Triple-helical Peptide Research Articles

A yeast two-hybrid system to obtain triple-helical ligands from combinatorial random peptide libraries

Many bioactive proteins interact with collagen, recognizing amino acid sequences displayed on the triple helix. We report here a selection strategy to obtain triple-helical peptides that interact with the proteins from a combinatorial random library constructed in yeast cells. This system enables us to select them using the standard two-hybrid protocol, detecting interactions between triple-helical peptides and target proteins fused to the GAL4-activating and binding domains, respectively. The library was constructed having triple-helical peptides with a “host-guest” design in which host helix-stabilizing regions flanked guest random sequences. Using this system, we selected peptides that bind to pigment epithelium-derived factor (PEDF), a collagen-binding protein that shows anti-angiogenic and neurotrophic activities, from the libraries. Two-step selections from the total random library and subsequently from the second focused library yielded new PEDF-binding sequences that exhibited a comparable affinity to or more potent than that of the native PEDF-binding sequence in collagen. The obtained sequences also contained a variant of the PEDF-binding motif that did not match the known motif identified from the native collagen sequences. This combinatorial library system allows the chemical space of triple-helical peptides to be screened more widely than that found in native collagen, thus increasing the expectation of obtaining more specific and high-affinity peptides.

From Collagen Mimetics to Collagen Hybridization and Back.

ConspectusFacilitated by the unique triple-helical protein structure, fibrous collagens, the principal proteins in animals, demonstrate a dual function of serving as building blocks for tissue scaffolds and as a bioactive material capable of swift renewal in response to environmental changes. While studies of triple-helical collagen mimetic peptides (CMPs) have been instrumental in understanding the molecular forces responsible for the folding and assembly of triple helices, as well as identifying bioactive regions of fibrous collagen molecules, single-strand CMPs that can specifically target and hybridize to denatured collagens (i.e., collagen hybridizing peptides, CHPs) have proven useful in identifying the remodeling activity of collagen-rich tissues related to development, homeostasis, and pathology. Efforts to improve the utility of CHPs have resulted in the development of new skeletal structures, such as dimeric and cyclic CHPs, as well as the incorporation of artificial amino acids, including fluorinated proline and N-substituted glycines (peptoid residues). In particular, dimeric CHPs were used to capture collagen fragments from biological fluid for biomarker study, and the introduction of peptoid-based collagen mimetics has sparked renewed interest in peptidomimetic research because peptoids enable a stable triple-helical structure and the presentation of an extensive array of side chain structures offering a versatile platform for the development of new collagen mimetics.This Account will cover the evolution of our research from CMPs as biomaterials to ongoing efforts in developing triple-helical peptides with practical theranostic potential in targeting denatured and damaged collagens. Our early efforts in functionalizing natural collagen scaffolds via noncovalent modifications led to the discovery of an entirely new use of CMPs. This discovery resulted in the development of CHPs that are now used by many different laboratories for the investigation of pathologies associated with changes in the structures of extracellular matrices including fibrosis, cancer, and mechanical damage to collagen-rich, load-bearing tissues. Here, we delve into the essential design features of CHPs contributing to their collagen binding properties and practical usage and explore the necessity for further mechanistic understanding of not only the binding processes (e.g., binding domain and stoichiometry of the hybridized complex) but also the biology of collagen degradation, from proteolytic digestion of fibrils to cellular processing of collagen fragments. We also discuss the strengths and weaknesses of peptoid-based triple-helical peptides as applied to collagen hybridization touching on thermodynamic and kinetic aspects of triple-helical folding. Finally, we highlight current limitations and future directions in the use of peptoid building blocks to develop bioactive collagen mimetics as new functional biomaterials.

Mapping the Binding Sites of MMPs on Types II and III Collagens Using Triple-Helical Peptide Toolkits.

