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Backbone dynamics of a bacterially expressed peptide from the receptor binding domain of Pseudomonas aeruginosa pilin strain PAK from heteronuclear 1H-15N NMR spectroscopy

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Abstract

The backbone dynamics of a 15N-labeled recombinant PAK pilin peptide spanning residues 128–144 in the C-terminal receptor binding domain of Pseudomonas aeruginosa pilin protein strain PAK (Lys128-Cys-Thr-Ser-Asp-Gln-Asp-Glu-Gln-Phe-Ile-Pro-Lys-Gly-Cys-Ser-Lys144) were probed by measurements of 15N NMR relaxation. This PAK(128–144) sequence is a target for the design of a synthetic peptide vaccine effective against multiple strains of P. aeruginosa infection. The 15N longitudinal (T1) and transverse (T2) relaxation rates and the steady-state heteronuclear {1H}-15N NOE were measured at three fields (7.04, 11.74 and 14.1 Tesla), five temperatures (5, 10, 15, 20, and 25 °C ) and at pH 4.5 and 7.2. Relaxation data was analyzed using both the `model-free' formalism [Lipari, G. and Szabo, A. (1982) J. Am. Chem. Soc., 104, 4546–4559 and 4559–4570] and the reduced spectral density mapping approach [Farrow, N.A., Szabo, A., Torchia, D.A. and Kay, L.E. (1995) J. Biomol. NMR, 6, 153–162]. The relaxation data, spectral densities and order parameters suggest that the type I and type II β-turns spanning residues Asp134-Glu-Gln-Phe137 and Pro139-Lys-Gly-Cys142, respectively, are the most ordered and structured regions of the peptide. The biological implications of these results will be discussed in relation to the role that backbone motions play in PAK pilin peptide immunogenicity, and within the framework of developing a pilin peptide vaccine capable of conferring broad immunity across P. aeruginosa strains.

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Authors and Affiliations

  1. Department of Medicinal Chemistry, School of Pharmacy, University of Washington, Seattle, WA, 98195, U.S.A.

    A. Patricia Campbell

  2. Department of Biochemistry, Canada

    Leo Spyracopoulos & Brian D. Sykes

  3. Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, AB, Canada, T6G 2H7

    Randall T. Irvin

  4. Protein Engineering Network of Centers of Excellence, University of Alberta, Edmonton, AB, Canada, T6G 2S2

    Randall T. Irvin & Brian D. Sykes

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  1. A. Patricia Campbell
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Campbell, A.P., Spyracopoulos, L., Irvin, R.T. et al. Backbone dynamics of a bacterially expressed peptide from the receptor binding domain of Pseudomonas aeruginosa pilin strain PAK from heteronuclear 1H-15N NMR spectroscopy. J Biomol NMR 17, 239–255 (2000). https://doi.org/10.1023/A:1008311319998

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