Folding Studies of pH‐Dependent Collagen Peptides

Abstract

Synthetic collagen model peptides containing carboxylate-modified hydroxyproline residues (P E ) have been shown to form a collagen triple helix at low pH (1). Based on the fact that P E -containing peptides unfold on demand by altering the pH, we investigated the folding kinetics of a series of these collagen-based peptides. Refolding data indicated that the introduction of P E residues within a peptide affects the stability and refolding of the collagen peptides through electrostatic interactions and steric hindrance. Moreover, the specific placement of the P E residues within the collagen peptides allowed for selective testing for possible nucleation sites at the termini or the center of the peptides. Specifically, the data demonstrated that collagen peptides may nucleate from either terminally or internally located repeating sequences of Pro-Hyp-Gly. These studies indicate that the P E residue may play a useful role as a tool for studying the folding mechanism of synthetic collagen peptides.Key words: collagen, peptide, pH-dependent, folding, nucleation Abbreviations: Hyp, hydroxyproline; CTH, collagen triple helix; NC, non-collagenous; CD, circular dichroism; TFA, trifluoroacetic acid. Received 29 October 2009, revised 18 November 2009 accepted for publication 18 November 2009Collagen is one of the most abundant proteins found in mammals and is a major component of tissue, bone and cartilage. The major structural motif found in fibril forming collagen is the collagen triple helix (CTH) (2). This CTH motif is composed of three chains, each forming a left-handed type II polyproline helix, that associate and are stabilized by interchain hydrogen bonding within the CTH (3-6). To accommodate close packing, the CTH requires a glycine (Gly) residue at every third position within the helix, thereby generating repeating sequences of Gly-Xaa-Yaa in various types of collagen. A high imino acid content is found in the Xaa and Yaa positions, such as proline (Pro) and hydroxyproline (Hyp), and these residues have also been shown to be necessary in stabilizing the CTH (7-10).In nature, collagen is synthesized at the membrane of the rough endoplasmic reticulum in a procollagen form containing N-and C-terminal non-collagenous globular propeptides [non-collagenous (NC) domains] flanking the triple helical (Gly-Xaa-Yaa) n central collagenous domain (2,11-13). The assembly of the NC C-terminal domains is necessary for proper chain selection, registration and nucleation to generate an aligned triple helix (14-16). Nucleation of triple helix at (Gly-Pro-Hyp) rich sequences within the (Gly-Xaa-Yaa) regions is initiated through the NC domain assembly and is propagated in a zipper-like manner (17) with cis-trans isomerization as the rate-limiting step (18). In collagen III and IV, folding of triple helix proceeds from the C-to N-terminus, while other types of collagen display a variance in the directionality of their folding and nucleation (18-26).Although controversy exists over the reversibility of collagen triple helix formation...

Morphological transformations in a dually thermoresponsive coil–rod–coil bioconjugate

“…In a previous study, the rate of refolding of collagen triple helical peptide was reported to decrease due to the steric hindrance after the hydroxyproline residue of the peptide was modified with the carboxylate moiety linked through the ethyl linker. 42 Altogether, our CD results indicate that the triple helical form is retained, the thermal unfolding is sustained, and helix-to-coil transition is reversible in the triblock hybrid context.…”

Proline Editing: A General and Practical Approach to the Synthesis of Functionally and Structurally Diverse Peptides. Analysis of Steric versus Stereoelectronic Effects of 4-Substituted Prolines on Conformation within Peptides

“…Acylation was particularly effective for the incorporation of polar amino acid side-chain functional groups. Functionalities added include ammonium (lysine mimetics ( 62 , 63 ), via β-alanine), guanidinium (arginine mimetics ( 64 , 65 ), via the guanidino acid ,, of β-alanine), and carboxylates (aspartic/glutamic acid mimetics ( 66 - 70 ), via maleic anhydride, succinic anhydride, or glutaric anhydride). , In addition, amino acids or peptides could be directly incorporated at the site of the hydroxyproline. This approach, via a β-alanine linker, allowed the incorporation of a cysteine residue (1,2 aminothiol functionality ( 73 )), for native chemical ligation, and of an RGD peptide ( 72 ), for cell surface recognition. , …”

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“…These findings indicate that the installation of termini modifications on the collagen-like peptides did not preclude the central POG Journal of the American Chemical Society COMMUNICATION core from homotrimerization, a result that has also been observed with collagen peptides containing three carboxylate moieties. 6 More significantly, these indicated that the metal binding ligands should be suitably positioned to participate in a trivalent interblock interaction at room temperature.…”