Interest in self-association of peptides and proteins is motivated by an interest in the mechanism of physiologically higher order assembly of proteins such as collagen as well as the mechanism of pathological aggregation such as ␤-amyloid formation. The triple helical form of (Pro-Hyp-Gly) 10 , a peptide that has proved a useful model for molecular features of collagen, was found to self-associate, and its association properties are reported here. Turbidity experiments indicate that the triple helical peptide self-assembles at neutral pH via a nucleationgrowth mechanism, with a critical concentration near 1 mM. The associated form is more stable than individual molecules by about 25°C, and the association is reversible. The rate of self-association increases with temperature, supporting an entropically favored process. After self-association, (ProHyp-Gly) 10 forms branched filamentous structures, in contrast with the highly ordered axially periodic structure of collagen fibrils. Yet a number of characteristics of triple helix assembly for the peptide resemble those of collagen fibril formation. These include promotion of fibril formation by neutral pH and increasing temperature; inhibition by sugars; and a requirement for hydroxyproline. It is suggested that these similar features for peptide and collagen self-association are based on common lateral underlying interactions between triple helical molecules mediated by hydrogen-bonded hydration networks involving hydroxyproline.There is increasing interest in the ability of proteins and peptides to self-associate into aggregates, both in normal and pathological processes. Normal self-association processes include fibril formation of collagen and polymerization of actin (1, 2), whereas pathological aggregation of amyloid peptides, ␣-synuclein, and prions is implicated in neurodegenerative diseases (3,4). Interest has focused on the nature of protein aggregation and the molecular and environmental determinants of the self-association process. The study of the ability of collagen-like peptides to aggregate offers an opportunity to characterize a unique system, which may relate to the physiological self-association of collagen molecules.Collagen, the major structural protein in the extracellular matrix, has a characteristic triple helical conformation, consisting of three polyproline II-like chains that are supercoiled around a common axis (5-7). The close packing of the three chains near the central axis generates a requirement for Gly as every third residue, (Gly-X-Y) n , whereas the high content of imino acids Pro and hydroxyproline (Hyp) stabilizes the individual polyproline II-like helices. Although imino acids are highly favorable for the triple helix, the post-translational modification of Pro to Hyp in the Y position confers an additional stabilizing contribution. This further stabilization of Hyp is likely to result from steroelectronic promotion of the more favorable exo ring pucker for the Y position and Hyp involvement in solvent-mediated hydrogen bonding...

No abstract

X-ray crystallography of collagen model peptides has provided high resolution structures of the basic triple-helical conformation and its water-mediated hydration network. Vibrational spectroscopy provides a useful bridge for transferring the structural information from x-ray diffraction to collagen in its native environment. The vibrational mode most useful for this purpose is the Amide I mode (mostly peptide bond C=O stretch) near 1650 cm −1 . The current study refines and extends the range of utility of a novel simulation method that accurately predicts the IR Amide I spectral contour from the three dimensional structure of a protein or peptide. The approach is demonstrated through accurate simulation of the experimental Amide I contour in solution for both a standard triple-helix, (Pro-ProGly) 10 , and a second peptide with a Gly → Ala substitution in the middle of the chain that models the effect of a mutation in the native collagen sequence. Monitoring the major Amide I peak as a function of temperature gives sharp thermal transitions for both peptides, similar to those obtained by circular dichroism spectroscopy, and the FTIR spectra of the unfolded states were compared with polyproline II.The simulation studies were extended to model early stages of thermal denaturation of (Pro-ProGly) 10 . Dihedral angle changes suggested by molecular dynamics simulations were made in a stepwise fashion to generate peptide unwinding from each end, which emulates the effect of increasing temperature. Simulated bands from these new structures were then compared to the experimental bands obtained as temperature was increased. The similarity between the simulated and experimental IR spectra lends credence to the simulation method, and paves the way for a variety of applications.

Missense mutations in the collagen triple helix that replace one Gly residue in the (Gly-X-Y) n repeating pattern by a larger amino acid have been shown to delay triple helix folding. One hypothesis is that such mutations interfere with the C-to N-terminal directional propagation and that the identity of the residues immediately N-terminal to the mutation site may determine the delay time and the degree of clinical severity. Model peptides are designed to clarify the role of tripeptide sequences N-terminal to the mutation site, with respect to length, stability, and nucleation propensity, to complete triple helix folding. Two sets of peptides with different N-terminal sequences, one with the natural sequence ␣1(I) 886 -900, which is just adjacent to the Gly 901 mutation, and one with a GPO(GAO) 3 sequence, which occurs at ␣1(I) 865-879, are studied by CD and NMR. Placement of the five tripeptides of the natural ␣1(I) collagen sequence N-terminal to the Gly to Ala mutation site results in a peptide that is folded only C-terminal to the mutation site. In contrast, the presence of the Hyp-rich sequence GPO(GAO) 3 N-terminal to the mutation allows complete refolding in the presence of the mutation. The completely folded peptide contains an ordered central region with unusual hydrogen bonding while maintaining standard triple helix structure at the N-and C-terminal ends. These peptide results suggest that the location and sequences of downstream regions favorable for renucleation could be the key factor in the completion of a triple helix N-terminal to a mutation.Abnormalities in protein folding are known to play a role in many diseases, including those arising from mutations in the collagen triple helix (1). The best characterized collagen disease is osteogenesis imperfecta (OI), 5 or brittle bone disease, in which there is defective mineralization of bones in type I collagen (2, 3). Missense mutations that change one Gly in the repetitive (Gly-X-Y) sequence are the most common mutations (4). Such Gly mutations are found all along the collagen chain, suggesting that the loss of a Gly at any site in the triple helix has pathological consequences. The phenotype of the disease varies widely, depending on the type of amino acid substitution and the site of the mutation (5, 6). There is evidence of abnormal folding of collagen in OI and other collagen diseases, which may relate to the pathology (7, 8).Folding of the triple helix is a complex, multistep process that includes association of three chains to form the supercoiled polyproline II triple helical structure (8). Collagen is synthesized in a procollagen form, with N-and C-terminal globular propeptides flanking the (Gly-X-Y) n central domain (9, 10). Posttranslational hydroxylation of Pro and Lys residues in the Y positions and further glycosylation of Hyl occur while the chains are unfolded (11, 12). Trimerization occurs through the association of three C-terminal propeptides, and then nucleation of the triple helix takes place at the (Gly-Pro-Hyp) n -rich sequence...