding of sytl to lipids via fluorescence spectrometry, lipids were made using 70% PC and 30% PS.
  • lipids were prepared using 1% NBD-PE, 1% Rho-PE, 20% PE, 48% PC, and 30% PS.
  • nanodiscs were made using 50% PC and 50% PG.
  • mGluR7 NDs were purified using Ni2+-NTA and anti-DYKDDDDK (Fisher) affinity resins as ELFN-1.
  • HCN1 NDs were purified using Streptactin affinity resin (IBA Life Sciences) and eluted in buffer D (50 mM Tris-HCl (pH 8), 300 mM NaCl, 10% glycerol, 2 mM DTT) plus 5 mM desthiobiotin.
  • buffer D 50 mM Tris-HCl (pH 8), 300 mM NaCl, 10% glycerol, 2 mM DTT
  • MalFGKo and mGluR7 NDs were further fractionated on a Superdex S200 3.2/300 column in buffer A, whereas HCN1 NDs were in buffer D.
  • VAMP2 VAMP2 into DeFrNDs from dense-core vesicles
  • samples were incubated with DeFrMSPs.
  • Extracted samples were clarified by centrifugation at 112,000 x g at 4°C for Ih and then analyzed by western blot using a VAMP2 antibody (Synaptic systems, 104211) and a secondary mouse IgG Fc binding protein (Santa Cruz Biotechnology, sc545209).
  • VAMP2 antibody Synaptic systems, 104211
  • a secondary mouse IgG Fc binding protein Santa Cruz Biotechnology, sc545209
  • MalFGFG proteoliposomes, protein-free liposomes, or HCN1 crude membranes were incubated (4 °C, O/N) with the indicated polymers at a final concentration of 1 % as recommended by the vendor (Curi Bio).
  • Formvar/carbon-coated copper grids (01754-F, Ted Pella, Inc.) were glow discharged (15 mA, 25 secs) using PELCO easiGlowTM (Ted Pella, Inc).
  • Nanodiscs (10 pg/ml) were applied onto the grids for 30 secs, followed by staining with 0.75% uranyl formate for 1 minute. Images were collected using a ThermoFisher Science Tecnai G2 TEM (100 kV) equipped with a Veleta CCD camera (Olympus). All TEM data were analyzed using Fiji to determine the nanodisc sizes.
  • Movies were captured at a magnification of 81,000x (pixel size of 1.058 A). A total of 12522 movies in the defocus range of -0.8 to -2.2 pM were recorded with a total accumulated dose of 55.80 e- /A.
  • Lipids were extracted from nanodiscs and characterized by liquid chromatography with Waters BEH C8 column (2.1 mm x 100 mm, 1.7 um) usingl6 min gradient.
  • Mobile phase A is 10 mM ammonium acetate with 5% methanol, 0.1% acetic acid in H2O and mobile phase B is 0.1% acetic acid in methanol.
  • Flowrate was set at 0.4 mL/min and column oven temperature at 30 °C. 5 pL of samples were injected into Vanquish UHPLC (Thermo Scientific) connected with Orbitrap ID-X Tribrid Mass Spectrometer (Thermo Scientific).
  • the 18 A-MalFGK2 nanodiscs were characterized using negative stain electron microscopy (EM). Although the purified particles from size-exclusion chromatography (SEC) show diameters of about 10-20 nm (FIG. IE), they were quite poly-disperse, consistent with the data from native PAGE (FIG. IB). Further, the structural feature of the MalFGK2 transporter was difficult to identify from raw images, highlighting deficiencies with 18 A.
  • SEC size-exclusion chromatography
  • N- and C-terminal modifications can enhance peptide insertion into membranes.
  • chemical modifications e.g., amide, acetylene, and different kinds of fatty acids
  • Hexanoic acid was the shortest fatty acid modification tested, yet it was the most effective one. It was initially expected that the longer fatty acids would be more effective as they are more hydrophobic and should be more active to destabilize bilayer and form nanodiscs. However, without wishing to be bound by any particular theory, the longer fatty acids may also make the peptide too hydrophobic to stay soluble in solution. Thus, peptides containing both N- terminal Hexanoic acid modification and C-terminal amidation were less active. Thus, peptides modified only with hexanoic acid at the N-terminus described herein were shown to be the best for nanodisc formation.
  • Example 3 - DeFrMSPs enclose stable and functional membranes for biochemical reconstitutions
  • a classic FRET assay was employed to determine if the lipids in DeFrMSP nanodiscs are stably embedded or rapidly diffused among individual nanodiscs 40 .
