Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

The biophysical requirements that govern the efficient endosomal escape of designed mini-proteins

Nature Chemistry volume 17, pages 1227–1235 (2025)Cite this article

Subjects

Abstract

The ZF5.3 mini-protein escapes endosomes efficiently to guide proteins into the cytosol and/or nucleus. However, other than the requirement for small or unfoldable cargo and an intact HOPS complex, little is known about how ZF5.3 traverses the limiting endocytic membrane. Here we characterize the requirements for efficient endosomal escape. We confirm that ZF5.3 remains folded at high temperatures and at pH values between 5.5 and 7.5. At lower pH, ZF5.3 unfolds cooperatively upon protonation of Zn(II)-binding His side chains whose pKa matches that of the late endolysosomal lumen. pH-induced unfolding of ZF5.3 is essential for endosomal escape, as an analogue that remains folded at low pH fails to efficiently reach the cytosol. Once unfolded, ZF5.3 interacts in a pH-dependent manner with bis(monoacylglycero)phosphate, a lipid present in the inner leaflet of late endolysosomal membranes. These data provide a biophysical model for HOPS-dependent endosomal escape.

Access through your institution
Buy or subscribe

This is a preview of subscription content, access via your institution

Access options

Access through your institution

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: ZF5.3 unfolds cooperatively at the pH of the late endolysosomal lumen.
Fig. 2: 15N–1H HSQC data provide evidence for pH-induced unfolding of ZF5.3.
Fig. 3: High-resolution NMR structure of ZF5.3 at pH 5.5.
Fig. 4: Residue-level characterization defines low-pH ZF5.3 conformer(s).
Fig. 5: The pH-insensitive ZF5.3 variant BBA5.3 fails to efficiently reach the cytosol.
Fig. 6: ZF5.3 displays pH-dependent binding to LUVs enriched in the late endosomal lipid BMP.

Similar content being viewed by others

Data availability

All data supporting this study are available in the main text or the Supplementary Information. The structural model of ZF5.3 was deposited in the RCSB Protein Data Bank (PDB) under accession number 9AZI. The structural model of zinc-finger protein 473 can be found in the PDB under accession number 2EOZ. Source data are provided with this paper.

Code availability

The MATLAB script used for FCS analysis is available on GitHub at https://github.com/schepartzlab/FCS (ref. 69).

References

  1. Bhardwaj, G. et al. Accurate de novo design of membrane-traversing macrocycles. Cell 185, 3520–3532.e26 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Doherty, G. J. & McMahon, H. T. Mechanisms of endocytosis. Annu. Rev. Biochem. 78, 857–902 (2009).

    Article  CAS  PubMed  Google Scholar 

  3. Mettlen, M., Chen, P.-H., Srinivasan, S., Danuser, G. & Schmid, S. L. Regulation of clathrin-mediated endocytosis. Annu. Rev. Biochem. 87, 871–896 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Frankel, A. D. & Pabo, C. O. Cellular uptake of the tat protein from human immunodeficiency virus. Cell 55, 1189–1193 (1988).

    Article  CAS  PubMed  Google Scholar 

  5. Green, M. & Loewenstein, P. M. Autonomous functional domains of chemically synthesized human immunodeficiency virus tat trans-activator protein. Cell 55, 1179–1188 (1988).

    Article  CAS  PubMed  Google Scholar 

  6. Zhao, Z., Ukidve, A., Kim, J. & Mitragotri, S. Targeting strategies for tissue-specific drug delivery. Cell 181, 151–167 (2020).

