Backbone dynamics of an alamethicin in methanol and aqueous detergent solution determined by heteronuclear ¹H−¹⁵N NMR spectroscopy

Summary

The ¹⁵N relaxation rates of the α-aminoisobutyric acid (Aib)-rich peptide alamethicin dissolved in methanol at 27°C and 5°C, and dissolved in aqueous sodium dodecylsulfate (SDS) at 27°C, were measured using inverse-detected one-and two-dimensional ¹H−¹⁵N NMR spectroscopy. Measurements of ¹⁵N longitudinal (R_N(N_z)) and transverse (R_N(N_x,y)) relaxation rates and the {¹H} ¹⁵N nuclear Overhauser enhancement (NOE) at 11.7 Tesla were used to calculate (quasi-) spectral density values at 0, 50, and 450 MHz for the peptide in methanol and in SDS. Spectral density mapping at 0, 50, 450, 500, and 550 MHz was done using additional measurements of the ¹H−¹⁵N lingitudinal two-spin order, R_NH(2H ^supN_infZ N_Z), two-spin antiphase coherence, R_NH(2H ^supZ_infN N_x,y), and the proton longitudinal relaxation rate, R_H(H ^supZ_infN ), for the peptide dissolved in methanol only. The spectral density of motions was also modeled using the three-parameter Lipari-Szabo function. The overall rotational correlation times were determined to be 1.1, 2.5, and 5.7 ns for alamethicin in methanol at 27°C and 5°C, and in SDS at 27°C, respectively. From the rotational correlation time determined in SDS the number of detergent molecules associated with the peptide was estimated to be about 40. The average order parameter was about 0.7 and the internal correlation times were about 70 ps for the majority of backbone amide ¹⁵N sites of alamethicin in methanol and in SDS. The relaxation data, spectral densities, and order parameters suggest that the peptide N-H vectors of alamethicin are not as highly constrained as the ‘core’ regions of folded globular proteins. However, the peptide backbone is clearly not as mobile as the most unconstrained regions of folded proteins, such as those found in the ‘frayed’ C-and N-termini of some proteins, or in randomcoil peptides. The data also suggest significant mobility at both ends of the peptide dissolved in methanol. In SDS the mobility in the middle and at the ends of the peptide is reduced. The implications of the results with respect to the sterically hindered Aib residues and the biological activities of the peptide are discussed.