Skip to main content
SpringerLink
Log in

Use of Fluorescent Nucleotides to Map RNA-Binding Sites on Protein Surface

  • Protocol
  • First Online:
RNA Spectroscopy

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2113))

Abstract

Currently, studies of RNA/protein interactions occupy a prominent place in molecular biology and medicine. The structures of RNA–protein complexes may be determined by X-ray crystallography or NMR for further analyses. These methods are time-consuming and difficult due to the versatility and dynamics of the RNA structure. Furthermore, due to the need to solve the “phase problem” for each dataset in crystallography, crystallographic structures of RNA are still underrepresented. Structure determination of single ribonucleotide–protein complexes is a useful tool to identify the position of single-stranded RNA-binding sites in proteins. We describe here a structural approach that incorporates affinity measurement of a protein for various single ribonucleotides, ranking the RNA/protein complexes according to their stability. This chapter describes how to perform these measurements, including a perspective for the analysis of RNA-binding sites in protein and single-nucleotide crystal soaking.

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

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Peng Y, Soper TJ, Woodson SA (2012) RNase Footprinting of protein binding sites on an mRNA target of small RNAs. In: Bacterial regulatory RNA. Humana Press, Totowa, pp 213–224

    Chapter  Google Scholar 

  2. Nilsen TW (2014) RNase Footprinting to map sites of RNA-protein interactions. Cold Spring Harb Protoc 2014:pdb.prot080788

    Article  Google Scholar 

  3. Ascano M, Hafner M, Cekan P, Gerstberger S, Tuschl T (2012) Identification of RNA-protein interaction networks using PAR-CLIP. Wiley Interdiscip Rev RNA 3:159–177

    Article  CAS  Google Scholar 

  4. Garzia A, Morozov P, Sajek M, Meyer C, Tuschl T (2018) PAR-CLIP for discovering target sites of RNA-binding proteins. Methods Mol Biol 1720:55–75

    Article  CAS  Google Scholar 

  5. Garber M, Gongadze G, Meshcheryakov V, Nikonov O, Nikulin A, Perederina A, Piendl W, Serganov A, Tishchenko S (2002) Crystallization of RNA/protein complexes. Acta Crystallogr D Biol Crystallogr 58:1664–1669

    Article  Google Scholar 

  6. Ke A, Doudna JA (2004) Crystallization of RNA and RNA-protein complexes. Methods 34:408–414

    Article  CAS  Google Scholar 

  7. Obayashi E, Oubridge C, Pomeranz Krummel D, Nagai K (2007) Crystallization of RNA-protein complexes. Methods Mol Biol 363:259–276

    Article  CAS  Google Scholar 

  8. Nikulin A, Mikhailina A, Lekontseva N, Balobanov V, Nikonova E, Tishchenko S (2017) Characterization of RNA-binding properties of the archaeal Hfq-like protein from Methanococcus jannaschii. J Biomol Struct Dyn 35:1615–1628

    Article  CAS  Google Scholar 

  9. Murina V, Lekontseva N, Nikulin A (2013) Hfq binds ribonucleotides in three different RNA-binding sites. Acta Crystallogr Sect D Biol Crystallogr 69:1504–1513

    Article  CAS  Google Scholar 

  10. Oerum S, Catala M, Atdjian C, Brachet F, Ponchon L, Barraud P, Iannazzo L, Droogmans L, Braud E, Ethève-Quelquejeu M, Tisné C (2019) Bisubstrate analogues as structural tools to investigate m 6 a methyltransferase active sites. RNA Biol 16:798–808

    Article  Google Scholar 

  11. Verlinde CLMJ, Fan E, Shibata S, Zhang Z, Sun Z, Deng W, Ross J, Kim J, Xiao L, Arakaki TL, Bosch J, Caruthers JM, Larson ET, Letrong I, Napuli A, Kelly A, Mueller N, Zucker F, Van Voorhis WC, Buckner FS, Merritt EA, Hol WGJ (2009) Fragment-based cocktail crystallography by the medical structural genomics of pathogenic protozoa consortium. Curr Top Med Chem 9:1678–1687

    Article  CAS  Google Scholar 

  12. Patel D, Bauman JD, Arnold E (2014) Advantages of crystallographic fragment screening: functional and mechanistic insights from a powerful platform for efficient drug discovery. Prog Biophys Mol Biol 116:92–100

    Article  CAS  Google Scholar 

  13. Schumacher MA, Pearson RF, Møller T, Valentin-Hansen P, Brennan RG (2002) Structures of the pleiotropic translational regulator Hfq and an Hfq-RNA complex: a bacterial Sm-like protein. EMBO J 21:3546–3556