Libraries of triple-helical collagen-like peptides (Collagen Toolkits) have helped to define collagens II and III binding specificities of numerous collagen-binding proteins. Here I describe a simple solid-phase binding assay utilizing a biotin-streptavidin system to screen the Collagen Toolkits for binding of two distinct matrix metalloproteinases (MMPs) implicated in cancer: the collagenolytic MMP1 (collagenase 1) and the non-collagenolytic MMP3 (stromelysin 1). The screening revealed markedly disparate binding footprints of these MMPs on collagens II and III, in line with their distinct biological activities. Analogous screening of other potentially collagen-binding proteases may shed light on their inherent tissue retention capabilities and their pro- or anti-metastatic potential.

Synthetic peptides activating discoidin domain receptor 2 and collagen-binding integrins cooperate to stimulate osteoblast differentiation of skeletal progenitor cells.

Skeletal progenitor: collagen interactions are critical for bone development and regeneration. Both collagen-binding integrins and discoidin domain receptors (DDR1 and DDR2) function as collagen receptors in bone. Each receptor is activated by a distinct collagen sequence; GFOGER for integrins and GVMGFO for DDRs. Specific triple helical peptides containing each of these binding domains were evaluated for ability to stimulate DDR2 and integrin signaling and osteoblast differentiation. GVMGFO peptide stimulated DDR2 Y740 phosphorylation and osteoblast differentiation as measured by induction of osteoblast marker mRNAs and mineralization without affecting integrin activity. In contrast, GFOGER peptide stimulated focal adhesion kinase (FAK) Y397 phosphorylation, an early measure of integrin activation, and to a lesser extent osteoblast differentiation without affecting DDR2-P. Significantly, the combination of both peptides cooperatively enhanced both DDR2 and FAK signaling and osteoblast differentiation, a response that was blocked in Ddr2-deficient cells. These studies suggest that the development of scaffolds containing DDR and integrin-activating peptides may provide a new route for promoting bone regeneration. STATEMENT OF SIGNIFICANCE: A method for stimulating osteoblast differentiation of skeletal progenitor cells is described that uses culture surfaces coated with a collagen-derived triple-helical peptide to selectively activate discoidin domain receptors. When this peptide is combined with an integrin-activating peptide, synergistic stimulation of differentiation is seen. This approach of combining collagen-derived peptides to stimulate the two main collagen receptors in bone (DDR2 and collagen-binding integrins) provides a route for developing a new class of tissue engineering scaffolds for bone regeneration.

Autoantibodies to Disease-Related Proteins in Joints as Novel Biomarkers for the Diagnosis of Rheumatoid Arthritis.

This study was undertaken to develop and characterize a multiplex immunoassay for detection of autoantibodies against peptides derived from proteins known to play a role in development of arthritis and that are also expressed in joints. We selected peptides from the human counterpart of proteins expressed in the joints, based on mouse models that showed these to be targeted by pathogenic or regulatory antibodies in vivo. Using bead-based flow immunoassays measuring IgG antibodies, we selected triple helical or cyclic peptides, containing the epitopes, to avoid collinear reactivity. We characterized the analytical performance of the immunoassay and then validated it in 3 independent rheumatoid arthritis (RA) cohorts (n=2,110), Swedish age- and sex-matched healthy controls, and patients with osteoarthritis (OA), patients with psoriatic arthritis (PsA), and patients with systemic lupus erythematosus (SLE). Screening assays showed 5 peptide antigens that discriminated RA patients from healthy controls with 99% specificity (95% confidence interval [CI] 98-100%). In our validation studies, we reproduced the discriminatory capacity of the autoantibodies in 2 other RA cohorts, showing that the autoantibodies had high discriminatory capacity for RA versus OA, PsA, and SLE. The novel biomarkers identified 22.5% (95% CI 19-26%) of early RA patients seronegative for anti-cyclic citrullinated peptide and rheumatoid factor. The usefulness of the biomarkers in identifying seronegative RA patients was confirmed in validation studies using 2 independent cohorts of RA patients and cohorts of patients with OA, PsA, and SLE. A multiplex immunoassay with peptides from disease-related proteins in joints was found to be useful for detection of specific autoantibodies in RA serum. Of note, this immunoassay had high discriminatory capacity for early seronegative RA.