  • donor nanodiscs were prepared with a FRET pair (NBD-PE and Rho-PE) and then incubated with acceptor DeFrMSP nanodiscs or liposomes that do not harbor fluorescent lipids 41, 42 . If the lipids in DeFrMSP nanodiscs are unstable, the FRET pair, upon incubation with acceptor nanodiscs or liposomes, will be diluted and separated from each other, resulting in the increase of the fluorescence of NBD-PE.
  • Nanodiscs prepared using the DeFrMSP described herein were assayed in the context of asymmetric membranes.
  • Asymmetric PC/PG liposomes were formed using a previously described pH-driven method 49 . These liposomes were incubated with DeFrMSPs to form nanodiscs. If membrane asymmetry is preserved in DeFrMSP nanodiscs, they should contain mainly PG lipids on one side and largely PC lipids on the other side. SecA protein, which binds strongly to PG lipids, was used to examine if SecA protein binds to both sides or only one side of DeFrMSP nanodiscs (FIG. 3D). SecA protein is a dimeric protein in solution and will dissociate into monomers upon binding to negatively charged lipids 50 .
  • ADAM10 is an abundant protease expressed in many cells that is activated by PS externalization 59 (FIG. 16D).
  • DeFrNDs were prepared from HEK293 cells to assay ADAM10 activities. The results showed that ADAM10 activities in DeFrNDs and crude membranes were quite similar.
  • ADAM10 activity of ADAM10 was significantly increased if vesicles were prepared from crude membranes using detergent-mediated reconstitution, probably because detergent completely disrupted native cell membranes and caused the redistribution of PS lipids between the two leaflets of reconstituted vesicles.
  • an advantage of the method described herein is the potential to stabilize membrane protein complexes with native lipids (FIG. 4A).
  • FIG. 4A To illustrate, crude membrane fractions from E. coli expressing MalFGK2 were incubated with Hex-18 A and affinity purification was performed. The results established that M&1FGK2 was directly isolated in Hexl 8A-encased native nanodiscs (FIG. 4B). However, the results also showed that Hcx-18A was much less efficient than the detergent DDM to extract MalFGK? from crude E. coli membranes. Additional peptides were generated to achieve a DeFrMSP with superior performance to Hex-18A.
  • the amino acid sequence of SEQ ID NO: 1 was modified with a combination of positively charged residues with hydrophobic ones. Additionally, Phe was replaced with Tip in various candidates, and impact on efficacy in extracting MalFGK2 into nanodiscs from proteoliposomes was characterized.
  • the tested peptides were all modified with a Hex group at the N-tennini, and were designated as “F1W, F2W, F3W, and F4W.”
  • the amphipathic repeat of SEQ ID NO: 1 was slightly increased to 20 or 22 amino acids (“20B and 22B”), resulting in stability enhancement of the nanodiscs.
  • HCN1 was functional in the peptide-encased native nanodiscs, retaining the activity to undergo cAMP- triggered conformational changes.
  • the kinetics and affinity of HCNl-cAMP interactions in native nanodiscs are very different from data obtained in detergent micelles, supporting the role of lipids in regulating the property of this ion channel. In contrast, it was not possible to efficiently extract them using amphipathic polymers, let alone maintain their activities.
  • the present disclosure provides engineered peptides which allow detergent-free reconstitution of membrane proteins into nanodiscs from native membranes.
  • the approach as described in the present disclosure bypasses the limitation of detergent-mediated reconstitution used in traditional methods, thereby enabling structural and functional studies of membrane protein complexes that may be previously unattainable.
  • the membranes in DeFrMSP nanodiscs can faithfully mimic native lipid environments for biophysical dissections of both integral and periphery membrane proteins involved in various transmembrane signaling pathways.
  • nanodisc scaffold peptide (NSP)-bascd peptides arc capable of forming stable nanodisc structures. However, they are much less effective in penetrating the lipid bilayers to allow for detergent-free nanodisc reconstitution.
  • the peptides described herein e.g., the peptides described herein having hexanoic acid modification
  • the DeFrMSPs described herein may extend the potential of nanodiscs for fundamental research of membrane biology.
  • An advantage of the platform disclosed in the present disclosure is the ability to reconstitute asymmetric membranes with synthetic and native lipids. Such advantage may allow for further uses with a variety of biophysical approaches to investigate how lipid asymmetry regulates the conformational dynamics of membrane protein complexes as observed in cells.
  • the size of DeFrNDs can be expanded to 30-50 nm when generated from protein-free liposomes (FIGs. 15A-15C), indicating that DeFrNDs can accommodate much larger membrane protein complexes than the ones assayed in the study described herein.
  • DeFrND Another advantage of DeFrND is the ability to reconstitute asymmetric membranes with synthetic and native lipids.
  • the DeFrND technology is useful to unravel the molecular mechanism underlying the regulation of myriad transmembrane signaling pathways by native cell membranes.