    Article  CAS  PubMed  Google Scholar 

  7. Appelbaum, J. S. et al. Arginine topology controls escape of minimally cationic proteins from early endosomes to the cytoplasm. Chem. Biol. 19, 819–830 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. LaRochelle, J. R., Cobb, G. B., Steinauer, A., Rhoades, E. & Schepartz, A. Fluorescence correlation spectroscopy reveals highly efficient cytosolic delivery of certain penta-Arg proteins and stapled peptides. J. Am. Chem. Soc. 137, 2536–2541 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Wissner, R. F., Steinauer, A., Knox, S. L., Thompson, A. D. & Schepartz, A. Fluorescence correlation spectroscopy reveals efficient cytosolic delivery of protein cargo by cell-permeant miniature proteins. ACS Cent. Sci. 4, 1379–1393 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Knox, S. L., Wissner, R., Piszkiewicz, S. & Schepartz, A. Cytosolic delivery of argininosuccinate synthetase using a cell-permeant miniature protein. ACS Cent. Sci. 7, 641–649 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Zoltek, M. et al. HOPS-dependent endosomal escape demands protein unfolding. ACS Cent. Sci. 10, 860–870 (2024).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Zhang, X. et al. Dose-dependent nuclear delivery and transcriptional repression with a cell-penetrant MeCP2. ACS Cent. Sci. 9, 277–288 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Shen, F. et al. A cell-permeant nanobody-based degrader that induces fetal hemoglobin. ACS Cent. Sci. 8, 1695–1703 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Steinauer, A. et al. HOPS-dependent endosomal fusion required for efficient cytosolic delivery of therapeutic peptides and small proteins. Proc. Natl Acad. Sci. USA 116, 512–521 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Mellman, I., Fuchs, R. & Helenius, A. Acidification of the endocytic and exocytic pathways. Annu. Rev. Biochem. 55, 663–700 (1986).

    Article  CAS  PubMed  Google Scholar 

  16. Krizek, B. A., Amann, B. T., Kilfoil, V. J., Merkle, D. L. & Berg, J. M. A consensus zinc finger peptide: design, high-affinity metal binding, a pH-dependent structure, and a His to Cys sequence variant. J. Am. Chem. Soc. 113, 4518–4523 (1991).

    Article  CAS  Google Scholar 

  17. Urbani, A. et al. The metal binding site of the hepatitis C virus NS3 protease: a spectroscopic investigation. J. Biol. Chem. 273, 18760–18769 (1998).

    Article  CAS  PubMed  Google Scholar 

  18. Bodenhausen, G. & Ruben, D. J. Natural abundance nitrogen-15 NMR by enhanced heteronuclear spectroscopy. Chem. Phys. Lett. 69, 185–189 (1980).

    Article  CAS  Google Scholar 

  19. Pelton, J. G. et al. Structures of active site histidine mutants of IIIGlc, a major signal-transducing protein in Escherichia coli: effects on the mechanisms of regulation and phosphoryl transfer. J. Biol. Chem. 271, 33446–33456 (1996).

    Article  CAS  PubMed  Google Scholar 

  20. Cavanagh, J., Fairbrother, W. J., Palmer, A. G., Rance, M. & Skelton, N. J. in Protein NMR Spectroscopy 2nd edn (eds Cavanagh, J. et al.) 781–817 (Academic, 2007).

  21. Grzesiek, S. & Bax, A. Correlating backbone amide and side chain resonances in larger proteins by multiple relayed triple resonance NMR. J. Am. Chem. Soc. 114, 6291–6293 (1992).

    Article  CAS  Google Scholar 

  22. Skinner, S. P. et al. CcpNmr AnalysisAssign: a flexible platform for integrated NMR analysis. J. Biomol. NMR 66, 111–124 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Klukowski, P., Riek, R. & Güntert, P. Rapid protein assignments and structures from raw NMR spectra with the deep learning technique ARTINA. Nat. Commun. 13, 6151 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Klukowski, P., Riek, R. & Güntert, P. NMRtist: an online platform for automated biomolecular NMR spectra analysis. Bioinformatics 39, btad066 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Shen, Y. & Bax, A. in Artificial Neural Networks (ed. Cartwright, H.) 17–32 (Springer, 2015).

