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Biomolecular NMR Assignments

, Volume 12, Issue 2, pp 339–343 | Cite as

NMR assignments for monomeric phage L decoration protein

  • Rebecca L. Newcomer
  • Helen B. Belato
  • Carolyn M. TeschkeEmail author
  • Andrei T. AlexandrescuEmail author
Article

Abstract

Phage L encodes a trimeric 43 kDa decoration protein (Dec) that noncovalently binds and stabilizes the capsids of the homologous phages L and P22 in vitro. At physiological pH Dec was unsuitable for NMR. We were able to obtain samples amenable for NMR spectroscopy by unfolding Dec to pH 2 and refolding it to pH 4. Our unfolding/refolding protocol converted trimeric Dec to a folded 14.4 kDa monomer. We verified that the acid-unfolding protocol did not perturb the secondary structure, or the capsid-binding function of refolded Dec. We were able to obtain complete 1H, 15N, and 13C assignments for the Dec monomer, as well as information on its secondary structure and dynamics based on chemical shift assignments. The assigned NMR spectrum is being used to determine the three-dimensional structure of Dec, which is important for understanding how the trimer binds phage capsids and for the use of the protein as a platform for phage-display nanotechnology.

Keywords

Bacteriophage Viral assembly Procapsid Nanomaterials Protein stabilization 

Notes

Acknowledgements

This work was supported by NIH Grant R01 GM076661 and a Grant from the UConn Research Excellence Program. We thank Prof. Dmitry Korzhnev (UConn Health) for help in setting up deuterium-decoupled experiments, Prof. Angela Gronenborn (U. Pittsburgh School of Medicine) for useful discussion, and Prof. Kristin Parent for providing protocols and assistance for the Dec purification and CsCl gradient experiments.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

Ethical standards

All experiments complied with all laws of the United States of America.

References

  1. Casjens S, Hendrix R (1988) Control mechanisms in dsDNA bacteriophage assembly. In: Caendar R (ed) The bacteriophages. Plemum Press, New York, pp 15–91CrossRefGoogle Scholar
  2. Dai W, Hodes A, Hui WH, Gingery M, Miller JF, Zhou ZH (2010) Three-dimensional structure of tropism-switching Bordetella bacteriophage. Proc Natl Acad Sci USA 107:4347–4352.  https://doi.org/10.1073/pnas.0915008107 Google Scholar
  3. Gilcrease EB, Winn-Stapley DA, Hewitt FC, Joss L, Casjens SR (2005) Nucleotide sequence of the head assembly gene cluster of bacteriophage L and decoration protein characterization. J Bacteriol 187:2050–2057.  https://doi.org/10.1128/JB.187.6.2050-2057.2005 CrossRefGoogle Scholar
  4. Hernando-Perez M, Lambert S, Nakatani-Webster E, Catalano CE, de Pablo PJ (2014) Cementing proteins provide extra mechanical stabilization to viral cages. Nat Commun 5:4520.  https://doi.org/10.1038/ncomms5520 ADSCrossRefGoogle Scholar
  5. Iwai H, Forrer P, Pluckthun A, Guntert P (2005) NMR solution structure of the monomeric form of the bacteriophage lambda capsid stabilizing protein gpD. J Biomol NMR 31:351–356.  https://doi.org/10.1007/s10858-005-0945-7 CrossRefGoogle Scholar
  6. Murzin AG (1993) OB(oligonucleotide/oligosaccharide binding)-fold: common structural and functional solution for non-homologous sequences. EMBO J 12:861–867Google Scholar
  7. Parent KN, Deedas CT, Egelman EH, Casjens SR, Baker TS, Teschke CM (2012) Stepwise molecular display utilizing icosahedral and helical complexes of phage coat and decoration proteins in the development of robust nanoscale display vehicles. Biomaterials 33:5628–5637.  https://doi.org/10.1016/j.biomaterials.2012.04.026 CrossRefGoogle Scholar
  8. Qin L, Fokine A, O’Donnell E, Rao VB, Rossmann MG (2010) Structure of the small outer capsid protein, Soc: a clamp for stabilizing capsids of T4-like phages. J Mol Biol 395:728–741.  https://doi.org/10.1016/j.jmb.2009.10.007 CrossRefGoogle Scholar
  9. Schwarz B et al (2015) Symmetry controlled, genetic presentation of bioactive proteins on the P22 virus-like particle using an external decoration protein. ACS Nano 9:9134–9147.  https://doi.org/10.1021/acsnano.5b03360 CrossRefGoogle Scholar
  10. Shen Y, Bax A (2015) Protein structural information derived from NMR chemical shift with the neural network program TALOS-N. Methods Mol Biol 1260:17–32.  https://doi.org/10.1007/978-1-4939-2239-0_2 CrossRefGoogle Scholar
  11. Shen Y, Vernon R, Baker D, Bax A (2009) De novo protein structure generation from incomplete chemical shift assignments. J Biomol NMR 43:63–78.  https://doi.org/10.1007/s10858-008-9288-5 CrossRefGoogle Scholar
  12. Tang L, Gilcrease EB, Casjens SR, Johnson JE (2006) Highly discriminatory binding of capsid-cementing proteins in bacteriophage L. Structure 14:837–845.  https://doi.org/10.1016/j.str.2006.03.010 CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  1. 1.Department of Molecular and Cell BiologyUniversity of ConnecticutStorrsUSA
  2. 2.Department of ChemistryUniversity of ConnecticutStorrsUSA

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