Mechanism of the Early Catalytic Events in the Collagenolysis by Matrix Metalloproteinase-1.

The front cover artwork is provided by Dr. Karabencheva-Christova's group at Michigan Technological University. The images show the initially formed and the catalytically productive conformations of MMP-1 complex with the Triple Helical Peptide (THP), the free energy profile connecting them as well as the coordination geometry of the catalytic zinc (II). The background shows the collagen macromolecule. Read the full text of the Research Article at 10.1002/cphc.202200649.

Front Cover: Mechanism of the Early Catalytic Events in the Collagenolysis by Matrix Metalloproteinase‐1 (ChemPhysChem 3/2023)

The Front Cover show the initially formed and the catalytically productive conformations of MMP-1 complex with the Triple Helical Peptide (THP), the free energy profile connecting them as well as the coordination geometry of the catalytic zinc (II). The background shows the collagen macromolecule. More information can be found in the Research Article by Tatyana G. Karabencheva-Christova and co-workers.

Peptide-triggered self-assembly of collagen mimetic peptides into nanospheres by electrostatic interaction and π-π stacking.

Collagen is the most abundant protein in various connective tissues, providing mechanical integrity as well as regulating cellular activities. Self-assembled peptides have been extensively explored to develop collagen mimetic materials, due to their attractive features such as easy synthesis, selective sequences and low immunogenicity. Metal ion-triggered self-assembly of collagen mimetic peptides has recently received increasing interests, since the addition of external stimuli offers programmable control of the self-assembly process. We have for the first time reported a peptide-stimulated self-assembly of collagen mimetic peptides into nanospheres by electrostatic interaction and π-π stacking. We have accidentally discovered that FAM-modified positively-charged triple helical peptide FAM-PRG was highly soluble, while the addition of a single-stranded negatively-charged peptide EOG-10 efficiently drove its self-assembly into well-ordered spherical nanomaterials. Peptide EOG-10 has been shown to mediate similar self-assembly of TPE-modified triple-helical peptide TPE-PRG into luminescent exquisite nanospheres, consistently demonstrating the robustness of this peptide-triggered strategy. Fluorescence monitoring of the interaction of EOG-10 and TPE-PRG at different ratios indicated that EOG-10 specifically binds to TPE-PRG to form a 3 : 1 complex. High salt concentration was shown to inhibit the self-assembly of TPE-PRG with EOG-10, suggesting that their self-assembly was controlled by electrostatic interaction. The self-assembly of TPE-PRG with EOG-10 has been further revealed to require the exact lengths of both peptides as well as complementary sequences without mutations, indicating a pairwise "side-by-side" binding mode. Notably, the identity of the N-terminal residues of X-PRG has been found to play a determinant role in the self-assembly, while non-aromatic residues lost the self-assembling capability, suggesting that π-π stacking and electrostatic interactions collectively modulate the self-assembly of X-PRG and EOG-10. To conclude, we have developed a highly biocompatible and programmably controlled peptide-triggered self-assembly approach to create novel collagen mimetic nanomaterials, which may have great potential in advanced functional materials.

Structural insights into collagen binding by platelet receptor glycoprotein VI

AbstractGlycoprotein VI (GPVI) mediates collagen-induced platelet activation after vascular damage and is an important contributor to the onset of thrombosis, heart attack, and stroke. Animal models of thrombosis have identified GPVI as a promising target for antithrombotic therapy. Although for many years the crystal structure of GPVI has been known, the essential details of its interaction with collagen have remained elusive. Here, we present crystal structures of the GPVI ectodomain bound to triple-helical collagen peptides, which reveal a collagen-binding site across the β-sheet of the D1 domain. Mutagenesis and binding studies confirm the observed binding site and identify Trp76, Arg38, and Glu40 as essential residues for binding to fibrillar collagens and collagen-related peptides (CRPs). GPVI binds a site on collagen comprising two collagen chains with the core formed by the sequence motif OGPOGP. Potent GPVI-binding peptides from Toolkit-III all contain OGPOGP; weaker binding peptides frequently contain a partial motif varying at either terminus. Alanine-scanning of peptide III-30 also identified two AGPOGP motifs that contribute to GPVI binding, but steric hindrance between GPVI molecules restricts the maximum binding capacity. We further show that no cooperative interactions could occur between two GPVI monomers binding to a stretch of (GPO)5 and that binding of ≥2 GPVI molecules to a fibril-embedded helix requires non-overlapping OGPOGP motifs. Our structure confirms the previously suggested similarity in collagen binding between GPVI and leukocyte-associated immunoglobulin-like receptor 1 (LAIR-1) but also indicates significant differences that may be exploited for the development of receptor-specific therapeutics.