  26. Schmidt, E. & Güntert, P. A new algorithm for reliable and general NMR resonance assignment. J. Am. Chem. Soc. 134, 12817–12829 (2012).

    Article  CAS  PubMed  Google Scholar 

  27. Güntert, P. Automated NMR structure calculation with CYANA. Methods Mol. Biol. 278, 353–378 (2004).

    PubMed  Google Scholar 

  28. Güntert, P. & Buchner, L. Combined automated NOE assignment and structure calculation with CYANA. J. Biomol. NMR 62, 453–471 (2015).

    Article  PubMed  Google Scholar 

  29. Schwieters, C. D., Kuszewski, J. J., Tjandra, N. & Clore, G. M. The Xplor-NIH NMR molecular structure determination package. J. Magn. Reson. 160, 65–73 (2003).

    Article  CAS  PubMed  Google Scholar 

  30. Schwieters, C. D., Bermejo, G. A. & Clore, G. M. Xplor‐NIH for molecular structure determination from NMR and other data sources. Protein Sci. Publ. Protein Soc. 27, 26–40 (2018).

    Article  CAS  Google Scholar 

  31. Meng, E. C. et al. UCSF ChimeraX: tools for structure building and analysis. Protein Sci. 32, e4792 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Williamson, M. P. in Modern Magnetic Resonance (ed. Webb, G. A.) 995–1012 (Springer, 2018).

  33. Vuister, G. W. & Bax, A. Quantitative J correlation: a new approach for measuring homonuclear three-bond J(HNHα) coupling constants in 15N-enriched proteins. J. Am. Chem. Soc. 115, 7772–7777 (1993).

    Article  CAS  Google Scholar 

  34. Struthers, M., Ottesen, J. J. & Imperiali, B. Design and NMR analyses of compact, independently folded BBA motifs. Fold. Des. 3, 95–103 (1998).

    Article  CAS  PubMed  Google Scholar 

  35. Ingólfsson, H. I. et al. Lipid organization of the plasma membrane. J. Am. Chem. Soc. 136, 14554–14559 (2014).

    Article  PubMed  Google Scholar 

  36. Kobayashi, T. et al. Separation and characterization of late endosomal membrane domains. J. Biol. Chem. 277, 32157–32164 (2002).

    Article  CAS  PubMed  Google Scholar 

  37. Chevallier, J. et al. Lysobisphosphatidic acid controls endosomal cholesterol levels. J. Biol. Chem. 283, 27871–27880 (2008).

    Article  CAS  PubMed  Google Scholar 

  38. Matsuo, H. et al. Role of LBPA and Alix in multivesicular liposome formation and endosome organization. Science 303, 531 (2004).

    Article  CAS  PubMed  Google Scholar 

  39. Pattanakitsakul, S. et al. Association of Alix with late endosomal lysobisphosphatidic acid is important for dengue virus infection in human endothelial cells. J. Proteome Res. 9, 4640–4648 (2010).

    Article  CAS  PubMed  Google Scholar 

  40. Roth, S. L. & Whittaker, G. R. Promotion of vesicular stomatitis virus fusion by the endosome-specific phospholipid bis(monoacylglycero)phosphate (BMP). FEBS Lett. 585, 865–869 (2011).

