Frame Shifts Affect the Stability of Collagen Triple Helices
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Abstract:
Collagen model peptides (CMPs), composed of proline–(2S,4R)-hydroxyproline–glycine (POG) repeat units, have been extensively used to study the structure and stability of triple-helical collagen─the dominant structural protein in mammals─at the molecular level. Despite the more than 50-year history of CMPs and numerous studies on the relationship between the composition of single-stranded CMPs and the thermal stability of the assembled triple helices, little attention has been paid to the effects arising from their terminal residues. Here, we show that frame-shifted CMPs, which share POG repeat units but terminate with P, O, or G, form triple helices with vastly different thermal stabilities. A melting temperature difference as high as 16 °C was found for triple helices from 20-mers Ac-OG[POG]6-NH2 and Ac-[POG]6PO-NH2, and triple helices of the constitutional isomers Ac-[POG]7-NH2 and Ac-[GPO]7-NH2 melt 10 °C apart. A combination of thermal denaturation, circular dichroism and NMR spectroscopic studies, and molecular dynamics simulations revealed that the stability differences originate from the propensity of the peptide termini to preorganize into a polyproline-II helical structure. Our results advise that care must be taken when designing peptide mimics of structural proteins, as subtle changes in the terminal residues can significantly affect their properties. Our findings also provide a general and straightforward tool for tuning the stability of CMPs for applications as synthetic materials and biological probes.Keywords:
Polyproline helix
Thermal Stability
Denaturation (fissile materials)
Hydroxyproline
Helix (gastropod)
Collagen helix
Polyproline helix
Collagen helix
Helix (gastropod)
Thermal Stability
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Abstract Collagens are the most abundant extracellular matrix proteins in multicellular animals. They all contain a name‐giving collagen triple helix, which connects their three chains and varying amounts of other noncollagenous protein domains. To form the triple helix a repeated sequence of ‐Gly‐Xaa‐Yaa‐ is required, where Xaa and Yaa can be any residue. Each chain forms a polyproline‐II like left‐handed helix. The three chains are staggered by one residue from each other, and form a right‐handed helix. Twenty eight types of collagen molecules have been identified in mammals. The stability of the collagen triple helix is based on the length and the amino acid sequence of each polypeptide chain, and also by the presence of interchain cross‐links and/or trimerization domains. The 4(R)‐hydroxylation of proline residues in the Yaa position significantly increases the stability of the collagen triple helix.
Collagen helix
Polyproline helix
Helix (gastropod)
Hydroxylation
Residue (chemistry)
Sequence (biology)
Side chain
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The individual chains in the triple helix of collagen occur in a conformation related to polyproline II because of the presence of large number of imino peptide bonds. However, these residues are not evenly distributed in the collagen molecule which also contains many non-imino residues. These non-imino regions of collagen may be expected to show preference for other than triple helical conformations. The appearance of several Raman bands in solution phase at 65 degrees C raises the possibility of non-uniform triple helical structure in collagen. Raman spectroscopic studies on collagen in the solid state and in solution at a temperature greater than its denaturation temperature, reported here suggest that denatured collagen may exhibit an ensemble of conformational states with yet unknown implications to the biochemical interactions of this important protein component of connective tissues.
Polyproline helix
Collagen helix
Denaturation (fissile materials)
Type I collagen
Helix (gastropod)
Imino acid
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The structure of a protein triple helix has been determined at 1.9 angstrom resolution by x-ray crystallographic studies of a collagen-like peptide containing a single substitution of the consensus sequence. This peptide adopts a triple-helical structure that confirms the basic features determined from fiber diffraction studies on collagen: supercoiling of polyproline II helices and interchain hydrogen bonding that follows the model II of Rich and Crick. In addition, the structure provides new information concerning the nature of this protein fold. Each triple helix is surrounded by a cylinder of hydration, with an extensive hydrogen bonding network between water molecules and peptide acceptor groups. Hydroxyproline residues have a critical role in this water network. The interaxial spacing of triple helices in the crystal is similar to that in collagen fibrils, and the water networks linking adjacent triple helices in the crystal structure are likely to be present in connective tissues. The breaking of the repeating (X-Y-Gly) n pattern by a Gly→Ala substitution results in a subtle alteration of the conformation, with a local untwisting of the triple helix. At the substitution site, direct interchain hydrogen bonds are replaced with interstitial water bridges between the peptide groups. Similar conformational changes may occur in Gly→X mutated collagens responsible for the diseases osteogenesis imperfecta, chondrodysplasias, and Ehlers-Danlos syndrome IV.