Synthetic Collagen-like Polymer That Undergoes a Sol-Gel Transition Triggered by O-N Acyl Migration at Physiological pH.

We previously reported an artificial collagen gel that can be used as a cell-culture substrate by end-to-end cross-linking of collagen-like triple-helical peptides via disulfide bonds. However, the gel had to be formed a priori by polymerizing the peptide in an acidic solution containing dimethyl sulfoxide for several days, which prevented its use as an injectable gel or three-dimensional (3D) scaffold for cell culture. In this study, we developed a collagen-like peptide polymer by incorporating an O–N acyl migration-triggered triple helix formation mechanism into a collagen-like peptide, which formed a gel within 10 min. We demonstrated that the collagen-like peptide polymer can be used as a 3D cell scaffold and that the 3D structure formation of cells can be controlled by collagen-derived bioactive sequences introduced into the peptide sequence.

Biomaterial Functionalization with Triple-Helical Peptides for Tissue Engineering

Biomaterial Functionalization with Triple-Helical Peptides for Tissue Engineering

Peptoid Residues Make Diverse, Hyperstable Collagen Triple-Helices.

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.

A synergy between the catalytic and structural Zn(II) ions and the enzyme and substrate dynamics underlies the structure-function relationships of matrix metalloproteinase collagenolysis.

Matrix metalloproteinases (MMPs) are Zn(II) dependent endopeptidases involved in the degradation of collagen. Unbalanced collagen breakdown results in numerous pathological conditions, including cardiovascular and neurodegenerative diseases and tumor growth and invasion. Matrix metalloproteinase-1 (MMP-1) is a member of the MMPs family. The enzyme contains catalytic and structural Zn(II) ions. Despite many studies on the enzyme, there is little known about the synergy between the two Zn(II) metal ions and the enzyme and substrate dynamics in MMP-1 structure-function relationships. We performed a computational study of the MMP-1•triple-helical peptide (THP) enzyme•substrate complex to provide this missing insight. Our results revealed Zn(II) ions' importance in modulating the long-range correlated motions in the MMP-1•THP complex. Overall, our results reveal the importance of the catalytic Zn(II) and the role of the structural Zn(II) ion in preserving the integrity of the enzyme active site and the overall enzyme-substrate complex synergy with the dynamics of the enzyme and the substrate. Notably, both Zn(II) sites participate in diverse networks of long-range correlated motions that involve the CAT and HPX domains and the THP substrate, thus exercising a complex role in the stability and functionality of the MMP-1•THP complex. Both the Zn(II) ions have a distinct impact on the structural stability and dynamics of the MMP-1•THP complex. The study shifts the paradigm from the "local role" of the Zn(II) ions with knowledge about their essential role in the long-range dynamics and stability of the overall enzyme•substrate (ES) complex.

