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.
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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.
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Newcomer, R.L., Belato, H.B., Teschke, C.M. et al. NMR assignments for monomeric phage L decoration protein. Biomol NMR Assign 12, 339–343 (2018). https://doi.org/10.1007/s12104-018-9836-1
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DOI: https://doi.org/10.1007/s12104-018-9836-1