Nitroxyl-induced T1 relaxation rates as a probe of conformation in peptide hormones

Proton spin-lattice relaxation rates in the N-H region of the n.m.r. spectra of aqueous LHRH and angiotensin II were measured in the presence of varying concentrations of 2,2,6,6-tetramethylpiperidinoxyl. At peptide concentrations of 3-7 mM and nitroxyl concentrations up to three times the peptide concentration, the relaxation rate is linearly dependent on nitroxyl concentration. Second order rate constants for nitroxyl induced relaxation in water are in the 200-1000s-1 M-1 range, and are dependent more on conformational factors than on side chain bulk. Exclusion of the radical from the hydration sphere of imidazolium ion appears to occur. The measurements for LHRH agree with earlier observations of the effect of the nitroxyl on linewidths. A two- to three-fold decrease in sensitivity of the amide protons of at least three of the residues in angiotensin II when the His6 imidazole is protonated indicates a conformational transition related to this ionization.