Skip to main content
SpringerLink
Log in

Femtosecond UV-laser pulses to unveil protein–protein interactions in living cells

  • Original Article
  • Published:
Cellular and Molecular Life Sciences Aims and scope Submit manuscript

Abstract

A hallmark to decipher bioprocesses is to characterize protein–protein interactions in living cells. To do this, the development of innovative methodologies, which do not alter proteins and their natural environment, is particularly needed. Here, we report a method (LUCK, Laser UV Cross-linKing) to in vivo cross-link proteins by UV-laser irradiation of living cells. Upon irradiation of HeLa cells under controlled conditions, cross-linked products of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were detected, whose yield was found to be a linear function of the total irradiation energy. We demonstrated that stable dimers of GAPDH were formed through intersubunit cross-linking, as also observed when the pure protein was irradiated by UV-laser in vitro. We proposed a defined patch of aromatic residues located at the enzyme subunit interface as the cross-linking sites involved in dimer formation. Hence, by this technique, UV-laser is able to photofix protein surfaces that come in direct contact. Due to the ultra-short time scale of UV-laser-induced cross-linking, this technique could be extended to weld even transient protein interactions in their native context.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Goodsell DS, Olson AJ (2000) Structural symmetry and protein function. Annu Rev Biophys Biomol Struct 29:105–153

    Article  PubMed  CAS  Google Scholar 

  2. Levy ED, Pereira-Leal JB, Chothia C, Teichmann SA (2006) 3D complex: a structural classification of protein complexes. PLoS Comput Biol 2:e155

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  3. Tang X, Bruce JE (2009) Chemical cross-linking for protein-protein interaction studies. Methods Mol Biol 492:283–293

    Article  PubMed  CAS  Google Scholar 

  4. Subbotin RI, Chait BT (2014) A pipeline for determining protein-protein interactions and proximities in the cellular milieu. Mol Cell Proteomics 13:2824–2835

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  5. Sinz A (2006) Chemical cross-linking and mass spectrometry to map three-dimensional protein structures and protein-protein interactions. Mass Spectrom Rev 25:663–682

    Article  PubMed  CAS  Google Scholar 

  6. Bruce JE (2012) In vivo protein complex topologies: sights through a cross-linking lens. Proteomics 12:1565–1575

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  7. Zhang H, Tang X, Munske GR, Tolic N, Anderson GA, Bruce JE (2009) Identification of protein-protein interactions and topologies in living cells with chemical cross-linking and mass spectrometry. Mol Cell Proteomics 8:409–420

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  8. Walzthoeni T, Leitner A, Stengel F, Aebersold R (2013) Mass spectrometry supported determination of protein complex structure. Curr Opin Struct Biol 23:252–260

    Article  PubMed  CAS  Google Scholar 

  9. Guerrero C, Tagwerker C, Kaiser P, Huang L (2006) An integrated mass spectrometry-based proteomic approach: quantitative analysis of tandem affinity-purified in vivo cross-linked protein complexes (QTAX) to decipher the 26 S proteasome-interacting network. Mol Cell Proteomics MCP 5:366–378

    Article  PubMed  CAS  Google Scholar 

  10. Sinz A (2014) The advancement of chemical cross-linking and mass spectrometry for structural proteomics: from single proteins to protein interaction networks. Expert Rev Proteomics 11:733–743

    Article  PubMed  CAS  Google Scholar 

  11. Gingras A, Gstaiger M, Raught B, Aebersold R, Raught RAB (2007) Analysis of protein complexes using mass spectrometry. Nat Rev Mol Cell Biol 8:645–654

    Article  PubMed  CAS  Google Scholar 

  12. Chavez JD, Weisbrod CR, Zheng C, Eng JK, Bruce JE (2013) Protein interactions, post-translational modifications and topologies in human cells. Mol Cell Proteomics 12:1451–1467

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  13. Zheng C, Yang L, Hoopmann MR, Eng JK, Tang X, Weisbrod CR et al (2011) Cross-linking measurements of in vivo protein complex topologies. Mol Cell Proteomics 10(M110):006841