Multimerin-1 Binds to Specific Motifs in Vessel Wall Collagens and Contributes to Thrombosis: Novel Insights Regarding the Mechanisms That Support Human and Mouse Platelet Adhesion

Multimerin-1 (MMRN1) is a homopolymeric glycoprotein stored in platelets and vascular endothelial cells for regulated release that enhances platelet adhesion to collagen. We tested Mmrn1 contributions to platelet adhesion ex vivo and in vivo using selective deficient mice and triple-helical collagen mimetic peptides to evaluate a putative Mmrn1-selective binding motif. Selective Mmrn1-deficient (Mmrn1-/-) mice were generated by crossing Mmrn1+/- “knockout-first allele” (Mmrn1flxneo/+ from European Mutant Mouse Archive) with flp deleter mice, and then with transgenic global cre deleter mice. The resultant heterozygous Mmrn1+/- mice were crossed with wild type (Mmrn1+/+) mice to remove the cre and flp transgenes. Inbred, gender-mixed, age-matched Mmrn1+/+ and Mmrn1-/- mice were then used for in vitro and in vivo assessments of platelet function. The following highly conserved GPAGPOGPX sequences in fibrillar collagens (that are distinct from collagen motifs that bind von Willebrand factor [VWF], platelet integrin alpha2beta1 or platelet GPVI) were identified to bind MMRN1: GPAGPOGPI (type I), GPAGPOGPV (type II), and GPAGPOGPQ (type III). In static adhesion assays with washed human platelets, triple-helical GPAGPOGPX peptides enhanced platelet adhesion when co-presented with the alpha2beta1-binding peptide GFOGER, like the VWF-binding peptide derived from type III collagen which was tested as a positive control. Static platelet adhesion to GPAGPOGPX peptides was also enhanced by agonists that induce MMRN1 release (p<0.02 for each comparison, where family-wise α=0.05, k=7), and co-presentation of GPAGPOGPX peptides with both GFOGER and III-23 maximized platelet adhesion (p<0.02 for each comparison, where family-wise α=0.05, k=5), providing insights on how MMRN1 and VWF synergistically enhance platelet adhesion. In high shear (1500 s-1) flow assays of platelet adhesion, platelet adhesion to triple-helical GPAGPOGPX peptides that were co-presented with GFOGER was greater than adhesion to GFOGER alone (p=0.015). Mmrn1-/- mice were viable and fertile, with normal blood cell counts and bleeding times (respective medians [ranges] for Mmrn1+/+ versus Mmrn1-/- mice: 300 [60-900] seconds vs. 330 [90-900] seconds, p=0.07), and confirmed Mmrn1 deficiency by Western blot analyses of platelets. In assays of platelet adhesion at high shear (1500 s-1), platelets from Mmrn1-/- mice showed a significant impairment in adhesion to full-length fibrillar collagen (% surface area covered by platelets ± SEM for Mmrn1+/+ versus Mmrn1-/- mice: 37 ± 3% vs. 18 ± 2%, p<0.001) and in collagen-induced aggregation in assays with washed platelets (20-30% reduction in maximal aggregation for Mmrn1-/- platelets compared to Mmrn1+/+ platelets, p<0.05). In assays of washed platelet adhesion, Mmrn1-/- platelets showed minimal static adhesionto the GPAGPOGPX peptide, unlike Mmrn1+/+ platelets(p<0.0001). Assays of platelet adhesion under conditions of flow indicated that co-presentation of GPAGPOGPX with GFOGER enhanced the adhesion of activated Mmrn1+/+ but not Mmrn1-/- platelets. Intravital microscopyexperiments indicated that Mmrn1-/- mice also had impaired platelet adhesion in arterioles treated with ferric chloride (platelets deposited per minute ± SEM for Mmrn1+/+ versus Mmrn1-/- mice: Mmrn1+/+ : 82 ± 4 vs. Mmrn1-/-: 42 ± 6, p<0.001), and the thrombi that formed in Mmrn1-deficient mice were smaller and less stable, with delayed vessel occlusion times (respective medians [ranges] for Mmrn1+/+ versus Mmrn1-/- mice: 17 [10-35] min vs. 35 [22-40] min, p<0.001). Taken together, our studies illustrate that multimerin-1 has functions in supporting and enhancing platelet adhesive function, in vivo and in vitro, and that fibrillar collagens contain highly conserved GPAGPOGPX motifs that bind Mmrn1 and contribute to platelet adhesion. DisclosuresNo relevant conflicts of interest to declare.