    Article  CAS  PubMed  Google Scholar 

  41. Erazo-Oliveras, A. et al. The late endosome and its lipid BMP act as gateways for efficient cytosolic access of the delivery agent dfTAT and its macromolecular cargos. Cell Chem. Biol. 23, 598–607 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Brock, D. J. et al. Mechanism of cell penetration by permeabilization of late endosomes: interplay between a multivalent TAT peptide and bis(monoacylglycero)phosphate. Cell Chem. Biol. 27, 1296–1307.e5 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Diaz, J. et al. Elucidating the impact of payload conjugation on the cell-penetrating efficiency of the endosomal escape peptide dfTAT: implications for future designs for CPP-based delivery systems. Bioconjug. Chem. 34, 1861–1872 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Yang, S.-T., Zaitseva, E., Chernomordik, L. V. & Melikov, K. Cell-penetrating peptide induces leaky fusion of liposomes containing late endosome-specific anionic lipid. Biophys. J. 99, 2525–2533 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Brock, D. J. et al. Efficient cell delivery mediated by lipid-specific endosomal escape of supercharged branched peptides. Traffic 19, 421–435 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Melo, A. M., Prieto, M. & Coutinho, A. in Fluorescence Spectroscopy and Microscopy: Methods and Protocols (eds Engelborghs, Y. & Visser, A. J. W. G.) 575–595 (Humana, 2014).

  47. Rhoades, E., Ramlall, T. F., Webb, W. W. & Eliezer, D. Quantification of α-synuclein binding to lipid vesicles using fluorescence correlation spectroscopy. Biophys. J. 90, 4692–4700 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Urbančič, I. et al. Lipid composition but not curvature is the determinant factor for the low molecular mobility observed on the membrane of virus-like vesicles. Viruses 10, 415 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  49. Middleton, E. R. & Rhoades, E. Effects of curvature and composition on α-synuclein binding to lipid vesicles. Biophys. J. 99, 2279–2288 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Ladokhin, A. S. & White, S. H. Folding of amphipathic alpha-helices on membranes: energetics of helix formation by melittin. J. Mol. Biol. 285, 1363–1369 (1999).

    Article  CAS  PubMed  Google Scholar 

  51. Wieprecht, T., Apostolov, O. & Seelig, J. Binding of the antibacterial peptide magainin 2 amide to small and large unilamellar vesicles. Biophys. Chem. 85, 187–198 (2000).

    Article  CAS  PubMed  Google Scholar 

  52. Cymer, F., von Heijne, G. & White, S. H. Mechanisms of integral membrane protein insertion and folding. J. Mol. Biol. 427, 999–1022 (2015).

    Article  CAS  PubMed  Google Scholar 

  53. Schnell, D. J. & Hebert, D. N. Protein translocons: multifunctional mediators of protein translocation across membranes. Cell 112, 491–505 (2003).

    Article  CAS  PubMed  Google Scholar 

  54. Lee, J., Oldham, M. L., Manon, V. & Chen, J. Principles of peptide selection by the transporter associated with antigen processing. Proc. Natl Acad. Sci. USA 121, e2320879121 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Ahlbach, C. L. et al. Beyond cyclosporine A: conformation-dependent passive membrane permeabilities of cyclic peptide natural products. Future Med. Chem. 7, 2121–2130 (2015).

    Article  CAS  PubMed  Google Scholar 

  56. Kino, T. et al. FK-506, a novel immunosuppressant isolated from a Streptomyces. II. Immunosuppressive effect of FK-506 in vitro. J. Antibiot. 40, 1256–1265 (1987).

    Article  CAS  Google Scholar 

  57. Kling, A. et al. Antibiotics. Targeting DnaN for tuberculosis therapy using novel griselimycins. Science 348, 1106–1112 (2015).

    Article  CAS  PubMed  Google Scholar 

  58. Bockus, A. T. et al. Probing the physicochemical boundaries of cell permeability and oral bioavailability in lipophilic macrocycles inspired by natural products. J. Med. Chem. 58, 4581–4589 (2015).

    Article  CAS  PubMed  Google Scholar 

  59. Vázquez-Maldonado, A. L., Chen, T., Rodriguez, D., Zoltek, M. & Schepartz, A. Hastened fusion-dependent endosomal escape improves activity of delivered enzyme cargo. ACS Cent. Sci. 11, 574–582 (2025).