Polyproline helix
Collagen helix
Helix (gastropod)
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The triple-helical structure of collagen, responsible for collagen’s remarkable biological and mechanical properties, has inspired both basic and applied research in synthetic peptide mimetics for decades. Since non-proline amino acids weaken the triple helix, the cyclic structure of proline has been considered necessary, and functional collagen mimetic peptides (CMPs) with diverse sidechains have been difficult to produce. Here we show that N-substituted glycines (N-glys), also known as peptoid residues, exhibit a general triple-helical propensity similar to or greater than proline, allowing synthesis of thermally stable triple-helical CMPs with unprecedented sidechain diversity. We found that the N-glys stabilize the triple helix by sterically promoting the preorganization of individual CMP chains into the polyproline-II helix conformation. Our findings were supported by the crystal structures of two atomic-resolution N-gly-containing CMPs, as well as experimental and computational studies spanning more than 30 N-gly-containing peptides. We demonstrated that N-gly sidechains with diverse exotic moieties including a ‘click’-able alkyne and a photo-sensitive sidechain can be incorporated into stable triple helices, enabling functional applications such spatio-temporal control of cell adhesion and migration on a gelatin matrix. The folding principles discovered in this study open up opportunities for a new generation of collagen mimetic therapeutics and materials with extraordinary properties.
Collagen helix
Polyproline helix
Peptoid
Helix (gastropod)
Folding (DSP implementation)
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As the only ribosomally encoded N-substituted amino acid, proline promotes distinct secondary protein structures. The high proline content in collagen, the most abundant protein in the human body, is crucial to forming its hallmark structure: the triple-helix. For over five decades, proline has been considered compulsory for synthetic designs aimed at recapitulating collagen's structure and properties. Here we describe that N-substituted glycines (N-glys), also known as peptoid residues, exhibit a general triple-helical propensity similar to or greater than proline, enabling synthesis of stable triple-helical collagen mimetic peptides (CMPs) with unprecedented side chain diversity. Supported by atomic-resolution crystal structures as well as circular dichroism and computational characterizations spanning over 30 N-gly-containing CMPs, we discovered that N-glys stabilize the triple-helix primarily by sterically preorganizing individual chains into the polyproline-II helix. We demonstrated that N-glys with exotic side chains including a "click"-able alkyne and a photosensitive side chain enable CMPs for functional applications including the spatiotemporal control of cell adhesion and migration. The structural principles uncovered in this study open up opportunities for a new generation of collagen-mimetic therapeutics and materials.
Peptoid
Helix (gastropod)
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Polyproline helix
Collagen helix
Helix (gastropod)
Globular protein
Folding (DSP implementation)
Type I collagen
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The triple-helical structure of collagen, responsible for collagen’s remarkable biological and mechanical properties, has inspired both basic and applied research in synthetic peptide mimetics for decades. Since non-proline amino acids weaken the triple helix, the cyclic structure of proline has been considered necessary, and functional collagen mimetic peptides (CMPs) with diverse sidechains have been difficult to produce. Here we show that N-substituted glycines (N-glys), also known as peptoid residues, exhibit a general triple-helical propensity similar to or greater than proline, allowing synthesis of thermally stable triple-helical CMPs with unprecedented sidechain diversity. We found that the N-glys stabilize the triple helix by sterically promoting the preorganization of individual CMP chains into the polyproline-II helix conformation. Our findings were supported by the crystal structures of two atomic-resolution N-gly-containing CMPs, as well as experimental and computational studies spanning more than 30 N-gly-containing peptides. We demonstrated that N-gly sidechains with diverse exotic moieties including a ‘click’-able alkyne and a photo-sensitive sidechain can be incorporated into stable triple helices, enabling functional applications such spatio-temporal control of cell adhesion and migration on a gelatin matrix. The folding principles discovered in this study open up opportunities for a new generation of collagen mimetic therapeutics and materials with extraordinary properties.
Polyproline helix
Collagen helix
Peptoid
Helix (gastropod)
Peptidomimetic
Folding (DSP implementation)
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Collagen mimetic peptides (CMPs) fold into a polyproline type II triple helix, allowing the study of the structure and function (or misfunction) of the collagen family of proteins. This Perspective will focus on recent developments in the use of CMPs toward understanding the structure and controlling the stability of the triple helix. Triple helix assembly is influenced by various factors, including the single amino acid propensity for the triple helix fold, pairwise interactions between these amino acids, and long-range effects observed across the helix, such as bend, twist, and fraying. Important progress in creating a comprehensive and predictive understanding of these factors for peptides with exclusively natural amino acids has been made. In contrast, several groups have successfully developed unnatural amino acids that are engineered to stabilize the triple helical structure. A third approach to controlling the triple helical structure includes covalent cross-linking of the triple helix to stabilize the assembly, which eliminates the problematic equilibrium of unfolding into monomers and enforces compositional control. Advances in all these areas have resulted in significant improvements to our understanding and control of this important class of protein, allowing for the design and application of more chemically complex and well-controlled collagen mimetic biomaterials.
Polyproline helix
Collagen helix
Helix (gastropod)
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