  • DeFrNDs have not exhibited preferred orientation as sometimes found in other membrane mimetic systems 66 .
  • This advantage may provide a useful alternative option for structural characterizations of membrane proteins in a near native lipid environment using single particle cryoEM.
  • the peptides described herein may also benefit therapeutic candidates based on nanodisc delivery to host cells.
  • Nanodiscs are very useful tools for vaccine development because of their nanoscale sizes, enhanced tissue penetration, and low immunogenicity. As such, these nanomaterials may avidly engage with immune cells and re-engineer the immune system to control cancer and viral infections.
  • the enhanced stability and efficiency of detergent-free nanodisc reconstitution by the peptides of the disclosure are advantages for therapeutic application in these areas.
  • Another application of the materials and methods described herein is the delivery of therapeutic protein complexes (e.g., genome editing reagents) to host cells.
  • amphipathic peptides can enclose protein therapeutics and facilitate their entry into the cells through the unconventional clathrin- independent endocytosis/macropinocytosis.
  • the results described herein demonstrate that nanodiscs obtained as described herein are useful reagents for the delivery of therapeutic proteins.
  • DeFrMSPs are compatible with various membrane proteins and vastly simplify the workflow for the reconstitution of nanodiscs compared to previous methods.
  • Current native nanodiscs are formed using different kinds of polymers that often require careful optimization 7, 27, such as the concentration of divalent ions and pH in the reconstitution buffer.
  • the compatibility of DeFrNDs with bivalent cations makes it a great complimentary system to detergent-free reconstitution systems based on polymers.
  • DeFrNDs are suitable for membrane proteins that are not active in polymer-based NDs (FIGs. 1A-3D and FIGs. 12A-12F).
  • a nanodisc includes a plurality of nanodiscs and equivalents thereof known to those skilled in the art, and so forth.
  • the term “about” signifies not more or less than 10 percent of the stipulated amount.
  • a diameter of about 11 nm may be interpreted to be inclusive of 9.9 nm to 12.1 nm.
  • Open-channel structure of a pentameric ligand-gated ion channel reveals a mechanism of leaflet- specific phospholipid modulation. Nature Communications 13, 7017 (2022). Weber, T. et al. SNAREpins: minimal machinery for membrane fusion. Cell 92, 759-772 (1998). Bao, H. et al. Exocytotic fusion pores are composed of both lipids and proteins. Nat Struct Mol Biol 23, 67-73 (2016). Bao, H. et al. Dynamics and number of trans-SNARE complexes determine nascent fusion pore properties. Nature 554, 260-263 (2016). Geppert, M. et al.
  • Synaptotagmin I a major Ca2+ sensor for transmitter release at a central synapse. Cell 79, 717-727 (1994). Jahn, R. & Sudhof, T.C. Synaptic Vesicles and Exocytosis. Annual Review of Neuroscience 17, 219-246 (1994). Tucker, W.C., Weber, T. & Chapman, E.R. Reconstitution of Ca2+-regulated membrane fusion by synaptotagmin and SNAREs. Science 304, 435-438 (2004). Bai, H. et al. Different states of synaptotagmin regulate evoked versus spontaneous release. Nature Communications 7 (2016). Lorent, J.H. et al.
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Abstract

La présente invention concerne un peptide comprenant une séquence d'acides aminés de formule (I) (DWX1KAFYDKX2AEKX3KEAX4 (SEQ ID NO : 41)) dans laquelle X1 est un acide aminé ayant une chaîne latérale hydrophobe ; X2 est choisi parmi V ou W ; X3 est choisi parmi L ou W ; X4 est choisi parmi F ou W ; et l'extrémité N-terminale du peptide est modifiée avec un acide gras. L'invention concerne également des procédés d'utilisation du peptide pour préparer un nanodisque.
PCT/US2025/010215 2024-01-04 2025-01-03 Peptides et procédé d'utilisation dans la préparation de nanodisques Pending WO2025147594A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030045460A1 (en) * 2000-08-24 2003-03-06 Fogelman Alan M. Orally administered peptides to ameliorate atherosclerosis
WO2016018665A1 (fr) * 2014-07-31 2016-02-04 Uab Research Foundation Peptides e-mimétiques d'apo ayant une puissance supérieure afin de dégager le taux de cholestérol plasmatique

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030045460A1 (en) * 2000-08-24 2003-03-06 Fogelman Alan M. Orally administered peptides to ameliorate atherosclerosis
WO2016018665A1 (fr) * 2014-07-31 2016-02-04 Uab Research Foundation Peptides e-mimétiques d'apo ayant une puissance supérieure afin de dégager le taux de cholestérol plasmatique

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