    Article  PubMed  PubMed Central  Google Scholar 

  60. Woods, B. et al. Confinement effect on hydrolysis in small lipid vesicles. Chem. Sci. 14, 2616–2623 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Grochmal, A., Prout, L., Makin-Taylor, R., Prohens, R. & Tomas, S. Modulation of reactivity in the cavity of liposomes promotes the formation of peptide bonds. J. Am. Chem. Soc. 137, 12269–12275 (2015).

    Article  CAS  PubMed  Google Scholar 

  62. van der Beek, J., de Heus, C., Sanza, P., Liv, N. & Klumperman, J. Loss of the HOPS complex disrupts early-to-late endosome transition, impairs endosomal recycling and induces accumulation of amphisomes. Mol. Biol. Cell 35, ar40 (2024).

    Article  PubMed  PubMed Central  Google Scholar 

  63. Markley, J. L. et al. Recommendations for the presentation of NMR structures of proteins and nucleic acids (IUPAC Recommendations 1998). Pure Appl. Chem. 70, 117–142 (1998).

    Article  CAS  Google Scholar 

  64. Findeisen, M., Brand, T. & Berger, S. A 1H-NMR thermometer suitable for cryoprobes. Magn. Reson. Chem. MRC 45, 175–178 (2007).

    Article  CAS  PubMed  Google Scholar 

  65. Stirling, D. R. et al. CellProfiler 4: improvements in speed, utility and usability. BMC Bioinformatics 22, 433 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  66. Rusu, L., Gambhir, A., McLaughlin, S. & Rädler, J. Fluorescence correlation spectroscopy studies of peptide and protein binding to phospholipid vesicles. Biophys. J. 87, 1044–1053 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Borenstain, V. & Barenholz, Y. Characterization of liposomes and other lipid assemblies by multiprobe fluorescence polarization. Chem. Phys. Lipids 64, 117–127 (1993).

    Article  CAS  PubMed  Google Scholar 

  68. Lakowicz, J. Principles of Fluorescence Spectroscopy (Springer, 2010).

  69. Cobb, G. & DeWitt, D. FCS. GitHub https://github.com/schepartzlab/FCS/blob/master/fcs_new_2022.m (2022).

Download references

Acknowledgements

This work was supported by the National Science Foundation CHE-2203903 (A.S.) and the National Institute of General Medical Sciences of the National Institutes of Health under award number GM134963 (A.S.). The UCSF NMR core facility is funded by an NIH Office of Research Infrastructure Programs (ORIP) High-End Instrumentation (HEI) grant under award number 1S10OD023455-01A1, and a Sandler Program for Breakthrough Biomedical Research TMC award. This material is based upon work supported by the National Science Foundation Graduate Research Fellowship Program under grant number DGE 2146752 (J.G.) and the National Science Foundation MPS-Ascend Postdoctoral Research Fellowship under grant number CHE-2213241 (D.B.B.). Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Author information

Author notes

  1. These authors contributed equally: Jonathan Giudice, Daniel D. Brauer.

Authors and Affiliations

  1. Department of Chemistry, University of California, Berkeley, Berkeley, CA, USA

    Jonathan Giudice, Daniel D. Brauer, Angel L. Vázquez-Maldonado, Neville Dadina & Alanna Schepartz

