CONFORMATIONAL STUDY OF A COLLAGEN PEPTIDE BY H-1-NMR SPECTROSCOPY - OBSERVATION OF THE N-14-H-1 SPIN-SPIN COUPLING OF THE ARG GUANIDINIUM MOIETY IN THE TRIPLE-HELIX STRUCTURE

CB2, a CNBr peptide of 36 residues from type I collagen alpha 1(I) cha in has been studied by NMR spectroscopy as a function of temperature. At low temperature, the guanidinium protons of Arg(9) showed sharp 1:1 :1 NMR triplets around 6.95 ppm, characteristic of N-14 coupled proton s ((1)J(NH) = 52 Hz) when the quadrupolar relaxation rate is drastical ly reduced. These spectral characteristics and the low temperature coe fficient of the 1:1:1 triplets (Delta delta/Delta T of -3.6 ppb/degree s C) suggest that the H atoms of the protonated guanidinium moiety of Arg(9) in the triple helix are slowly exchanging with bulk water, most likely involved in hydrogen bonds. On the basis of conformational ene rgy computations on a model segment of type I collagen (Vitagliano, L. , Nemethy, G., Zagari, A. and Scheraga, H.A. (1993) Biochemistry 32, 7 354-7359), similar to CB2, our data could indicate that the guanidiniu m group of Arg(9) form hydrogen bonds with a backbone carbonyl of an a djacent chain probably by using the N-epsilon hydrogen, leaving the fo ur N-eta hydrogens bound to water molecules that must be in slow excha nge with bulk water and that could therefore be considered structural elements of the trimeric alpha 1(I) CB2 triple helix, The behaviour of Arg(9) has been investigated also in terms of equilibrium between ran dom monomer and helical trimer conformations controlled by temperature , The thermal unfolding process was found to be reversible and the mel ting point resulted to be 17 degrees C. (C) 1998 Federation of Europea n Biochemical Societies.