    PubMed  Google Scholar 

  14. Kaake RM, Wang X, Burke A, Yu C, Kandur W, Yang Y et al (2014) A new in vivo cross-linking mass spectrometry platform to define protein-protein interactions in living cells. Mol Cell Proteomics 13:3533–3543

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  15. Lougheed KE, Bennett MH, Williams HD (2014) An in vivo crosslinking system for identifying mycobacterial protein-protein interactions. J Microbiol Methods 105:67–71

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  16. Suchanek M, Radzikowska A, Thiele C (2005) Photo-leucine and photo-methionine allow identification of protein-protein interactions in living cells. Nat Methods 2:261–267

    Article  PubMed  CAS  Google Scholar 

  17. Lin S, He D, Long T, Zhang S, Meng R, Chen PR (2014) Genetically encoded cleavable protein photo-cross-linker. J Am Chem Soc 136:11860–11863

    Article  PubMed  CAS  Google Scholar 

  18. Leo G, Altucci C, Bourgoin-Voillard S, Gravagnuolo AM, Esposito R, Marino G et al (2013) Ultraviolet laser-induced cross-linking in peptides. Rapid Commun Mass Spectrom 27:1660–1668

    Article  PubMed  CAS  Google Scholar 

  19. Petrotchenko EV, Borchers CH (2010) Crosslinking combined with mass spectrometry for structural proteomics. Mass Spectrom Rev 29:862–876

    Article  PubMed  CAS  Google Scholar 

  20. Keskin O, Gursoy A, Ma B, Nussinov R (2008) Principles of protein-protein interactions: what are the preferred ways for proteins to interact? Chem Rev 108:1225–1244

    Article  PubMed  CAS  Google Scholar 

  21. Jones S, Thornton JM (1996) Principles of protein-protein interactions. Proc Natl Acad Sci 93:13–20

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  22. Mezei M (2015) Statistical properties of protein-protein interfaces. Algorithms 8:92–99

    Article  Google Scholar 

  23. Falchi F, Caporuscio F, Recanatini M (2014) Structure-based design of small-molecule protein–protein interaction modulators: the story so far. Future Med Chem 6:343–357

    Article  PubMed  CAS  Google Scholar 

  24. Sirover MA (2014) Structural analysis of glyceraldehyde-3-phosphate dehydrogenase functional diversity. Int J Biochem Cell Biol 57:20–26

    Article  PubMed  CAS  Google Scholar 

  25. Henderson B, Martin AC (2014) Protein moonlighting: a new factor in biology and medicine. Biochem Soc Trans 42:1671–1678

    Article  PubMed  CAS  Google Scholar 

  26. Chuang DM, Ishitani R (1996) A role for GAPDH in apoptosis and neurodegeneration. Nat Med 2:609–610b

    Article  PubMed  CAS  Google Scholar 

  27. Jenkins JL, Tanner JJ (2006) High-resolution structure of human d-glyceraldehyde-3-phosphate dehydrogenase. Acta Crystallogr D Biol Crystallogr 62:290–301

    Article  PubMed  CAS  Google Scholar 

  28. Boyd RW (2008) Nonlinear optics. Elsevier Academic Press, ISBN:0123694701

  29. Valadan M, D’Ambrosio D, Gesuele F, Velotta R, Altucci C (2015) Temporal and spectral characterization of femtosecond deep-UV chirped pulses. Laser Phys Lett 12: 025302. IOP Select http://iopscience.iop.org/1612-202X/12/2/025302/article

  30. Altucci C, Nebbioso A, Benedetti R, Esposito R, Carafa V, Conte M et al (2012) Nonlinear protein—nucleic acid crosslinking induced by femtosecond UV laser pulses in living cells. Laser Phys Lett 9:234–239

    Article  CAS  Google Scholar 

  31. Plowman JE, Deb-Choudhury S, Grosvenor AJ, Dyer JM (2013) Protein oxidation: identification and utilisation of molecular markers to differentiate singlet oxygen and hydroxyl radical-mediated oxidative pathways. Photochem Photobiol Sci 12:1960–1967