  2. Department of Molecular and Cellular Biology, University of California, Berkeley, Berkeley, CA, USA

    Madeline Zoltek & Alanna Schepartz

  3. School of Pharmacy, University of California, San Francisco, San Francisco, CA, USA

    Mark Kelly

  4. California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, Berkeley, CA, USA

    Alanna Schepartz

  5. Chan Zuckerberg Biohub, San Francisco, CA, USA

    Alanna Schepartz

  6. Arc Institute, Palo Alto, CA, USA

    Alanna Schepartz

Authors
  1. Jonathan Giudice
    View author publications

    Search author on:PubMed Google Scholar

  2. Daniel D. Brauer
    View author publications

    Search author on:PubMed Google Scholar

  3. Madeline Zoltek
    View author publications

    Search author on:PubMed Google Scholar

  4. Angel L. Vázquez-Maldonado
    View author publications

    Search author on:PubMed Google Scholar

  5. Neville Dadina
    View author publications

    Search author on:PubMed Google Scholar

  6. Mark Kelly
    View author publications

    Search author on:PubMed Google Scholar

  7. Alanna Schepartz
    View author publications

    Search author on:PubMed Google Scholar

Contributions

J.G., D.D.B., M.K. and A.S. designed the project. J.G. led NMR experiments and experiments involving BBA5.3. D.D.B. led experiments with liposomes. M.Z. designed and performed the CD experiments. A.L.V.-M. synthesized and delivered Rho-ZF5.3. N.D. oversaw the STED experiments. M.K. oversaw all the NMR experiments. J.G., D.B.B., M.K. and A.S. analysed the experiments and prepared the manuscript.

Corresponding authors

Correspondence to Mark Kelly or Alanna Schepartz.

Ethics declarations

Competing interests

A.S. receives research support from Merck, Amgen and Novo Nordisk. A.S. is a science advisory board member for both Lycia and Evozyne. The remaining authors declare no competing interests.

Peer review

Peer review information

Nature Chemistry thanks the anonymous reviewers for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Additional data in support of Fig. 1.

Deconvoluted mass spectra of ZF5.3 generated a, recombinantly or b, by solid phase synthesis. c, CD spectra of recombinant ZF5.3 in Reconstitution Buffer at pH 7.5 (20 mM Tris–HCl, 150 mM KCl, 1 mM TCEP, and 100 µM ZnCl2) at concentrations between 25 µM and 250 µM. d, CD spectra of recombinant ZF5.3 in Reconstitution Buffer at pH 7.5 after experiencing a pH 4.5 buffer without the presence of any excess ZnCl2. e, Deconvoluted mass spectrum of Rho-ZF5.3 (pink, used for confocal microscopy, flow cytometry, and FCS experiments) prepared using solid-phase peptide synthesis as described in Methods. f, Deconvoluted mass spectrum of AF488-ZF5.3 (green, used for confocal microscopy) prepared using solid-phase peptide synthesis as described in Methods. g, Sequence of Rho-ZF5.3. h, Sequence of AF488-ZF5.3.

Source data

Extended Data Fig. 2 Additional data in support of Fig. 2.

a-c,15N-1H heteronuclear quantum coherence (HSQC) spectroscopy at pH 7.5, 6.5, and 3.5. 15N-1H HSQC of ZF5.3 (800 µM) was acquired in a 20 mM citrate–phosphate buffer containing 10% D2O, 100 mM NaCl, 2 mM TCEP and 1.6 mM ZnCl2 at pH 7.5, 6.5, and 3.5.

Extended Data Fig. 3 Assignment of His tautomers in ZF5.3 at pH 5.5.

a, Results of hbCBcgcdHD experiments that provide the 13Cα and 13Cꞵ chemical shifts of each of the three His residues in ZF5.3, His15, His19, and His23. b, Results of a 2D CBHD experiment that correlates the chemical shift of each 13Cꞵ with the corresponding 1Hδ. c, 13C-CT HSQC provides the chemical shift of 13Cδ and 1Hδ. If the chemical shift of 13Cδ is less than 122 ppm the His residue is said to be in the α-tautomeric state and if the chemical shift of 13Cδ is greater than 122 ppm it is in the ꞵ-tautomeric state. d, Assignment of His15 as the α-tautomer and Zn(II) coordinating His19 and His23 as the ꞵ-tautomer.

Extended Data Fig. 4 2D [15N-1H]- HMBC spectra provide evidence for His protonation at low pH.

a-f, [15N-1H] heteronuclear multiple bond quantum coherence spectroscopy (HMBC) at pH 7.5, 6.5, 5.5, 5.0, 4.5, and 3.5 provides the chemical shifts of 15N and 1H atoms in His residues in ZF5.3.

