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

The backbone dynamics of a N-15-labeled recombinant PAK pilin peptide spann ing residues 128-144 in the C-terminal receptor binding domain of Pseudomon as aeruginosa pilin protein strain PAK (Lys(128)-Cys-Thr-Ser-Asp-Gln-Asp-Gl u-Gln-Phe-Ile-Pro-Lys-Gly-Cys-Ser-Lys(144)) were probed by measurements of N-15 NMR relaxation. This PAK(128-144) sequence is a target for the design of a synthetic peptide vaccine effective against multiple strains of P. aer uginosa infection. The N-15 longitudinal (T-1) and transverse (T-2) relaxat ion rates and the steady-state heteronuclear {H-1}-N-15 NOE were measured a t three fields (7.04, 11.74 and 14.1 Tesla), five temperatures (5, 10, 15, 20, and 25 degrees 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. A m. Chem. Soc., 104, 4546-4559 and 4559-4570] and the reduced spectral densi ty 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 beta-turns spannin g residues Asp(134)-Glu-Gln-Phe(137) and Pro(139)-Lys-Gly-Cys(142), respect ively, are the most ordered and structured regions of the peptide. The biol ogical implications of these results will be discussed in relation to the r ole that backbone motions play in PAK pilin peptide immunogenicity, and wit hin the framework of developing a pilin peptide vaccine capable of conferri ng broad immunity across P. aeruginosa strains.