    Article  PubMed  CAS  Google Scholar 

  32. Williams TJ, Reay AJ, Whitwood AC, Fairlamb IJ (2014) A mild and selectivePd-mediated methodology for the synthesis of highly fluorescent 2-arylated tryptophans and tryptophan-containing peptides: a catalytic role for Pd(0) nanoparticles? Chem Commun (Camb) 50:3052–3054

    Article  CAS  Google Scholar 

  33. Pappenberger G, Schurig H, Jaenicke R (1997) Disruption of an ionic network leads to accelerated thermal denaturation of d-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima. J Mol Biol 274:676–683

    Article  PubMed  CAS  Google Scholar 

  34. He RQ, Li YG, Wu XQ, Li L (1995) Inactivation and conformation changes of the glycated and non-glycated d-glyceraldehyde-3-phosphate dehydrogenase during guanidine-HCl denaturation. Biochim Biophys Acta 1253:47–56

    Article  PubMed  Google Scholar 

  35. Pattison DI, Rahmanto AS, Davies MJ (2011) Photo-oxidation of proteins. Photochem Photobiol Sci 11:38–53

    Article  PubMed  Google Scholar 

  36. Fasman GD (1996) Circular dichroism and the conformational analysis of biomolecules. Springer Science & Business Media, New York

    Book  Google Scholar 

  37. Nakajima H, Amano W, Fukuhara A, Kubo T, Misaki S, Azuma YT et al (2009) An aggregate-prone mutant of human glyceraldehyde-3-phosphate dehydrogenase augments oxidative stress-induced cell death in SH-SY5Y cells. Biochem Biophys Res Commun 390:1066–1071

    Article  PubMed  CAS  Google Scholar 

  38. Nakajima H, Amano W, Kubo T, Fukuhara A, Ihara H, Azuma YT et al (2009) Glyceraldehyde-3-phosphate dehydrogenase aggregate formation participates in oxidative stress-induced cell death. J Biol Chem 284:34331–34341

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  39. Kryukov P, Letokhov V, Nikogosyan D, Borodavkin A, Budowsky E, Simukova N (1979) Multiquantum photoreactions of nucleic acid components in aqueous solution by powerful ultraviolet picosecond radiation. Chem Phys Lett 61:375–379

    Article  CAS  Google Scholar 

  40. Nikogosyan DN (1990) Two-quantum UV photochemistry of nucleic acids: comparison with conventional low-intensity UV photochemistry and radiation chemistry. Int J Radiat Biol 57:233–299

    Article  PubMed  CAS  Google Scholar 

  41. Klockenbusch C, Kast J (2010) Optimization of formaldehyde cross-linking for protein interaction analysis of non-tagged integrin B1. J Biomed Biotechnol 2010:927585

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  42. Gu L, Li C, Aach J, Hill DE, Vidal M, Church GM (2014) Multiplex single-molecule interaction profiling of DNA-barcoded proteins. Nature 515:554–557

    Article  PubMed  CAS  PubMed Central  Google Scholar 

Download references

Acknowledgments

This research was partially supported by “Programma STAR” of University of Naples Federico II, “Compagnia di San Paolo” and “Istituto Banco di Napoli—Fondazione”. We acknowledge the STRAIN PROJECT (POR Campania FSE 2007/2013 CUP B25B0900000000), which provided a postdoctoral fellowship to M.C. The authors are deeply indebted to Professor Gennaro Marino for his interest in this work and very useful comments and suggestions.