Extended Data Fig. 5 Alignment of ZF5.3 ensemble at pH 5.5 with ZF473.

a, Overlay of a single ZF473 conformer (mint) with the ZF5.3 conformer (pink) that resulted in the lowest pruned atom pair RMSD value of 0.586 Å. b, Overlay of a single ZF473 conformer (mint) with ZF5.3 conformer (pink) with the lowest RMSD value across all 27 pairs of 0.812 Å.

Extended Data Fig. 6 Additional data in support of Fig. 5.

Deconvoluted mass spectra of HPLC-purified a, BBA5.3 and b, Rho-BBA5.3 c, AF488-BBA5.3. d, Overlay of the wavelength-dependent CD spectra of BBA5.3 at concentrations between 50 and 125 µM in a Resuspension Buffer without Zn(II) containing 20 mM Tris, 150 mM KCl, and 1 mM TCEP at 37 °C. e, Plot of the mean residue ellipticity at 222 nm of 125 µM BBA5.3 in Resuspension Buffer without Zn(II) at pH 7.5 (teal) or pH 4.5 (red) at every 2 °C between 5 °C and 95 °C. f, Wavelength-dependent CD spectra of 125 µM (0.3 mg/mL) BBA5.3 at pH 7.5 and 4.5 in Resuspension Buffer without Zn(II) at 37 °C. g, Wavelength-dependent CD spectra of BBA5.3 (125 µM) before and after being heated from 5 to 95 °C. The sequences of h, Rho-BBA5.3 and i, AF488-BBA5.3 prepared using solid phase synthesis. j, Flow cytometry histograms for Lissamine rhodamine B signal from Saos-2 cells treated with media (black), Rho-ZF5.3 (pink), or Rho-BBA5.3 (blue) at specified concentrations.

Source data

Extended Data Fig. 7 LC-HRMS analysis of ZF5.3 and BBA5.3 integrity post-delivery into Saos-2 cells.

a, Primary sequences of dBio-ZF5.3 and dBio-BBA5.3, each of which carries a desthiobiotin affinity tag. b, m/z values of dBio-ZF5.3 (pink) or c, dBio-BBA5.3 (blue) eluted from Streptavidin-conjugated magnetic beads in vitro (dotted), extracted from Saos-2 cells postdelivery (solid), and extracted ion chromatogram for starred m/z value for an in vitro sample (dotted), post-delivery sample (solid), non-treated in vitro sample (dotted black), and a non-treated post-delivery sample (solid black).

Source data

Extended Data Fig. 8 Confocal images and extent of colocalization of Saos-2 cells expressing GFP-tagged organelle markers with either Rho-ZF5.3 or Rho-BBA5.3.

Representative confocal images of Saos-2 cells indicating the extent of colocalization between the signals due to a, Rho-ZF5.3 (magenta) or b, Rho-BBA5.3 and GFP organelle markers (green) for early endosomes (Rab5), late endosomes (Rab7), lysosomes (Lamp1), and mitochondria (PDH). Regions of the cell in which the magenta and green signals overlap appear white. Scale bars = 10 μm. c, Plot illustrating the calculated Manders’ overlap correlation coefficients (mean ± SEM) for each organelle for two biological replicates. Outliers were removed using the ROUT method (Q = 0.1%). For Rab5 n = 83 and 48 cells, Rab7 n = 92 and 55 cells, Lamp1 n = 114 and 88 cells, and for PDH n = 68 and 93 cells for Rho-ZF5.3 and Rho-BBA5.3 respectively.