Modulating hESC-derived cardiomyocyte and endothelial cell function with triple-helical peptides for heart tissue engineering

In this study, we investigated the role of cardiomyocyte (CM) and endothelial cell (EC) specific interactions with collagen in the assembly of an operational myocardium in vitro. Engineered cardiac patches represent valuable tools for myocardial repair following infarction and are generally constituted of a suitable biomaterial populated by CMs and supportive cell types. Among those, ECs are required for tissue vascularization and positively modulate CM function. To direct the function of human embryonic stem cell (hESC)-derived CM and EC seeded on biomaterials, we replicated cell-collagen interactions, which regulate cellular behaviour in the native myocardium, using triple-helical peptides (THPs) that are ligands for collagen-binding proteins. THPs enhanced proliferation and activity of CMs and ECs separately and in co-culture, drove CM maturation and enabled coordinated cellular contraction on collagen films. These results highlight the importance of collagen interactions on cellular response and establish THP-functionalized biomaterials as novel tools to produce engineered cardiac tissues.

Synthesis of an Array of Triple-Helical Peptides from Type II Collagen for Multiplex Analysis of Autoantibodies in Rheumatoid Arthritis.

Type II collagen (CII) is the most abundant protein in joint cartilage. Antibodies to CII appear around the clinical onset of the autoimmune disease rheumatoid arthritis (RA) in a subset of patients. They target specific epitopes on CII and can be pathogenic or protective. Assays for early detection of such autoantibodies may provide new opportunities for selecting effective treatment strategies of RA. We report the efficient and reproducible assembly of an array of covalently branched native and citrullinated triple helical peptides (THPs) from CII that contain defined autoantibody epitopes. Both monoclonal antibodies and sera from experimental mouse models show a unique reactivity toward the THPs, compared to cyclic peptides containing the epitopes, revealing the importance that the epitopes are displayed in a triple-helical conformation. Importantly, antibodies against three of the THPs that contain major CII epitopes were found to be increased in sera from patients with RA, compared to control persons. These results indicate that such synthetic THPs should be included in multiplex analysis of autoantibodies that are uniquely occurring in individuals with early RA, to provide valuable information on disease prognosis and on what type of therapy should be chosen for individual patients.

Collagen scaffolds functionalized with triple-helical peptides support 3D HUVEC culture.

Porous biomaterials which provide a structural and biological support for cells have immense potential in tissue engineering and cell-based therapies for tissue repair. Collagen biomaterials that can host endothelial cells represent promising tools for the vascularization of engineered tissues. Three-dimensional collagen scaffolds possessing controlled architecture and mechanical stiffness are obtained through freeze–drying of collagen suspensions, followed by chemical cross-linking which maintains their stability. However, cross-linking scaffolds renders their biological activity suboptimal for many cell types, including human umbilical vein endothelial cells (HUVECs), by inhibiting cell–collagen interactions. Here, we have improved crucial HUVEC interactions with such cross-linked collagen biomaterials by covalently coupling combinations of triple-helical peptides (THPs). These are ligands for collagen-binding cell-surface receptors (integrins or discoidin domain receptors) or secreted proteins (SPARC and von Willebrand factor). THPs enhanced HUVEC adhesion, spreading and proliferation on 2D collagen films. THPs grafted to 3D-cross-linked collagen scaffolds promoted cell survival over seven days. This study demonstrates that THP-functionalized collagen scaffolds are promising candidates for hosting endothelial cells with potential for the production of vascularized engineered tissues in regenerative medicine applications.

Modulation of receptor binding to collagen by glycosylated 5-hydroxylysine: Chemical biology approaches made feasible by Carpino's Fmoc group.

