Application and development of NMR spectroscopy to study the conformation and dynamics of collagen-like triple helical peptides

This work describes the NMR conformational and dynamic characterization of collagen-like triple helical peptides. The unique features of triple helices result in limited interproton distance information and the lack of long range contacts, so the solution structure of triple helical peptides can not be solved by NMR. Alternative strategies have been developed and applied on selectively 13C/15N doubly labeled synthetic peptides, to allow the visualization of peptide solution models. This approach, including the stagger chain identification, measurement of NMR conformational parameters, molecular modeling incorporating NMR data, and dynamic and hydrogen bonding investigation, provided the detailed structural and dynamic characterization of the triple helix system.;This method is applied to two families of peptides: peptides that model natural interruptions in the non-fibrillar collagen (Gly-X-Y)n repeats and peptides that model Gly to X substitutions in fibrillar collagen disease. NMR studies are presented to define the structural and dynamic effects that small breaks have on triple-helix structure, and allow the visualization of peptide models with two most common types of breaks with different lengths. Both breaks allow continuation of rod-like helices and maintenance of the 1-residue stagger, and have a highly localized perturbation in dihedral angle and hydrogen bonding. However, the conformation of residues within the breaks is different for the two kinds of breaks. This work has provided clues on the molecular basis of collagen binding at specific recognition sites.;The NMR methodology was also applied on peptides that model Gly to X mutations in fibrillar collagen diseases. Mutation-specific equilibrium states were identified for peptides with different Gly to X substitutions, indicating mutation specific abilities for the peptides to fold around the substitution. The first visualization of the solution conformation of a model peptide with a Gly to Ser substitution in a physiological sequence context was obtained, demonstrating the disturbed conformation and dynamic features and hydrogen bonding at the substitution site. It suggested that the substitution could alter the appearance of the exterior of the triple helix molecule as it is seen by binding partners, and represented a first step in clarifying how mutations could result in collagen disease.