Source data

Extended Data Fig. 9 Localization of Rho-ZF5.3 and Rho-BBA5.3 with lysosomal marker LAMP1-SiR using STED microscopy.

a, Whole cell STED images of Rho-ZF5.3 and LAMP1-SiR show the sub localization of Rho-ZF5.3 within the lysosomal lumen. b, Similarly, whole cell STED images of Rho-BBA5.3 and LAMP1-SiR depict a similar sub-organellar localization. Regions of interest across 6 different cells (3 for Rho-ZF5.3 and 3 for Rho-BBA5.3) were imaged at a higher spatial sampling rate to determine differences in sub-organellar organization of c, Rho-ZF5.3 and d, Rho-BBA5.3. Line profiles across vesicles of interest were plotted. An assortment of phenotypes is seen for both Rho-ZF5.3 and Rho-BBA5.3. Scale bar 5 µm (a, b), and 1 µm (c, d). Images are representative of two biological replicates per condition.

Source data

Extended Data Fig. 10 pH- and BMP-dependent binding of Rho-ZF5.3 to large unilamellar vesicles is observed via fluorescence anisotropy.

a, Equilibrium binding curves of BMP + LE/LY LUVs and b, BMP − LE/LY LUVs to 100 nM Rho-ZF5.3 and Rho-BBA5.3 at pH 4.5 and 7.5. Each point represents the mean ± SEM of anisotropy, n = 3 replicate wells.

Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1–4 and Tables 1–9.

Reporting Summary

Supplemental Video 1

Video of time-lapse images corresponding to Fig. 5d,e: 1 µM AF488-ZF5.3 (green) and 2 µM Rho-BBA5.3 treatment condition is shown.

Supplemental Video 2

Video of time-lapse image corresponding to Fig. 5d,e: 1 µM Rho-ZF5.3 (magenta) and 2 µM AF488-BBA5.3 (green) treatment condition is shown.

Supplemental Data 1

Source data for plots presented in Supplementary Fig. 1.

Supplemental Data 2

Source data for plots presented in Supplementary Fig. 4.

Source data

Source Data Fig. 1

Each tab in the Excel file corresponds to the panel in the figure. Tabs b–e contain raw CD data. Tab c also contains statistical source data.

Source Data Fig. 2

The chemical shift values in panel d are included as an Excel file.

Source Data Fig. 4

Each tab in the Excel files corresponds to the panel in the figure. Tab a contains the raw chemical shift values for high and low pH conformers as well as the calculated CSP which is the final data represented in the figure. Tabs c and d contain the NOEs measured at high and low pH used to create panels c and d. Tab e contains the chi-1 side chain torsional angles measured in Fig. 4e.

Source Data Fig. 5

Source data for panels e–g are included as individual tabs in the Excel file.

Source Data Fig. 6

Each tab contains the source data for the corresponding panel of Fig. 6b–e. The tab for Fig. 6c contains relevant statistical testing information.

Source Data Extended Data Fig. 1

Each tab in the Excel file corresponds to the panel in the figure. Tabs a,b,e,f contain mass spectra data and tabs c and d contain CD data.

Source Data Extended Data Fig. 6

The source data for Extended Data Fig. 6a–g are included in an Excel file, with each panel as a separate tab.

Source Data Extended Data Fig. 7

The source data for panel c is included as an Excel file with the statistical source data as well.

Source Data Extended Data Fig. 8

Each tab contains the intensity information for the line profile of each corresponding image. Samples are numbered 1–6, with 1–3 corresponding to the top row of images and 4–6 corresponding to the bottom row.

Source Data Extended Data Fig. 9

Each tab in the Excel file corresponds to the panel in the figure. Tabs b,c,f contain mass spectra data.

Source Data Extended Data Fig. 10

Each tab contains the source data for Extended Data Fig. 10a,b. An additional tab contains statistical information and fitting parameters for each curve.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Giudice, J., Brauer, D.D., Zoltek, M. et al. The biophysical requirements that govern the efficient endosomal escape of designed mini-proteins. Nat. Chem. 17, 1227–1235 (2025). https://doi.org/10.1038/s41557-025-01846-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue date:

  • DOI: https://doi.org/10.1038/s41557-025-01846-4

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research