Conformational study of a collagen peptide by 1H NMR spectroscopy: observation of the 14N-1H spin-spin coupling of the Arg guanidinium moiety in the triple-helix structure.

CB2, a CNBr peptide of 36 residues from type I collagen alpha1(I) chain has been studied by NMR spectroscopy as a function of temperature. At low temperature, the guanidinium protons of Arg9 showed sharp 1:1:1 NMR triplets around 6.95 ppm, characteristic of 14N coupled protons (1J(NH)=52 Hz) when the quadrupolar relaxation rate is drastically reduced. These spectral characteristics and the low temperature coefficient of the 1:1:1 triplets (delta delta/delta T of -3.6 ppb/degrees C) suggest that the H atoms of the protonated guanidinium moiety of Arg9 in the triple helix are slowly exchanging with bulk water, most likely involved in hydrogen bonds. On the basis of conformational energy computations on a model segment of type I collagen (Vitagliano, L., Némethy, G., Zagari, A. and Scheraga, H.A. (1993) Biochemistry 32, 7354-7359), similar to CB2, our data could indicate that the guanidinium group of Arg9 form hydrogen bonds with a backbone carbonyl of an adjacent chain probably by using the N(epsilon) hydrogen, leaving the four N(eta) hydrogens bound to water molecules that must be in slow exchange with bulk water and that could therefore be considered structural elements of the trimeric alpha1(I) CB2 triple helix. The behaviour of Arg9 has been investigated also in terms of equilibrium between random monomer and helical trimer conformations controlled by temperature. The thermal unfolding process was found to be reversible and the melting point resulted to be 17 degrees C.