The creation of the 9-fluorenylmethoxycarbonyl (Fmoc) group by the Carpino laboratory facilitated the synthesis of peptides containing acid-sensitive groups, such as O-linked glycosides. To fully investigative collagen biochemistry, one needs to assemble peptides that possess glycosylated 5-hydroxylysine (Hyl). A convenient method for the synthesis of Fmoc-Hyl(ε-tert-butyloxycarbonyl (Boc),O-tert-butyldimethylsilyl (TBDMS)) and efficient methods for the synthesis of Fmoc-Hyl[ε-Boc,O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)] have been developed. Glycosylated Fmoc-Hyl derivatives were used to construct a series of types I-IV collagen-model triple-helical peptides (THPs) that incorporated known or proposed receptor binding sites. Glycosylation of Hyl was found to strongly down-regulate the binding of CD44 and the α3β1 integrin to collagen, while the impact on α2β1 integrin binding was more modest. Molecular modeling of integrin binding indicated that Hyl glycosylation directly impacted the association between the α3β1 integrin metal ion-dependent adhesion site (MIDAS) and the receptor binding site within type IV collagen. The Fmoc solid-phase strategy ultimately allowed for chemical biology approaches to be utilized to study tumor cell interactions with glycosylated collagen sequences and document the modulation of receptor interactions by Hyl posttranslational modification.

Rules for the design of aza-glycine stabilized triple-helical collagen peptides.

The stability of the triple-helical structure of collagen is modulated by a delicate balance of effects including polypeptide backbone geometry, a buried hydrogen bond network, dispersive interfacial interactions, and subtle stereoelectronic effects. Although the different amino acid propensities for the Xaa and Yaa positions of collagen's repeating (Glycine–Xaa–Yaa) primary structure have been described, our understanding of the impact of incorporating aza-glycine (azGly) residues adjacent to varied Xaa and Yaa position residues has been limited to specific sequences. Here, we detail the impact of variation in the Xaa position adjacent to an azGly residue and compare these results to our study on the impact of the Yaa position. For the first time, we present a set of design rules for azGly-stabilized triple-helical collagen peptides, accounting for all canonical amino acids in the Xaa and Yaa positions adjacent to an azGly residue, and extend these rules using multiple azGly residues. To gain atomic level insight into these new rules we present two high-resolution crystal structures of collagen triple helices, with the first peptoid-containing collagen peptide structure. In conjunction with biophysical and computational data, we highlight the critical importance of preserving the triple helix geometry and protecting the hydrogen bonding network proximal to the azGly residue from solvent. Our results provide a set of design guidelines for azGly-stabilized triple-helical collagen peptides and fundamental insight into collagen structure and stability.

Photo-responsive azobenzene interactions promote hierarchical self-assembly of collagen triple-helical peptides to various higher-order structures.

Collagen is an essential structural protein in animal tissues and plays key roles in cellular modulation. We investigated methods to discover collagen model peptides (CMPs) that would self-assemble into triple helices and then grow into supramolecular organizations with diverse morphological features, which would be valuable as biomaterials. This challenging undertaking was achieved by placing azobenzene groups on the ends of the CMPs, (GPO)n (n = 3–10), Azo-(GPO)n. In a dilute aqueous solution (80 μM), CD spectra indicated that the Azo-(GPO)n (n > 4) formed triple helices due to strong hydrophobic azobenzene interactions, and that helix stability was increased with the peptide segment length. The resulting triple helices induced a specific azobenzene orientation through turned and twisted configurations as shown by CD spectra. TEM observations for the same solutions disclosed the morphologies for the Azo-CMPs. Azo-(GPO)3, having the shortest peptide segment, showed no nanostructure, both Azo-(GPO)4 and Azo-(GPO)5 provided consistent well-developed nanofiber structures resembling the natural collagen fibers, and Azo-(GPO)ns (n = 6–10) grew into flexible rod-like micelle fibers. In addition, alkyl chain-attached CmAzo-(GPO)5 displayed a toroidal morphology, and Azp-deg-(GPO)5 having a hydrophilic spacer assembled into a bilayer vesicle structure. These diverse morphological features are considered to be due to the characteristics of the pre-organized triple helix units. Photo-isomerization of the azobenzene moiety brought about the disappearance of such characteristic nano-architectures. When the solution concentration was increased up to 1 wt%, only Azo-(GPO)4 and Azo-(GPO)5 spontaneously formed hydrogels exhibiting a satisfactory gel-to-sol transition upon UV irradiation.