Backbone Dynamics of (Pro-Hyp-Gly)₁₀ and a Designed Collagen-like Triple-Helical Peptide by ¹⁵N NMR Relaxation and Hydrogen-Exchange Measurements

The backbone dynamics of specific residues in two collagen-like triple-helical peptides with (X-Y-Gly)_n sequences have been investigated using two-dimensional inverse-detected ¹⁵N NMR relaxation measurements and hydrogen-exchange experiments. One peptide, (POG)₁₀, has the highest possible imino acid content and is considered to be a very stable prototype of a triple helix. The second peptide, (POG)₃ITGARGLAGPOG(POG)₃ (denoted T3-785), models an imino acid poor region of type III collagen and contains 12 residues from near the unique collagenase cleavage site. ¹⁵N relaxation parameters and hydrogen-exchange data were obtained for a glycine residue in the center of (POG)₁₀ and for the tripeptide unit Gly-Leu-Ala in the middle of T3-785. Analysis of the relaxation data of the rodlike triple-helical peptides required the assumption of anisotropic overall motion, and the model-free approach of Lipari and Szabo (1982) was used to derive overall motional parameters and the order parameter, S², that describes the amplitudes of the internal motion. First the mobilities of the Gly, Leu, and Ala residues in peptide T3-785 were compared. Both hydrogen-exchange methods and relaxation measurements indicated that the residue in the Y position (Ala) is more mobile than residues in the Gly and X positions (Leu). The slower exchange rates of Gly and Leu compared to that of Ala are consistent with the two-hydrogen-bonded model for the triple helix. Then the backbone mobilities of the central Gly residue were compared for the two peptides (POG)₁₀ and T3-785. In this case, ¹⁵N relaxation measurements give different results from hydrogen exchange. The glycine residues in the trimer form of both T3-785 and (POG)₁₀ have high values for the order parameter (near 0.85), suggesting similar small-amplitude internal motions and rigid backbones in both peptides. In contrast to the similar values of the order parameters, hydrogen-exchange data indicate that the central Gly exchanges at a faster rate in the trimer form of T3-785 than in (POG)₁₀. These results suggest that a Gly in the imino acid rich environment of (POG)₁₀ is dynamically different from a Gly in the imino acid poor environment of T3-785 and that the difference lies in the slower motion related to stability, rather than the faster motion on the picosecond time scale. This sequence-dependent difference in dynamical properties may have important consequences for recognition processes in collagen.