Author information

Author notes

    Authors and Affiliations

    1. Department of Chemical Sciences, University of Naples Federico II, 80126, Naples, Italy

      Francesco Itri, Daria M. Monti, Roberto Vinciguerra, Marco Chino, Angelina Lombardi, Leila Birolo, Renata Piccoli & Angela Arciello

    2. Istituto Nazionale di Biostrutture e Biosistemi (INBB), Rome, Italy

      Daria M. Monti, Renata Piccoli & Angela Arciello

    3. Department of Physics, University of Naples Federico II, 80126, Naples, Italy

      Bartolomeo Della Ventura, Felice Gesuele, Raffaele Velotta & Carlo Altucci

    4. Consorzio Nazionale Interuniversitario per le Scienze Fisiche della Materia (CNISM), UdR, Naples, Italy

      Raffaele Velotta & Carlo Altucci

    Authors
    1. Francesco Itri
      View author publications

      You can also search for this author in PubMed Google Scholar

    2. Daria M. Monti
      View author publications

      You can also search for this author in PubMed Google Scholar

    3. Bartolomeo Della Ventura
      View author publications

      You can also search for this author in PubMed Google Scholar

    4. Roberto Vinciguerra
      View author publications

      You can also search for this author in PubMed Google Scholar

    5. Marco Chino
      View author publications

      You can also search for this author in PubMed Google Scholar

    6. Felice Gesuele
      View author publications

      You can also search for this author in PubMed Google Scholar

    7. Angelina Lombardi
      View author publications

      You can also search for this author in PubMed Google Scholar

    8. Raffaele Velotta
      View author publications

      You can also search for this author in PubMed Google Scholar

    9. Carlo Altucci
      View author publications

      You can also search for this author in PubMed Google Scholar

    10. Leila Birolo
      View author publications

      You can also search for this author in PubMed Google Scholar

    11. Renata Piccoli
      View author publications

      You can also search for this author in PubMed Google Scholar

    12. Angela Arciello
      View author publications

      You can also search for this author in PubMed Google Scholar

    Corresponding author

    Correspondence to Angela Arciello.

    Ethics declarations

    Conflict of interest

    The authors declare no competing financial interests.

    Additional information

    F. Itri, D.M. Monti were contributed equally to the paper.

    Electronic supplementary material

    Below is the link to the electronic supplementary material.

    18_2015_2015_MOESM1_ESM.tif

    Supplementary material 1 Electronic supporting material_Figure 1. Western blot analyses with anti-GAPDH antibodies of total proteins extracted from untreated cells (control) and from cells irradiated under the experimental conditions detailed in ESM_Table 1 (lanes 1-24). Protein samples in a, b and c refer to experimental conditions tested at constant energy dose (4.0 J). Protein samples in d and e were obtained by varying total irradiation energy between 2.4 and 4.4 J.(TIFF 2143 kb)

    18_2015_2015_MOESM2_ESM.tif

    Supplementary material 2 Electronic supporting material_Figure 2. SDS-PAGE analyses of UV-laser irradiated and control HeLa cell extracts immuno-selected by anti-GAPDH antibodies. Gel was cut into 12 slices (denoted by boxes 1–12) to be analysed by mass spectrometry; the results of slice-by-slice protein identification are reported in ESM_Table 3. M, molecular mass markers. (TIFF 3359 kb)

    18_2015_2015_MOESM3_ESM.tif

    Supplementary material 3 Electronic supporting material_Figure 3. Aromatic residues distance matrix as measured from human GAPDH crystal structure (PDB ID 1U8F). Distances are reported in angstroms and are highlighted in a heat map (blue/close, red/far) for visualization clarity. (TIFF 4461 kb)

    Supplementary material 4 (DOCX 20 kb)

    Supplementary material 5 (DOCX 24 kb)

    Supplementary material 6 (DOCX 19 kb)

    Rights and permissions

    Reprints and permissions

    About this article

    Check for updates. Verify currency and authenticity via CrossMark

    Cite this article

    Itri, F., Monti, D.M., Della Ventura, B. et al. Femtosecond UV-laser pulses to unveil protein–protein interactions in living cells. Cell. Mol. Life Sci. 73, 637–648 (2016). https://doi.org/10.1007/s00018-015-2015-y

    Download citation

    • Received:

    • Revised:

    • Accepted:

    • Published:

    • Issue Date:

    • DOI: https://doi.org/10.1007/s00018-015-2015-y

    Keywords

    Search

    Navigation