Conformational analysis of the type II and type III collagen alpha-1 chain N-telopeptides by 1H-NMR spectroscopy and restrained molecular mechanics calculations.

Abstract

The type II and type III collagen alpha-1 chain N-telopeptides are a nonadecamer with the sequence pEMAGGFDEKAGGAQLGVMQ-NH2 and a tetradecamer with the sequence pEYEAYDVKSGVAGG-NH2, respectively. Their conformations have been studied in CD3OH/H2O (60/40) solution by means of two-dimensional proton nmr spectroscopy. Based on double quantum filtered correlation spectroscopy, total correlation spectroscopy, rotating frame nuclear Overhauser enhancement (ROE) spectroscopy, and nuclear Overhauser enhancement (NOE) spectroscopy experiments, all resonances were assigned and the conformational properties were analyzed in terms of vicinal NH-H alpha coupling constants, sequential and medium-range NOEs (ROEs), and amide proton temperature coefficients. The NOE distance constraints as well as dihedral constraints based on the vicinal NH-H alpha coupling constants were used as input parameters for restrained molecular mechanics, consisting of restrained molecular dynamics and restrained energy minimization calculations. The type II N-telopeptide's conformation is dominated by a fused beta gamma-turn between Phe6 and Ala10, stabilized by three hydrogen bonds and a salt bridge between the side-chain end groups of Glu8 and Lys9. The first 5 amino acids are extended with a much higher degree of conformational freedom. The 2 Gly residues following the turns were found to be highly flexible (hinge-like), leaving the spatial position of the second half of the molecule relative to the fused beta gamma-turn undefined. In the type III telopeptide, a series of sequential NH(i)-NH(i + 1) ROEs were observed between the amino acids Tyr2 and Ser9, indicating that a fraction of the conformational space is helical. However, the absence of medium-range ROEs and the lack of regularity of the effects associated with alpha-helices suggest the presence of a nascent rather than a complete helix.