    Article  CAS  Google Scholar 

  14. Linke P, Amaning K, Maschberger M, Vallee F, Steier V, Baaske P, Duhr S, Breitsprecher D, Rak A (2016) An automated microscale thermophoresis screening approach for fragment-based lead discovery. J Biomol Screen 21:414–421

    Article  CAS  Google Scholar 

  15. Wang S, Poon GMK, Wilson WD (2015) Quantitative investigation of protein-nucleic acid interactions by biosensor surface Plasmon resonance. Methods Mol Biol 1334:313–332

    Article  CAS  Google Scholar 

  16. Nemchinova M, Balobanov V, Nikonova E, Lekontseva N, Mikhaylina A, Tishchenko S, Nikulin A (2017) An experimental tool to estimate the probability of a nucleotide presence in the crystal structures of the nucleotide–protein complexes. Protein J 36:157–165

    Article  CAS  Google Scholar 

  17. Hwang W, Arluison V, Hohng S (2011) Dynamic competition of DsrA and rpoS fragments for the proximal binding site of Hfq as a means for efficient annealing. Nucleic Acids Res 39:5131–5139

    Article  CAS  Google Scholar 

  18. Hulme EC, Trevethick MA (2010) Ligand binding assays at equilibrium: validation and interpretation. Br J Pharmacol 161:1219–1237

    Article  CAS  Google Scholar 

  19. Wlodawer A, Dauter Z, Jaskolski M (eds) (2017) Protein crystallography. Springer, New York

    Google Scholar 

  20. Ennifar E (ed) (2016) Nucleic acid crystallography. Springer, New York

    Google Scholar 

  21. Sauter C, Basquin J, Suck D (2003) Sm-like proteins in eubacteria: the crystal structure of the Hfq protein from Escherichia coli. Nucleic Acids Res 31:4091–4098

    Article  CAS  Google Scholar 

  22. Nikulin A, Stolboushkina E, Perederina A, Vassilieva I, Blaesi U, Moll I, Kachalova G, Yokoyama S, Vassylyev D, Garber M, Nikonov S (2005) Structure of Pseudomonas aeruginosa Hfq protein. Acta Crystallogr D Biol Crystallogr 61:141–146

    Article  Google Scholar 

  23. Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60:2126–2132

    Article  Google Scholar 

  24. Debreczeni JÉ, Emsley P (2012) Handling ligands with coot. Acta Crystallogr D Biol Crystallogr 68:425–430

    Article  CAS  Google Scholar 

  25. Pozharski E, Deller MC, Rupp B (2017) Validation of protein-ligand crystal structure models: small molecule and peptide ligands. Methods Mol Biol 1607:611–625

    Article  CAS  Google Scholar 

  26. Hwang H, Myong S (2014) Protein induced fluorescence enhancement (PIFE) for probing protein-nucleic acid interactions. Chem Soc Rev 43:1221–1229

    Article  CAS  Google Scholar 

  27. Wang ZX, Kumar NR, Srivastava DK (1992) A novel spectroscopic titration method for determining the dissociation constant and stoichiometry of protein-ligand complex. Anal Biochem 206:376–381

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by RFBR grant #18-04-00222. We thank V. Arluison and R. Lease for their constructive comments on the paper.

Author information

Authors and Affiliations

  1. Institute of Protein Research Russian Academy of Sciences, Pushchino, Moscow Region, Russia

    V. Balobanov, N. Lekontseva, A. Mikhaylina & A. Nikulin

Authors
  1. V. Balobanov
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. N. Lekontseva
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. A. Mikhaylina
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. A. Nikulin
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to V. Balobanov .

Editor information

Editors and Affiliations

  1. Université de Paris, Paris, France, Laboratoire Léon Brillouin LLB, CEA, CNRS UMR12, Université Paris Saclay, Gif-sur-Yvette, France

    Véronique Arluison

  2. Synchrotron SOLEIL L’Orme des Merisiers Saint Aubin, Gif-sur-Yvette, France

    Frank Wien

Rights and permissions

Reprints and permissions

Copyright information

© 2020 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Balobanov, V., Lekontseva, N., Mikhaylina, A., Nikulin, A. (2020). Use of Fluorescent Nucleotides to Map RNA-Binding Sites on Protein Surface. In: Arluison, V., Wien, F. (eds) RNA Spectroscopy. Methods in Molecular Biology, vol 2113. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0278-2_17

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-0278-2_17

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0277-5

  • Online ISBN: 978-1-0716-0278-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation