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Synthesis of (Pro-Hyp-Gly) n of defined molecular weights. Evidence for the stabilization of collagen triple helix by hydroxypyroline
Abstract
(Pro-Hyp-Gly)5 and (Pro-Hyp-Gly)10 were synthesized by repeated condensation of tert-butyloxycarbonyl(Boc)-Pro-Hyp[benzyl(Bzl)]-Gly on a Merrifield resin followed by cleavage of the peptides from the resin with HF. The solution properties of the peptides were then compared with those of (Pro-Pro-Gly)5 and (Pro-Pro-Gly)10 synthesized previously. The peptides containing hydroxyproline were similar to those that did not, in that they formed triple-helical structures analogous to the triple-helical structure of collagen. At low temperatures the peptides showed a high degree of negative optical rotation and there was a relatively sharp change in optical rotation as the temperature was increased. The was about 5 °C for (Pro-Hyp-Gly)5 and about 58 °C for (Pro-Hyp-Gly)10. Ultracentrifugation studies were consistent with the conclusion that the temperature-dependent changes in optical rotation reflected a transition between triple-stranded and single-stranded forms of the peptides. The major difference between the peptides containing hydroxyproline and those that did not was that the values for the hydroxyproline-containing peptides were higher by about 35 °C. The results indicated that the hydroxyprolyl residues preceding glycyl residues in the peptides stabilize the triple-helical structure, and they suggested therefore that hydroxyproline may perform a similar function in collagen.
... [33][34][35][36] First CMPs primarily consisted of the canonical sequences (Pro-Pro-Gly) n and (Pro-Hyp-Gly) n . 37,38 These CMPs have critically contributed to understanding the fine molecular differences determining collagen structure and hydration patterns, 16,[39][40][41][42][43][44] folding, 45-47 thermodynamic stability, 37,38,46,[48][49][50] and higher-order assembly 51 as well as the specific role of Hyp in determining the above-mentioned properties. 16,38,40,[49][50][51][52] Later, so-called host-guest CMPs have been introduced, where either individual amino acids or tripeptide units were modified in the central part of the peptide sequence. ...
... [33][34][35][36] First CMPs primarily consisted of the canonical sequences (Pro-Pro-Gly) n and (Pro-Hyp-Gly) n . 37,38 These CMPs have critically contributed to understanding the fine molecular differences determining collagen structure and hydration patterns, 16,[39][40][41][42][43][44] folding, 45-47 thermodynamic stability, 37,38,46,[48][49][50] and higher-order assembly 51 as well as the specific role of Hyp in determining the above-mentioned properties. 16,38,40,[49][50][51][52] Later, so-called host-guest CMPs have been introduced, where either individual amino acids or tripeptide units were modified in the central part of the peptide sequence. ...
... 37,38 These CMPs have critically contributed to understanding the fine molecular differences determining collagen structure and hydration patterns, 16,[39][40][41][42][43][44] folding, 45-47 thermodynamic stability, 37,38,46,[48][49][50] and higher-order assembly 51 as well as the specific role of Hyp in determining the above-mentioned properties. 16,38,40,[49][50][51][52] Later, so-called host-guest CMPs have been introduced, where either individual amino acids or tripeptide units were modified in the central part of the peptide sequence. 13,[53][54][55] With this strategy, also disease-related mutations have been investigated 13 as well as segments of biochemically active sequences, such as protease cleavage and integrin binding sites. ...
Native collagen molecules usually contract upon dehydration, but the details of their interaction with water are poorly understood. Previous molecular modeling studies indicated a spatially inhomogeneous response, with a combination of local axial expansion and contraction. Such sequence-dependent effects are difficult to study with native collagen. In this article, we use collagen-mimetic peptides (CMPs) to investigate the effect of osmotic pressure on several collagen-mimetic sequences. Synchrotron x-ray diffraction combined with molecular dynamics simulations shows that CMPs pack differently depending on osmotic pressure and exhibit changes in the helical rise per residue of individual molecules. Infrared spectroscopy reveals that osmotic pressure affects the stability of the triple helix through changes in triple helix-stabilizing hydrogen bonds. Surprisingly, CMPs with the canonical collagen sequence glycine–proline–hydroxyproline are found to elongate upon dehydration, while sequence modifications are able to reverse this tendency. This strongly suggests that the overall contraction of native collagen molecules is not programmed into the canonical sequence but is specific to local amino acids that substitute for proline or hydroxyproline along the protein chain. Collagen is an essential protein in mammalian extracellular tissues and a better understanding of its mechanical function is important both from a materials science and from a biomedical viewpoint. Recently, collagen has been shown to contract along the fibre direction when subjected to osmotic stress, a process that could play important roles in strengthening bone and in developing tissue tension during extracellular matrix development. The present work uses collagen-like short peptides to show that the canonical collagen sequence is not responsible for this contraction. The conclusion is that the collagen amino acid sequence must have evolved to include guest sequences within the canonical glycine-proline-hydroxyproline repeat that provide the observed contractility.
Impact statement
Collagen is an essential protein in mammalian extracellular tissues and a better understanding of its mechanical function is important both from a materials science and from a biomedical viewpoint. Recently, collagen has been shown to contract along the fibre direction when subjected to osmotic stress, a process that could play important roles in strengthening bone and in developing tissue tension during extracellular matrix development. The present work uses collagen-like short peptides to show that the canonical collagen sequence is not responsible for this contraction. The conclusion is that the collagen amino acid sequence must have evolved to include guest sequences within the canonical glycine-proline-hydroxyproline that provide the observed contractility.
Graphic Abstract
... Ces cassures sont également accompagnées du dépliement de la triple hélice et de la dénaturation du collagène. Concernant le peptide hydroxylé, des études ont montré qu'en phase condensée ces hydroxyprolines favorisaient la stabilité de la triple hélice de collagène [213,214]. ...
... Caractérisation de la structure de peptides modèles du collagène par spectrométrie de mobilité ionique L'origine de la stabilité et de la structure en triple hélice du collagène est source de débats depuis de nombreuses années. Des modèles peptidiques de la triple hélice du collagène ont été étudiés de nombreuses fois en phase condensée, afin notamment d'identifier les séquences qui reproduisent le mieux cette structure particulière en triple hélice [205,218,219], mais aussi pour sonder le rôle de l'hydroxylation des prolines dans sa stabilisation [213,214,216,220]. Deux hypothèses ont alors émergé dans la littérature concernant ce dernier point. ...
... Ces DTA peuvent ensuite être converties en sections efficaces de collision à l'aide de la relation 2.3.5, détaillée dans le chapitre 2. La quasi-abscence de dimères est cohérente avec le fait que les oligomères produits en phase gazeuse sont déjà pré-existants en solution et ne sont pas le résultat d'une agrégation survenant lors du processus de désolvatation. Des études ont en effet montré que ces peptides, initialement sous forme de poudre, possèdent cette structure particulière en triple hélice [214], conservée en phase liquide [213]. ...
Le collagène est la protéine la plus abondante dans les mammifères, et le constituant principal de la matrice extracellulaire du cartilage. Les propriétés mécaniques de ce tissu sont dues à la structure particulière du collagène : la triple hélice. Lors de cette thèse, nous nous sommes intéressés à des peptides modèles de la triple hélice du collagène en phase gazeuse, ce qui permet l’étude de leurs propriétés intrinsèques, dont les processus fondamentaux induits par des rayonnements ionisants. Une étude structurale de ces systèmes par spectrométrie de mobilité ionique a permis de s’assurer qu’ils conservent bien leurs propriétés structurales et de stabilité en l’absence de solvant. De plus, cette stabilité se manifeste aussi lors de l’irradiation par photons ionisants dans un piège à ions. Par ailleurs, nous avons observé, grâce à la spectrométrie de masse, une transition entre photo-excitation et photo-ionisation lorsque l’énergie du photon absorbé augmente dans la gamme VUV-X. Une partie de cette énergie est également redistribuée dans les modes de vibration du système, croît avec l’énergie du photon, et induit la fragmentation inter puis intramoléculaire de la triple hélice. Nous avons également irradié pour la première fois des peptides en phase gazeuse par un faisceau d’ions carbones à l’énergie cinétique pertinente dans le contexte de l’hadronthérapie. Un processus non-observé avec les photons a été mis en évidence : le détachement de proton. Enfin, la validation d’un nouveau dispositif expérimental dédié à l’irradiation de protéines et brins d’ADN par des ions en faisceaux croisés, ainsi que les premiers résultats obtenus, seront abordés.
... In particular, the post-translational hydroxylation converting Pro into Hyp is assumed to be essential for providing stability to the collagen triple helix structure through the hydrogen-bonded network formation with encapsulated water molecules. 24 The thermal stability of triple-helical collagen is reported to be improved by the hydroxyl group on the pyrrolidine ring of the Hyp residues as demonstrated for (Pro−Pro−Gly) 10 (25°C) versus (Pro− Hyp−Gly) 10 (58°C). 24 However, this hydration-based stabilization model has been challenged by other studies. ...
... 24 The thermal stability of triple-helical collagen is reported to be improved by the hydroxyl group on the pyrrolidine ring of the Hyp residues as demonstrated for (Pro−Pro−Gly) 10 (25°C) versus (Pro− Hyp−Gly) 10 (58°C). 24 However, this hydration-based stabilization model has been challenged by other studies. For example, replacing the central Hyp of (Pro−Hyp−Gly) 10 with (2S,4R)-4-fluoroproline (Flp) was found to greatly increase collagen stability due to favorable inductive effects. ...
The potential of ultra-short peptides to self-assemble into well-ordered functional nanostructures makes them promising minimal components for mimicking the basic ingredient of nature and diverse biomaterials. However, selection and modular design of perfect de novo sequences are extremely tricky due to their vast possible combinatorial space. Moreover, a single amino acid substitution can drastically alter the supramolecular packing structure of short peptide assemblies. Here, we report the design of rigid hybrid hydrogels produced by sequence engineering of a new series of ultra-short collagen-mimicking tripeptides. Connecting glycine with different combinations of proline and its post-translational product 4-hydroxyproline, the single triplet motif, displays the natural collagen-helix-like structure. Improved mechanical rigidity is obtained via co-assembly with the non-collagenous hydrogelator, fluorenylmethoxycarbonyl (Fmoc) diphenylalanine. Characterizations of the supramolecular interactions that promote the self-supporting and self-healing properties of the co-assemblies are performed by physicochemical experiments and atomistic models. Our results clearly demonstrate the significance of sequence engineering to design functional peptide motifs with desired physicochemical and electromechanical properties and reveal co-assembly as a promising strategy for the utilization of small, readily accessible biomimetic building blocks to generate hybrid biomolecular assemblies with structural heterogeneity and functionality of natural materials.
... 41 The resulting triple helix possesses a characteristic melting temperature (T m ) that is dependent on both the number of G-P-O repeats and the amino acid sequence; the effects of such parameters on T m have been rigorously investigated by others. [42][43][44][45] CLPs have been tested for their potential in diagnostic applications, 46 their ability to form supramolecular structures, [47][48][49][50] and their properties as components in hydrogels 39,51 and cell adhesion scaffolds, 52, 53 but CLPs have infrequently been studied as thermoresponsive elements in materials. ...
... Given that the resulting vesicles formed from this conjugate underwent complete thermal dissociation only at relatively high temperatures (70°C),61 we employed in the current study CLPs that have previously been shown to possess T m values lower than that of the N 3 -(GPO) 4 GFOGER(GPO) 4 GG sequence.41,47,63,67 It is well established that CLP sequences that possess few hydroxyproline residues (O) in the Y AA position, as well as sequences that have relatively short repeat lengths (< 8), have lower T m values than CLP sequences that are high in O content and/or are relatively long.41,42,44,67 For these reasons, four CLPs with an N-terminal azide (N 3 ) (N 3 -(GPO) 6 GG, N 3 -(GPO) 7 GG, N 3 -(GPO) 3 GFOGER(GPO) 3 GG, and N 3 -(GPP) 10 GG) ...
Materials that respond to temporally defined exogenous cues continue to be an active pursuit of research towards on‐demand nanoparticle drug delivery applications, and using one or more exogenous temperature stimuli could significantly expand the application of nanoparticle‐based drug delivery formulations under both hyperthermal and hypothermal conditions. Previously we have reported the development of a biocompatible and thermoresponsive elastin‐b‐collagen‐like polypeptide (ELP‐CLP) conjugate that is capable of self‐assembling into vesicles and encapsulating small molecule therapeutics that can be delivered at different rates via a single temperature stimulus. Herein we report the evaluation of multiple ELP‐CLP conjugates, demonstrating that the inverse transition temperature (Tt) of the ELP‐CLPs can be manipulated by modifying the melting temperature (Tm) of the CLP domain, and that the overall hydrophilicity of the ELP‐CLP conjugate also may alter the Tt. Based on these design parameters, we demonstrate that the ELP‐CLP sequence (VPGFG)6‐(GPO)7GG can self‐assemble into stable vesicles at 25°C and dissociate at elevated temperatures by means of the unfolding of the CLP domain above its Tm. We also demonstrate here for the first time the ability of this ELP‐CLP vesicle to dissociate via a hypothermic temperature stimulus by means of exploiting the inverse transition temperature (Tt) phenomena found in ELPs. The development of design rules for manipulating the thermal properties of these bioconjugates will enable future modifications to either the ELP or CLP sequences to more finely tune the transitions of the conjugates for specific biomedical applications. This article is protected by copyright. All rights reserved.
... Collagen in general was considered 'noncrystallizable' and it was not until the introduction of solid-phase synthesis of collagen model peptides that this perception changed. Physicochemical analysis confirmed that these collagen peptides were trimeric in dilute aqueous solution, formed triple helical structures in which the three chains were parallel and in register, and showed sharp thermal transitions corresponding to the denaturation of the triple helices [47][48][49][50][51][52][53]. Chemically synthesized collagen peptides such as (PPG) 10 or (POG) 10 are homogeneous in molecular mass, have defined length and chemical composition, and thus are amenable to producing single crystals ( Figure 1B). ...
... Importantly, lack of prolyl hydroxylation in animals results in collagens that are not stable at physiological temperature, and removal of P4H activity is lethal for both vertebrate and invertebrate animal models [99,100]. The very first collagen model peptides provided further evidence for the effect of Hyp on thermal stability: (POG) 10 had a temperature of denaturation 30 • C higher than that of the homologous peptide (PPG) 10 [52]. The impact of Hyp and many related Pro derivatives has since been studied extensively using collagen model peptides, and several reviews of these studies have been published [44,[101][102][103][104][105]. ...
The main features of the triple helical structure of collagen were deduced in the mid-1950s from fibre X-ray diffraction of tendons. Yet, the resulting models only could offer an average description of the molecular conformation. A critical advance came about 20 years later with the chemical synthesis of sufficiently long and homogeneous peptides with collagen-like sequences. The availability of these collagen model peptides resulted in a large number of biochemical, crystallographic and NMR studies that have revolutionized our understanding of collagen structure. High-resolution crystal structures from collagen model peptides have provided a wealth of data on collagen conformational variability, interaction with water, collagen stability or the effects of interruptions. Furthermore, a large increase in the number of structures of collagen model peptides in complex with domains from receptors or collagen-binding proteins has shed light on the mechanisms of collagen recognition. In recent years, collagen biochemistry has escaped the boundaries of natural collagen sequences. Detailed knowledge of collagen structure has opened the field for protein engineers who have used chemical biology approaches to produce hyperstable collagens with unnatural residues, rationally designed collagen heterotrimers, self-assembling collagen peptides, etc. This review summarizes our current understanding of the structure of the collagen triple helical domain (COLx3) and gives an overview of some of the new developments in collagen molecular engineering aiming to produce novel collagen-based materials with superior properties.
... The fundamental molecular structure is a right-handed helix comprising three left-handed helical chains. Proline and hydroxyproline together comprise 22% of all amino acids in most fibrillar collagens [41]. Proline is amphiphilic, able to interact with water and lipids, and it interacts strongly with water in its immediate environment but in such a way that does not disturb the bulk water structure [42,43]. ...
The importance, and the difficulty, of generating biosynthetic articular cartilage is widely recognized. Problems arise from obtaining sufficient stiffness, toughness and longevity in the material and integration of new material into existing cartilage and bone. Much work has been done on chondrocytes and tissue macromolecular components while water, which comprises the bulk of the tissue, is largely seen as a passive component; the ‘solid matrix’ is believed to be the main load-bearing element most of the time. Water is commonly seen as an inert filler whose restricted flow through the tissue is believed to be sufficient to generate the properties measured. We propose that this model should be turned on its head. Water comprises 70–80% of the matrix and has a bulk modulus considerably greater than that of cartilage. We suggest that the macromolecular components structure the water to support the loads applied. Here, we shall examine the structure and organization of the main macromolecules, collagen, aggrecan and hyaluronan, and explore how water interacts with their polyelectrolyte nature. This may inform the biosynthetic process by identifying starting points to enable developing tissue properties to guide the cells into producing the appropriate macromolecular composition and structure.
... The triplehelical conformation is responsible for the complexity and hierarchy of fibers and networks that collagens participate in. That said, hydroxyprolines occupying the second amino acid position of the pattern significantly enhance the thermal stability of the triple helix [25,26], as long as they do not occupy at the same time the first amino acid position of the pattern or the hydroxyl group is not in the 4S form as in (2S,4S)-4-hydroxyproline [27,28]. ...
Collagen is a major component of the extracellular matrix (ECM) and has an imminent role in fibrosis, in, among others, chronic kidney disease (CKD). Collagen alpha-1(I) (col1a1) is the most abundant collagen type and has previously been underlined for its contribution to the disease phenotype. Here, we examined 5000 urinary peptidomic datasets randomly selected from healthy participants or patients with CKD to identify urinary col1a1 fragments and study their abundance, position in the main protein, as well as their correlation with renal function. We identified 707 col1a1 peptides that differed in their amino acid sequence and/or post-translational modifications (hydroxyprolines). Well-correlated peptides with the same amino acid sequence, but a different number of hydroxyprolines, were combined into a final list of 503 peptides. These 503 col1a1 peptides covered 69% of the full col1a1 sequence. Sixty-three col1a1 peptides were significantly and highly positively associated (rho > +0.3) with the estimated glomerular filtration rate (eGFR), while only six peptides showed a significant and strong, negative association (rho < −0.3). A similar tendency was observed for col1a1 peptides associated with ageing, where the abundance of most col1a1 peptides decreased with increasing age. Collectively the results show a strong association between collagen peptides and loss of kidney function and suggest that fibrosis, potentially also of other organs, may be the main consequence of an attenuation of collagen degradation, and not increased synthesis.
... Thus, one metric to evaluate collagen's mechanical integrity is from its melting point or T m , defined as the midpoint during the temperature window in which the triple helix unfolds. Several researchers have examined various biological species to discover or design collagen peptides with greater thermal stability (Burjanadze, 1979;Gekko and Koga, 1983;Rigby, 1968;Mohs et al., 2007;Inouye et al., 1982;Sakakibara et al., 1973a). Others have tried to add various additives to increase T m (Gekko and Koga, 1983). ...
Collagen is the most abundant structural protein in humans, with dozens of sequence variants accounting for over 30% of the protein in an animal body. The fibrillar and hierarchical arrangements of collagen are critical in providing mechanical properties with high strength and toughness. Due to this ubiquitous role in human tissues, collagen-based biomaterials are commonly used for tissue repairs and regeneration, requiring chemical and thermal stability over a range of temperatures during materials preparation ex vivo and subsequent utility in vivo. Collagen unfolds from a triple helix to a random coil structure during a temperature interval in which the midpoint or Tm is used as a measure to evaluate the thermal stability of the molecules. However, finding a robust framework to facilitate the design of a specific collagen sequence to yield a specific Tm remains a challenge, including using conventional molecular dynamics modeling. Here we propose a de novo framework to provide a model that outputs the Tm values of input collagen sequences by incorporating deep learning trained on a large data set of collagen sequences and corresponding Tm values. By using this framework, we are able to quickly evaluate how mutations and order in the primary sequence affect the stability of collagen triple helices. Specifically, we confirm that mutations to glycines, mutations in the middle of a sequence, and short sequence lengths cause the greatest drop in Tm values.
... In this case, the tissue fragility was linked to the decreased stability of the collagen triple helix due to the lack of 4Hyp. Studies using triple helical peptides have firmly established the significant stabilizing effects of a 4Hyp in the Y-position compared to that of a Pro [30][31][32][33][34]. The Hyl related glycosylation and cross-links were also considered an important part of the fibril stability, and the extent of the modification increases with the advance of the developmental stages [35][36][37]. ...
Collagen is the major protein in the extracellular matrix and plays vital roles in tissue development and function. Collagen is also one of the most processed proteins in its biosynthesis. The most prominent post-translational modification (PTM) of collagen is the hydroxylation of Pro residues in the Y-position of the characteristic (Gly-Xaa-Yaa) repeating amino acid sequence of a collagen triple helix. Recent studies using mass spectrometry (MS) and tandem MS sequencing (MS/MS) have revealed unexpected hydroxylation of Pro residues in the X-positions (X-Hyp). The newly identified X-Hyp residues appear to be highly heterogeneous in location and percent occupancy. In order to understand the dynamic nature of the new X-Hyps and their potential impact on applications of MS and MS/MS for collagen research, we sampled four different collagen samples using standard MS and MS/MS techniques. We found considerable variations in the degree of PTMs of the same collagen from different organisms and/or tissues. The rat tail tendon type I collagen is particularly variable in terms of both over-hydroxylation of Pro in the X-position and under-hydroxylation of Pro in the Y-position. In contrast, only a few unexpected PTMs in collagens type I and type III from human placenta were observed. Some observations are not reproducible between different sequencing efforts of the same sample, presumably due to a low population and/or the unpredictable nature of the ionization process. Additionally, despite the heterogeneous preparation and sourcing, collagen samples from commercial sources do not show elevated variations in PTMs compared to samples prepared from a single tissue and/or organism. These findings will contribute to the growing body of information regarding the PTMs of collagen by MS technology, and culminate to a more comprehensive understanding of the extent and the functional roles of the PTMs of collagen.
... X and Y are often proline (Pro) or hydroxylated proline, 3-hydroxyproline (3-Hyp) or 4-hydroxyproline (4-Hyp), respectively (35,36). While 4-Hyp in position Y of the Gly-X-Y repeat is frequently found in all collagen types and wellestablished as a major contributor to collagen thermodynamic stability (37)(38)(39)(40)(41), 3-Hyp has so far only been unambiguously detected in very few defined X positions of Gly-X-Y in collagen chains of type I, II, IV, and V, and 3-Hyp function is much less understood (42)(43)(44)(45)(46). Frequent non-collagenous (NC) domains, e.g., in FACIT, beaded-filament forming and anchoring fibril collagens, are fibronectin type III, von Willebrand, thrombospondin (TSP) and Kunitz domains. The physiological function of these domains is incompletely understood. ...
In addition to providing a macromolecular scaffold, the extracellular matrix (ECM) is a critical regulator of cell function by virtue of specific physical, biochemical, and mechanical properties. Collagen is the main ECM component and hence plays an essential role in the pathogenesis and progression of chronic lung disease. It is well-established that many chronic lung diseases, e.g., chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF) primarily manifest in the elderly, suggesting increased susceptibility of the aged lung or accumulated alterations in lung structure over time that favour disease. Here, we review the main steps of collagen biosynthesis, processing, and turnover and summarise what is currently known about alterations upon lung ageing, including changes in collagen composition, modification, and crosslinking. Recent proteomic data on mouse lung ageing indicates that, while the ER-resident machinery of collagen biosynthesis, modification and triple helix formation appears largely unchanged, there are specific changes in levels of type IV and type VI as well as the two fibril-associated collagens with interrupted triple helices (FACIT), namely type XIV and type XVI collagens. In addition, levels of the extracellular collagen crosslinking enzyme lysyl oxidase are decreased, indicating less enzymatically mediated collagen crosslinking upon ageing. The latter contrasts with the ageing-associated increase in collagen crosslinking by advanced glycation endproducts (AGEs), a result of spontaneous reactions of protein amino groups with reactive carbonyls, e.g., from monosaccharides or reactive dicarbonyls like methylglyoxal. Given the slow turnover of extracellular collagen such modifications accumulate even more in ageing tissues. In summary, the collective evidence points mainly toward age-induced alterations in collagen composition and drastic changes in the molecular nature of collagen crosslinks. Future work addressing the consequences of these changes may provide important clues for prevention of lung disease and for lung bioengineering and ultimately pave the way to novel targeted approaches in lung regenerative medicine.
... The thermal stability of collagen arises from many different factors, such as interstrand hydrogen bonding between Gly and amino acids at the Xaa position and stereochemical restrictions by the imino acid rings of Pro and Hyp [5]. Since substitution of Pro with 4Hyp at the Yaa position increased the stability [6] and provided additional water bridges in model peptides [7], it had been thought that collagen is stabilized mainly by hydrogen bonding between the 4-hydroxyl groups and main-chain oxygens. However, Holmgren et al. [8] reported that the stability of collagen was also increased by (2S,4R)-4-fluoroproline whose side chain does not form hydrogen bonds, which indicates that water bridges do not contribute significantly to the stabilization in solution [9,10]. ...
There is a general consensus that collagen stability is largely maintained by Pro and its major hydroxylated form, 4-hydroxyproline (4Hyp). However, positional difference in their stabilizing effect at the Xaa or Yaa position of collagenous Gly-Xaa-Yaa sequences has remained inconclusive. Here, we position-specifically evaluated the correlation of imino acid contents to denaturation temperature (Td) of collagen among various vertebrate and invertebrate species, using a recently developed LC–MS methodology. 4Hyp at the Yaa position showed the highest positive correlation with Td, followed by Pro at the Xaa position, which was even further increased by excluding invertebrates. We confirmed that Gly-Pro-4Hyp liberated after bacterial collagenase digestion was highly positively correlated with Td. Furthermore, other tripeptides with Yaa position 4Hyp also had comparable positive correlation, excepting negative correlation of Gly-Gly-4Hyp, while tripeptides with Xaa position Pro did not. These data provide evidence that 4Hyp dominantly contributes to thermal stability of collagen depending on its sequence position, especially in vertebrates.
... An early approach was to use chemical cross-links at the C-terminus to bring about the correct association of the three polypeptide chains [70,71]. Later, it was found that peptides with repeating sequences of (Gly-Pro-Hyp) n with n > 6 can self-assemble into a stable triple helix without the need of cross-links, and the thermal stability increases with an increase in the number of tripeptide units [72]. Thus, while the triple helix (Gly-Pro-Hyp) 6 was only marginally stable with a melting temperature (T m ) of 10 • C, that of a (Gly-Pro-Hyp) 10 can reach 68 • C. Peptides with (Gly-Pro-Pro) n repeating sequences also form a triple helix, but have a much lower thermal stability; in comparison with (Gly-Pro-Hyp) 10 , the thermal stability of (Gly-Pro-Pro) 10 is only about 27 • C [73,74]. ...
Since their first synthesis in the late 1960s, collagen mimetic peptides (CMPs) have been used as a molecular tool to study collagen, and as an approach to develop novel collagen mimetic biomaterials. Collagen, a major extracellular matrix (ECM) protein, plays vital roles in many physiological and pathogenic processes. Applications of CMPs have advanced our understanding of the structure and molecular properties of a collagen triple helix—the building block of collagen—and the interactions of collagen with important molecular ligands. The accumulating knowledge is also paving the way for developing novel CMPs for biomedical applications. Indeed, for the past 50 years, CMP research has been a fast-growing, far-reaching interdisciplinary field. The major development and achievement of CMPs were documented in a few detailed reviews around 2010. Here, we provided a brief overview of what we have learned about CMPs—their potential and their limitations. We focused on more recent developments in producing heterotrimeric CMPs, and CMPs that can form collagen-like higher order molecular assemblies. We also expanded the traditional view of CMPs to include larger designed peptides produced using recombinant systems. Studies using recombinant peptides have provided new insights on collagens and promoted progress in the development of collagen mimetic fibrillar self-assemblies.
... Detailed bioand physicochemical studies determined that these stabilising bonds are present in glycine between the N-H bonds and in the C-O bonds of the amino acids present in the subsequent strands [15]. Additionally, reports suggest that the hydroxyl (OH) group present in the hydroxyproline and the water molecule networks surrounding triple helices also contribute to thermal and conformation stabilisation [106]. Studies reported by Kramer et al. calculated the effect of water molecules using a collagen-like peptide structure. ...
Grand challenges facing humanity today are closely linked to the rapid exhaustion of natural resources in conjunction with the massive growth of industrial production that sustains the booming world population. The processing of animal skin waste to create collagen-based materials has the potential to provide an eco-friendly method to develop multifunctional materials such as films, sponges/scaffolds, fibers, gels, etc., that could contribute to technological advancements in different sectors. Hence in this review, we present methods for potential improvements in the development of collagen-based materials from a materials science perspective. We explored different possible approaches for utilizing collagen to generate multifunctional materials that exhibit outstanding properties, in combination with mechanical robustness and chemical stability. In sum, this review will present collagen as an eco-friendly resource that can be used to produce multifunctional, recyclable, biocompatible, and biodegradable materials that are ideal for new technologies in materials science, biomedicine, and environmental remediation.
... X and Y are usually Pro and Hyp, respectively. The presence of Hyp gives more stability and rigidity to the backbone via hydrogen bonding and the melting temperature (Tm) of collagen significantly increases [101]. A total of 109 and 78 HyP residues were predicted for α1 (F6SSG3_HORSE) and α2 chain (F6RTI8_HORSE) by bioinformatic analysis [102], respectively. ...
Type I collagen is the most abundant protein of the human body. Due to its favourable properties, collagen extracted from animal tissues is adopted to manufacture a wide range of devices for biomedical applications. Compared to bovine and porcine collagens, which are the most largely used, equine collagen is free from the risk of zoonosis, has no reported immune reactions, and has not religious constraints. In this work, a recently available type I collagen extracted from horse tendon was evaluated and compared with a commercially available collagen isoform derived from the same species and tissue. Detailed physical, chemical and biological investigations were performed, in agreement with the requirements of the current standard for the characterization of type I collagen to be used for the manufacture of Tissue Engineering Medical Products. To the best of our knowledge, this is the first report on the complete primary structure of the investigated collagen.
... As reported by Abdelhedi et al, imino acids content for black-barred halfbeak and commercial gelatins were 24.35% and 27%, respectively (Abdelhedi et al., 2017). Hyp is the main residue responsible for the thermal stability of collagen triple-helix through the hydrogen bonding ability of its hydroxyl group, that is why the UAE gelatin showed a higher melting point at the highest level (Sakakibara et al., 1973). However, Cys had the lowest percentage in all three gelatins. ...
The effects of ultrasound‐assisted extraction (UAE) and microwave‐assisted extraction (MAE) methods on molecular and physicochemical characteristics of the resultant gelatin were examined. Before extraction procedure, we investigated the optimum pH for swelling of Common carp by‐products which is an important pretreatment for gelatin production. The highest swelling yield was achieved at pH 13 among pH 1‐14 with unit intervals. Results indicated that the UAE gelatin has a higher gel strength, viscosity, melting point, and gelling point. The power and time of sonication showed a reverse relation with these characteristics. In addition, as the time of microwave heating was raised, the gel strength, viscosity, melting point, and gelling point were decreased. The FT‐IR spectra showed similar peaks but the amide B in UAE gelatin slightly vanished. The electrophoretic pattern also revealed the higher gel strength and viscosity of UAE gelatin due to the higher intensity of α and β chains compared to MAE gelatin. It can be concluded from all of the results of this study that the produced gelatin using these procedures can be a good source of gelatin in food and drug industries.
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... Synthetic collagen-like peptides have been used to study the structure, the stability, and the biological function of collagen. The earliest works focused on synthesizing simple triple-helical tripeptide repeats such as (PPG) n [77] and (POG) n [78]. As more techniques to prepare collagenlike peptides and methods to characterize triple helices are being developed, our understanding of collagen and its interactions with other proteins has grown dramatically. ...
The discoidin domain receptors, DDR1 and DDR2, are a subfamily of receptor tyrosine kinases that are activated upon binding to collagen. DDR–collagen interactions play an important role in cell proliferation and migration. Over the past few decades, synthetic peptides and recombinant collagen have been developed as tools to study the biophysical characteristics of collagen and various protein–collagen interactions. Herein we review how these techniques have been used to understand DDR–collagen interactions. Using synthetic collagen-like peptides, the GVM-GFO motif has been found to be the major binding site on collagens II and III for DDR1 and DDR2. An X-ray co-crystal structure of the DDR2 DS domain bound to a synthetic collagen-like peptide containing the GVM-GFO motif further provides molecular details of the DDR–collagen interactions. Recombinant collagen has also been used to provide further validation of the GVM-GFO binding motif. Although GVM-GFO has been defined as the minimal binding site, in synthetic peptide studies at least two triplets N-terminal to the essential GVM-GFO binding motif in collagen III sequence are needed for DDR2 activation at high peptide concentrations.
... However, fusion to an abiological, short triple-helical domain (and expression using an in vitro system) may obfuscate the normal role of the C-Pro in initiating collagen trimerization, instead allowing the triple helix itself or the N-Pro domain to drive trimerization in certain cases. Indeed, even much shorter triple-helical sequences are able to assemble in vitro in the complete absence of a C-Pro domain 32 , whereas proper assembly of full-length collagen requires the C-Pro. Differences in the melting temperature of the Colα2(I) versus Colα1(III) mini-triple-helical domains used may also complicate data interpretation. ...
Fundamental questions regarding collagen biosynthesis, especially with respect to the molecular origins of homotrimeric versus heterotrimeric assembly, remain unanswered. Here, we demonstrate that the presence or absence of a single cysteine in type-I collagen’s C-propeptide domain is a key factor governing the ability of a given collagen polypeptide to stably homotrimerize. We also identify a critical role for Ca²⁺ in non-covalent collagen C-propeptide trimerization, thereby priming the protein for disulfide-mediated covalent immortalization. The resulting cysteine-based code for stable assembly provides a molecular model that can be used to predict, a priori, the identity of not just collagen homotrimers, but also naturally occurring 2:1 and 1:1:1 heterotrimers. Moreover, the code applies across all of the sequence-diverse fibrillar collagens. These results provide new insight into how evolution leverages disulfide networks to fine-tune protein assembly, and will inform the ongoing development of designer proteins that assemble into specific oligomeric forms.
... Moreover, proline and other aa within the triple helix remain accessible to solvent and, thus, their numerous known PTMs are possible without disturbing the native helix structure. For example, probably the most important PTM is the 4(R)-hydroxylation of proline residues position Y, because it substantially increases the thermal stability of the collagen triple helix (Sakakibara et al., 1973). Although all proline and hydroxyproline residues are essential for the stability of a PPII helix, ∼65% of aa at these positions can vary in the native triple helix, while still maintaining its stability. ...
The cellular microenvironment, characterized by an extracellular matrix (ECM), played an essential role in the transition from unicellularity to multicellularity in animals (metazoans), and in the subsequent evolution of diverse animal tissues and organs. A major ECM component are members of the collagen superfamily -comprising 28 types in vertebrates - that exist in diverse supramolecular assemblies ranging from networks to fibrils. Each assembly is characterized by a hallmark feature, a protein structure called a triple helix. A current gap in knowledge is understanding the mechanisms of how the triple helix encodes and utilizes information in building scaffolds on the outside of cells. Type IV collagen, recently revealed as the evolutionarily most ancient member of the collagen superfamily, serves as an archetype for a fresh view of fundamental structural features of a triple helix that underlie the diversity of biological activities of collagens. In this Opinion, we argue that the triple helix is a protein structure of fundamental importance in building the extracellular matrix, which enabled animal multicellularity and tissue evolution.
... The melting temperature (Tm) of collagen significantly increases due to the presence of Hyp. 27 Yang et al. unexpectedly observed Hyp residues in the Xaa-position of Gly-Xaa-Yaa triplets, which resulted in Gly-Hyp-Val and Gly-Hyp-Ala triplets at certain positions in bovine placental collagen. 28 There was uncertainty as to the form of the Hyp in the above triplets as 3-Hyp is believed to be exclusive to the Xaa position of the triplet Gly-Pro-Hyp, with 4-Hyp exclusive to the Yaa position. ...
Flexible and protease resistant (G4S)n linkers are used extensively in protein engineering to connect various protein domains. Recently, a number of groups have observed xylose-based O-glycosylation at linker Ser residues that yield unwanted heterogeneity and may affect product quality. Because of this, an engineering effort was implemented to explore different linker sequence constructs. Here, we demonstrate the presence of an unexpected hydroxylation of a prolyl residue in the linker, made possible through the use of high-resolution mass spectrometry (HR-MS) and MSn. The discovery started with the detection of a poorly resolved ∼+17 Da mass addition at the reduced protein chain level of an Fc-fusion construct by liquid chromatography-MS. Upon further investigation at the peptide level using HR-MS, the mass increase was determined to be +15.99 Da and was localized to the linker peptide SLSLSPGGGGGPAR [210-223]. This peptide corresponds to the C-terminus of Fc [210-216], the G4P linker [217-221], and first two amino acids of a growth factor [222-223]. The linker peptide was first subjected to MS (2) with collision-induced dissociation (CID) activation. The fragmentation profile localized the modification to the GGGPA [218-222] portion of the peptide. Accurate mass measurement indicated that the modification is an addition of an oxygen and cannot be CH4, thus eliminating a number of possibilities such as Pro→Leu. However, other possibilities cannot be ruled out. Higher-energy collision-induced dissociation (HCD)-MS (2) and MS (3) using CID/CID were both unable to differentiate between Ala222→ Ser222 or Pro221→ Hyp221. Finally, MS (3) using high-resolution CID/HCD confirmed the mass increase to be a Pro221→Hyp221 post-translational modification.
... Pro and Hyp are very abundant in connective tissues (Sato, 1993). Hyp is the main residue responsible for the thermal stability of collagen triple-helix through the hydrogen bonding ability of its hydroxyl group (Sakakibara et al., 1973). Nevertheless, His, Tau (derived from Cys), Met and Ile were present in very low amounts in both gelatins. ...
In the current study, gelatin was extracted from black-barred halfbeak (Hemiramphus far) skin by successive alkaline and acid treatments and then hydrolyzed with Purafect®. The black-barred halfbeak gelatin (BG) and its hydrolysate (BGH) were characterized and compared to the commercial bovine gelatin (CG). Samples were evaluated for their antioxidant, antibacterial and angiotensin I-converting enzyme (ACE) inhibitory activities. Results obtained using size exclusion chromatography showed that BG contained lower level of high molecular weight proteins, compared to CG. In addition, the amino acids composition revealed that BG contained lower level of imino acids (Pro+Hpx), compared to CG. These differences reflect the variations observed in the gel strength and gelling and melting temperatures of both skin gelatins. Furthermore, high similarities were observed between CG and BG in terms of their Fourier transform infrared (FTIR) spectra, while their amino acid compositions were quite different. BGH, with a degree of hydrolysis of 12.5%, showed high antioxidant potential that was assessed by the scavenging activity, reducing power, oxygen radical absorbance capacity, β-carotene bleaching protection and lipid per-oxidation assays. In addition, BGH sample exhibited antibacterial activity against different Gram+ and Gram- bacteria. The ACE-inhibitory activity was also investigated. BGH showed an inhibitory effect of 80.76%, while BG inhibited the ACE only by 36.51% at 1 mg/ml. Thus, black-barred halfbeak gelatin represents a promising source of antioxidant, ACE-inhibitory and antimicrobial peptides that might prevent human from several diseases.
... 56 The IL-2-treated wounds had higher levels of hydroxyproline, which is indicative of collagen crosslinking and may elude to increased ECM deposition or scarring in response to IL-2. 57 The authors of this study interpret these results to mean that lymphocyte activation by IL-2, rather than IL-2 action on skin cells, mediates increased strength following IL-2 treatment, although this study does not directly test that hypothesis. However, this study does hint that the role of IL-2 in altering the course of wound healing may involve systemic actions. ...
Ineffective skin wound healing is a significant source of morbidity and mortality. Roughly 6.5 million Americans experience chronically open wounds and the cost of treating these wounds numbers in the billions of dollars annually. In contrast, robust wound healing can lead to the development of either hypertrophic scarring or keloidosis, both of which can cause discomfort and can be cosmetically undesirable. Appropriate wound healing requires the interplay of a variety of factors, including the skin, the local microenvironment, the immune system, and the external environment. When these interactions are perturbed, wounds can be a nidus for infection, which can cause them to remain open an extended period of time, or can scar excessively. Interleukin-2, a cytokine that directs T-cell expansion and phenotypic development, appears to play an important role in wound healing. The best-studied role for Interleukin-2 is in influencing T-cell development. However, other cell types, including fibroblasts, the skin cells responsible for closing wounds, express the Interleukin-2 receptor, and therefore may respond to Interleukin-2. Studies have shown that treatment with Interleukin-2 can improve the strength of healed skin, which implicates Interleukin-2 in the wound healing process. Furthermore, diseases that involve impaired wound healing, such as diabetes and systemic lupus erythematosus, have been linked to deficiencies in Interleukin-2 or defects Interleukin-2-receptor signaling. The focus of this review is to summarize the current understanding of the role of Interleukin-2 in wound healing, to highlight diseases in which Interleukin-2 and its receptor may contribute to impaired wound healing, and to assess Interleukin-2-modulating approaches as potential therapies to improve wound healing.
... The ensuing creation of (2S,4R)-4-hydroxyproline (Hyp) residues is necessary for the formation of stable collagen triple helices. 3,4 Molecular oxygen (O 2 ), α-ketoglutarate, and Fe(II) are required for the catalytic activity of CP4H. 5 During catalysis, α-ketoglutarate is decarboxylated oxidatively to produce succinate and CO 2 . 6 CP4H can decarboxylate α-ketoglutarate without effecting the hydroxylation of proline residues. ...
Collagen is the most abundant protein in animals. The posttranslational hydroxylation of proline residues in collagen contributes greatly to its conformational stability. Deficient hydroxylation is associated with a variety of disease states, including scurvy. The hydroxylation of proline residues in collagen is catalyzed by an Fe(II)- and α-ketoglutarate-dependent dioxygenase, collagen prolyl 4-hydroxylase (CP4H). CP4H has long been known to suffer oxidative inactivation during catalysis, and the cofactor ascorbate (vitamin C) is required to reactivate the enzyme by reducing its iron center from Fe(III) to Fe(II). Herein, we report on the discovery of the first synthetic activators of CP4H. Specifically, we find that 2,2′-bipyridine-4 carboxylate and 2,2′ bipyridine-5-carboxylate serve as ligands for the iron center in human CP4H that enhance the rate of ascorbate-dependent reactivation. This new mode of CP4H activation is available to other biheteroaryl compounds but does not necessarily extend to other prolyl 4 hydroxylases. As collagen is weakened in many indications, analogous activators of CP4H could have therapeutic benefit.
The application of non-absorbable hernia mesh in the repair of abdominal abnormalities has been associated with a range of problems and a considerable loss in quality of life. The purpose of this study is to develop a nanofiber hernia mesh coated with a growth factor, to evaluate its effect on abdominal defect repair in a rat model, and to show that this new mesh is superior. The coaxial electrospinning technique was used to synthesize the polycaprolactone and polyvinyl alcohol (PCL/PVA) mesh coated with vascular endothelial growth Factor165 (VEGF165) and fibroblast growth factor-21 (FGF-21), and the CCK8 assay was used to determine the proliferation rate of NIH3T3 co-cultured with the synthetic product. Sixteen healthy male Sprague–Dawley rats with hernias were randomly assigned to receive mesh made of PCL/PVA or polypropylene (PP). The tensile strength of meshes was determined. The tissue was stained to determine the amount of inflammation and angiogenesis using a microscope. Polymerase chain reaction (PCR) was used to detect the expression of inflammatory factors. Our results demonstrated that the PCL/PVA mesh had no effect on cell proliferation in vitro. The experimental rats tolerated PCL/PVA mesh well, and there was no difference in breaking strength between the PCL/PVA mesh and PP mesh groups. Foreign body giant cells were substantially more abundant in the PCL/PVA mesh group than in the PP mesh group. The PCL/PVA mesh group demonstrated considerably more angiogenesis than the PP mesh group. The PCL/PVA mesh group had higher levels of colony-stimulating factor 2 (CSF2), interleukin-2 (IL-2), and transforming growth factor-β (TGF-β) and lower levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) than the PP mesh group. This study showed that the PCL/PVA mesh coated with VEGF165 and FGF-21 can be well tolerated and promote angiogenesis in hernia repair. It may be a potential mesh for clinical applications.
Collagen is the most abundant structural protein in humans, providing crucial mechanical properties, including high strength and toughness, in tissues. Collagen-based biomaterials are, therefore, used for tissue repair and regeneration. Utilizing collagen effectively during materials processing ex vivo and subsequent function in vivo requires stability over wide temperature ranges to avoid denaturation and loss of structure, measured as melting temperature (T m ). Although significant research has been conducted on understanding how collagen primary amino acid sequences correspond to T m values, a robust framework to facilitate the design of collagen sequences with specific T m remains a challenge. Here, we develop a general model using a genetic algorithm within a deep learning framework to design collagen sequences with specific T m values. We report 1,000 de novo collagen sequences, and we show that we can efficiently use this model to generate collagen sequences and verify their T m values using both experimental and computational methods. We find that the model accurately predicts T m values within a few degrees centigrade. Further, using this model, we conduct a high-throughput study to identify the most frequently occurring collagen triplets that can be directly incorporated into collagen. We further discovered that the number of hydrogen bonds within collagen calculated with molecular dynamics (MD) is directly correlated to the experimental measurement of triple-helical quality. Ultimately, we see this work as a critical step to helping researchers develop collagen sequences with specific T m values for intended materials manufacturing methods and biomedical applications, realizing a mechanistic materials by design paradigm.
Collagen is one of the most important structural proteins in biology, and its structural hierarchy plays a crucial role in many mechanically important biomaterials. Here, we demonstrate how transformer models can be used to predict, directly from the primary amino acid sequence, the thermal stability of collagen triple helices, measured via the melting temperature Tm. We report two distinct transformer architectures to compare performance. First, we train a small transformer model from scratch, using our collagen data set featuring only 633 sequence-to-Tm pairings. Second, we use a large pretrained transformer model, ProtBERT, and fine-tune it for a particular downstream task by utilizing sequence-to-Tm pairings, using a deep convolutional network to translate natural language processing BERT embeddings into required features. Both the small transformer model and the fine-tuned ProtBERT model have similar R2 values of test data (R2 = 0.84 vs 0.79, respectively), but the ProtBERT is a much larger pretrained model that may not always be applicable for other biological or biomaterials questions. Specifically, we show that the small transformer model requires only 0.026% of the number of parameters compared to the much larger model but reaches almost the same accuracy for the test set. We compare the performance of both models against 71 newly published sequences for which Tm has been obtained as a validation set and find reasonable agreement, with ProtBERT outperforming the small transformer model. The results presented here are, to our best knowledge, the first demonstration of the use of transformer models for relatively small data sets and for the prediction of specific biophysical properties of interest. We anticipate that the work presented here serves as a starting point for transformer models to be applied to other biophysical problems.
The hydrogen bond strength and stabilization energy of hydroxyproline–water complexes were investigated by performing density-functional theory calculations. In particular, the hydrogen bond formation between carbonyl groups serving as proton acceptors and amino groups as proton donors in the hydroxyproline–water was examined. Hydroxyproline-water exhibit higher energy of their hydrogen bond when the carbonyl groups of their hydroxyproline moieties acts as a proton acceptor. Furthermore, the infrared spectra of isolated water and hydroxyproline molecules were compared with those of the hydroxyproline–water complexes, and the observed frequency shifts were discussed.
In the growing field of tissue engineering, providing cells in biomaterials with the adequate biological cues represents an increasingly important challenge. Yet, biomaterials with excellent mechanical properties often are often biologically inert to many cell types. To address this issue, researchers resort to functionalization, i.e. the surface modification of a biomaterial with active molecules or substances. Functionalization notably aims to replicate the native cellular microenvironment provided by the extracellular matrix, and in particular by collagen, its major component. As our understanding of biological processes regulating cell behaviour increases, functionalization with biomolecules binding cell surface receptors constitutes a promising strategy. Amongst these, triple-helical peptides (THPs) that reproduce the architectural and biological properties of collagen are especially attractive. Indeed, THPs containing binding sites from the native collagen sequence have successfully been used to guide cell response by establishing cell-biomaterial interactions. Notably, the GFOGER motif recognising the collagen-binding integrins is extensively employed as a cell adhesive peptide. In biomaterials, THPs efficiently improved cell adhesion, differentiation and function on biomaterials designed for tissue repair (especially for bone, cartilage, tendon and heart), vascular graft fabrication, wound dressing, drug delivery or immunomodulation. This review describes the key characteristics of THPs, their effect on cells when combined to biomaterials and their strong potential as biomimetic tools for regenerative medicine.
Statement of significance
This review article describes how triple-helical peptides constitute efficient tools to improve cell-biomaterial interactions in tissue engineering. Triple helical peptides are bioactive molecules that mimic the architectural and biological properties of collagen. They have been successfully used to specifically recognize cell-surface receptors and provide cells seeded on biomaterials with controlled biological cues. Functionalization with triple-helical peptides has enabled researchers to improve cell function for regenerative medicine applications, such as tissue repair. However, despite encouraging results, this approach remains limited and under-exploited, and most functionalization strategies reported in the literature rely on biomolecules that are unable to address collagen-binding receptors. This review will assist researchers in selecting the correct tools to functionalize biomaterials in efforts to guide cellular response.
Organ fibrosis is characterized by epithelial injury and aberrant tissue repair, where activated effector cells, mostly fibroblasts and myofibroblasts, excessively deposit collagen into the extracellular matrix. Fibrosis frequently results in organ failure and has been estimated to contribute to at least one third of all global deaths. Also lung fibrosis, in particular idiopathic pulmonary fibrosis (IPF), is a fatal disease with rising incidence worldwide. As current treatment options targeting fibrogenesis are insufficient, there is an urgent need for novel therapeutic strategies. During the last decade, several studies have proposed to target intra- and extracellular components of the collagen biosynthesis, maturation, and degradation machinery. This includes intra- and extracellular targets directly acting on collagen gene products, but also such that anabolize essential building blocks of collagen, in particular glycine and proline biosynthetic enzymes. Collagen, however, is a ubiquitous molecule in the body and fulfils essential functions as a macromolecular scaffold, growth factor reservoir, and receptor binding site in virtually every tissue. This review summarizes recent advances and future directions in this field. Evidence for the proposed therapeutic targets and where they currently stand in terms of clinical drug development for treatment of fibrotic disease is provided. The drug targets are furthermore discussed in light of (1) specificity for collagen biosynthesis, maturation and degradation, and (2) specificity for disease-associated collagen. As therapeutic success and safety of these drugs may largely depend on targeted delivery, different strategies for specific delivery to the main effector cells and to the extracellular matrix are discussed.
Collagen is a major structural protein, and abnormalities in collagen structure can lead to several connective tissue diseases such as osteoporosis. We report the preparation of a collagen sensor using a synthetic peptide as proof of concept for detecting the collagen like peptides. The synthetic peptide 9-fluorenylmethyloxycarbonyl (Fmoc)-(prolyl-prolyl-glycine)7-OH was coupled to thiazolidine, which gets adsorbed on metal surfaces. Fmoc-(prolyl-prolyl-glycine)7-thiazolidine was immobilized on the surface of a quartz crystal microbalance (QCM) electrode used as a sensor probe. The collagen model peptide (prolyl-prolyl-glycine)10 could be detected, and the model peptide was directly adsorbed onto the surface of the electrode and was not removed by washing with hot water. Additionally, it was proved that the sensitivity of the probe could be enhanced to nanogram order by immobilizing the blocking reagent, Fmoc-prolyl-prolyl-glycine, within the gap of sensor probes on the electrode. The detectable mass of the model peptide decreased as the probe gap became narrower because of self-association of the probes. Moreover, the sensitivity of sensor probes also decreases as the gap between the probes becomes wider. Therefore, the optimum distance between the immobilized probes was determined from the simulation based on the experimental values. The association rate of the model peptide with sensor probes could be quantitatively determined when the distance between the probes was optimum, and this result suggested that most sensor probes could form a triple helical structure with the model peptide.
In present study, title compound Trans-4-Hydroxy-L-Proline is investigated theoretically and experimentally by FT-IR, NMR and UV–Vis spectra. The theoretical optimized geometrical parameters, vibrational analysis were performed by Density Functional Theory (DFT) with the B3LYP method at 6-311++G(d,p) basis set. Hirshfeld surface analysis has been performed and studied the nature of intermolecular interactions within the crystal structure via 3D and 2D surfaces, and demonstrated the H-bonding with close contacts in crystal via dnorm surface. Local chemical activity was studied via molecular electrostatic potential (MEP) and analyzed the reactive areas of the titled molecule, and local chemical reactivity was studied by population analysis and Fukui function analysis. FMOs investigation given various chemical activity parameters like- chemical hardness, chemical softness and electrophilicity index. NLO has been performed and analyzed the hyperpolarizability value and polarity of molecule. Natural Bond Orbital (NBO) analysis was carried out, reporting the hybridization of atoms that form bonds. The charge transfer of title molecule have been examined by TD-DFT method. The compound was docked with (A) 1TKZ, (B) 4WKE, (C) 5KK7, (D) 5OSC, (E) 6B6G, (F) 6HSN, and (G) 6YW0 Protein receptors to find the best ligand–protein interactions, the best and lowest binding energy obtained was −5.4 Kcal/mol. Drug likeness was also performed on titled molecule and its different derivatives for the confirmation of drug-like character of titled molecule.
Collagen is the major protein in the extracellular matrix and plays vital roles in tissue development and function. Collagen is also one of the most processed proteins in its biosynthesis. The most prominent post-translational modification (PTM) of collagen is the hydroxylation of Pro residues in the Y-position of the characteristic (Gly-Xaa-Yaa) repeating amino acid sequence of a collagen triple helix. Recent studies using mass-spectrometry (MS) and tandem MS sequencing (MS/MS) have revealed unexpected hydroxylation of Pro residues in the X-positions (X-Hyp). The newly identified X-Hyp residues appear to be highly heterogeneous in location and percent occupancy. In order to understand the dynamic nature of the new X-Hyps and their potential impact on applications of MS and MS/MS for collagen research, we sampled four different collagen samples using standard MS and MS/MS techniques. We found considerable variations in the degree of PTMs of the same collagen from different organisms and/or tissues. The rat tail tendon type I collagen is particularly variable in terms of both over-hydroxylation of Pro in the X-position and under-hydroxylation of Pro in the Y-position. In contrast, only a few unexpected PTMs in collagens type I and type III from human placenta were observed. The reproducibility of the different sequencing efforts of the same sample is also limited especially when the modified species are present at a low population, presumably due to the unpredictable nature of the ionization process. Additionally, despite the heterogeneous preparation and sourcing, collagen samples from commercial sources do not show elevated variations in PTMs compared to samples prepared from a single tissue and/or organism. These findings will contribute to the growing body of information regarding the PTMs of collagen by MS technology, and culminate to a more comprehensive understanding of the extent and the functional roles of the PTMs of collagen.
Abbreviations page
Both the single letter and the three letter abbreviations of an amino acid will be used with the following additions: Hyp or O stands for 4R-hydroxylated proline and 3Hyp stands for 3- hydroxylated proline. When needed for clarity, the lower case single letter abbreviation will be used to represent the genomic DNA sequence, and upper case ones the sequence seen in the peptides.
We present our recent work on development in coarse-grained models to investigate materials made of synthetic and biological polymers with chemistries that have specific and directional molecular interactions. These coarse-grained models address an existing need within the field of polymer simulations for models that can capture the small scale, specific and directional interactions alongside longer, macromolecular/polymeric length and time scales. In particular, we describe our recent work developing and using coarse-grained models that allow us to simulate experimentally relevant systems of oligomers of nucleic acids (e.g., DNA, RNA, etc.), collagen-mimetic peptides or collagen-like peptides and polymer nanocomposites. We organize this chapter in three sections with each section independently focused on each of these three topics. In each section, we present a background subsection where we describe briefly relevant literature from other researchers on the topic, followed by subsections describing our model, simulation and analyses methods, a subsection describing key results achieved using simulations with these models, and conclude with a limitations and potential future directions subsection.
The formation of disulfide bonds is probably the most influential modification of peptides and proteins. An elaborate set of cellular machinery exists to catalyze and guide this process. In recent years, significant developments have been made in both our understanding of the in vivo situation and the in vitro manipulation of disulfide bonds. This is the first monograph to provide a comprehensive overview of this exciting and rapidly developing area. It offers in-depth insights into the mechanisms of in vivo and in vitro oxidative folding of proteins as well as mono- and multiple-stranded peptides. Procedures applied for laboratory and industrial purposes are also discussed by top experts in the field. The book describes the enzymes involved in the correct oxidative folding of cysteine-containing proteins in prokaryotes and eukaryotes. It then goes on to discuss the mimicking of these enzymes for successful in vitro folding of proteins (including synthetic replicates) and to deal with important issues concerning cysteine-rich peptides. The ability of natural bioactive peptides to fold correctly, and in high yields, to form defined structural motifs using cysteine sequence patterns is still puzzling. With this in mind, synthetic procedures for establishing native cysteine frameworks are discussed using selected examples, such as the potential of selenocysteines. The biotechnological and pharmaceutical relevance of proteins, peptides, their variants and synthetic replicates is continuously increasing. Consequently, this book is invaluable for peptide and protein chemists involved in related research and production.
Collagen, characterized by the unique triple helical structure, is a main component of the extracellular matrix, and exists in almost all parts of the body such as skin and bone. It is considered that denatured collagen, which has unfolded portions in the triple helical molecule, is produced and accumulated during the degradation process in the tissues surrounding malignant tumors. Collagen-mimetic peptide (CMP) is a generic name for chemically synthesized peptides that mimic the triple helical structure of collagen. A single-stranded CMP can hybridize to the loose triple helical parts of collagen by its inherent triple helix-forming propensity. In this review, we summarize studies on the collagen-hybridizing CMPs, and introduce a new DDS concept that targets changes in the structure of extracellular matrices. Although the relationship between collagen denaturation and diseases is not well understood, the collagen-hybridizing CMP would be useful for understanding of pathological conditions, and development of new diagnostic and therapeutic strategies.
The primary structure of collagen, the major protein in connective tissue of mammals, comprises of repeating triads [(LPro‐LHyp‐Gly)n, P1, LHyp being 4R‐hydroxy‐lProline)] in a single strand that adopts left‐handed polyproline II type helix. Three such single stranded helices wind around each another and held together by interchain H‐bonds to form right‐handed triple helix. This manuscript reports on collagen derived from its mirror image triad [(DPro‐DHyp‐Gly)n, P2, DHyp being 4S‐hydroxy‐DProline) and its 4‐amino analogue (DPro‐DAmp‐Gly)n P4, DAmp being 4S‐amino‐DProline that form corresponding speigelmeric triplexes. The amino L‐collagen peptide (LPro‐LAmp‐Gly)n P3 and its D‐analogue P4 show higher thermal stabilities compared to 4‐hydroxy‐lProline collagen peptides P1 and P2. The enantiomeric peptide pairs show mirror image CD profiles and identical thermal stability, with ionizable 4‐amino group in P3 and P4 imparting pH dependent triplex stability. Upon cold mixing of the L‐ and D‐collagen peptides, different morphological nanostructures arise from inter triplex peptide association. When the peptides are hot mixed (annealed), the inter peptide association occurs via interaction of single stranded peptide chains of opposite handedness leading to networked gel formation in P1 and P2, while the charged peptides P3 and P4 show more ordered nanofibers, different from the enantiomerically pure peptides. The nanocomposites of such chiral hybrid peptides may have not only interesting physicomorphology, but also biological properties that need exploration.
Elastin-like polypeptides (ELPs) are thermoresponsive biopolymers that undergo an LCST-like phase transition in aqueous solutions. The temperature of this LCST-like transition, Tt , can be tuned by varying the number of repeat units in the ELP, sequence and composition of the repeat units, the solution conditions, and via conjugation to other biomacromolecules. In this study, we show how and why the choice of guest (X) residue in the VPGXG pentad repeat tunes the Tt of short ELPs, (VPGXG)4, in the free state and when conjugated to collagen-like peptides (CLPs). In experiments, the (VPGWG)4 chain (in short, WWWW) has a Tt < 278 K, while (VPGFG)4 or FFFF has a Tt > 353 K in both free ELP and ELP–CLP systems. The Tt for the FWWF ELP sequence decreases from being >353 K for free ELP to <278 K for the corresponding ELP–CLP system. The decrease in Tt upon conjugation to CLP has been shown to be due to the crowding of ELP chains that decreases the entropic loss upon ELP aggregation. Even though the net hydrophobicity of ELP has been reasoned to drive the Tt , the origins of lower Tt of WWWW compared to FFFF are unclear, as there is disagreement in hydrophobicity scales in how phenylalanine (F) compares to tryptophan (W). Motivated by these experimental observations, we use a combination of atomistic and coarse-grained (CG) molecular dynamics simulations. Atomistic simulations of free and tethered ELPs show that WWWW are more prone to acquire β-turn structures than FFFF at lower temperatures. Also, the atomistically informed CG simulations show that the increased local stiffness in W than F due to the bulkier side chain in W compared to F, alone does not cause the shift in the transition of WWWW versus FFFF. The experimentally observed lower Tt of WWWW than FFFF is achieved in CG simulations only when the CG model incorporates both the atomistically informed local stiffness and stronger effective attractions localized at the W position versus the F position. The effective interactions localized at the guest residue in the CG model is guided by our atomistically observed increased propensity for β-turn structure in WWWW versus FFFF and by past experimental work of Urry et al. quantifying hydrophobic differences through enthalpy of association for W versus F.
Bacterial collagen-like proteins differ from vertebrate collagens in that they do not contain hydroxyproline, which is seen as a characteristic of the vertebrate collagens, and which provides a significant contribution to the stability of the collagen triple-helix at body temperature. Despite this difference, the bacterial collagens are stable at around body temperature through inclusion of other stabilising sequence elements. Another difference is the lack of aggregation, and certain vertebrate collagen binding domains that can be introduced into the bacterial sequence lack full function when hydroxyproline is absent. In the present study we have demonstrated that a simple method utilising co-translational incorporation during fermentation can be used to incorporate hydroxyproline into the recombinant bacterial collagen. The presence and amount of hydroxyproline incorporation was shown by amino acid analysis and by mass spectrometry. A small increase in thermal stability was observed using circular dichroism spectroscopy.
Statement of significance:
Recombinant bacterial collagens provide a new opportunity for biomedical materials as they are readily produced in large quantity in E. coli. Unlike animal collagens, they are stable without the need for inclusion of a secondary modification system for hydroxyproline incorporation. In animal collagens, however, introduction of hydroxyproline is essential for stability and is also important for functional molecular interactions within the mammalian extracellular matrix. The present study has shown that hydroxyproline can be readily introduced into recombinant S. pyogenes bacterial collagen through direct co-translational incorporation of this modified imino acid during expression using the codons for proline in the introduced gene construct. This hydroxylation further improves the stability of the collagen and is available to enhance any introduced molecular functions.
The origin of the triple‐helix structure and high stability of collagen has been debated for many years. As models of the triple helix and building blocks for new biomaterials, collagen mimetic peptide (CMP) assemblies have been deeply studied in the condensed phase. In particular, it was found that hydroxylation of proline, an abundant post‐translational modification in collagen, increases its stability. Two main hypotheses emerged to account for this behavior: 1) intra‐helix stereoelectronic effects, and 2) the role of water molecules H‐bound to hydroxyproline side‐chains. However, in condensed‐phase investigations, the influence of water cannot be fully removed. Therefore, we employed a combination of tandem ion mobility and mass spectrometries to assess the structure and stability of CMP assemblies in the gas phase. Our results show a conservation of the structure and stability properties of triple helix models in the absence of solvent, supporting an important role of stereoelectronic effects. Moreover, we give evidence that small triple helix assemblies with controlled stoichiometry can be studied in the gas phase, which opens new perspectives in the understanding of the first steps of collagen fiber growth.
In this paper, we present the development of a phenomenological coarse-grained model that represents single strands of collagen-like peptides (CLPs) as well as CLP triple helices. The goal of this model development is to enable coarse-grained molecular simulations of solutions of CLPs and conjugates of CLPs with other macromolecules and to predict trends in the CLP melting temperature with varying CLP design, namely CLP length and composition. Since the CLP triple helix is stabilized primarily by hydrogen bonds between amino acids in adjacent strands, for modeling CLP melting we get inspiration from a recent coarse-grained (CG) model that was used to capture specific and directional hydrogen-bonding interactions in base-pair hybridization within oligonucleotides and reproduced known DNA melting trends with DNA sequence and composition in implicit water. In this paper, we systematically describe the changes we make to this original CG model and then show that these improvements reproduce the known melting trends of CLPs seen in past experiments. Specifically, the CG simulations of CLP solutions at experimentally relevant concentrations show increasing melting temperature with increasing CLP length and decreasing melting temperature with incorporation of charged residues in place of uncharged residues in the CLP, in agreement with past experimental observations. Finally, results from simulations of CLP triple helices conjugated with elastin like peptides (ELPs), using this new CG model of CLP, reproduce the same trends in ELP aggregation as seen in past experiments.
Researchers have recently questioned the role hydroxylated prolines play in stabilizing the collagen triple helix. To address these issues, we have developed new molecular mechanics parameters for the simulation of peptides containing 4(R)-fluoroproline (Flp), 4(R)-hydroxyproline (Hyp), and 4(R)-aminoproline (Amp). Simulations of peptides based on these parameters can be used to determine the components that stabilize hydroxyproline over proline in the triple helix. The dihedrals F–C–C–N, O–C–C–N, and N–C–C–N were built using a N-β-ethyl amide model. One nanosecond simulations were performed on the trimers [(Pro–Pro–Gly)10]3, [(Pro–Hyp–Gly)10]3, [(Pro–Amp–Gly)10]3, [(Pro–Amp1+–Gly)10]3, and [(Pro–Flp–Gly)10]3 in explicit solvent. The results of our simulations suggest that pyrrolidine ring conformation is mediated by the strength of the gauche effect and classical electrostatic interactions. © 2002 Wiley Periodicals, Inc. Biopolymers 64: 63–71, 2002
We report on an experimental single-photon absorption study on gas-phase protonated collagen peptides employing a combination of mass spectrometry and synchrotron radiation. The partial ion yields for the main photoabsorption products vary steadily with photon energy over the range from 14 to 545 eV. At low energy, non-dissociative photoionisation competes with neutral molecule loss from the precursor ion, whereas fragmentation of the peptide backbone dominates at soft X-ray energies. Neutral molecule losses from the ionised peptide are found to have low energy barriers and most likely involve amino-acid residue side-chains with radical character, in particular aspartic acid. A particularly interesting finding is photoinduced loss of proline hydroxylation. The loss of this typical collagen post-translational modification might play a destabilizing role for the collagen structure.
Collagen is the most abundant protein in the human body. Cartilage and tendons owe their special mechanical properties to the fibrous collagen structure. These strong fibrils are aggregates of a sub-unit consisting of three collagen proteins wound around each other in a triple helix. The response of this protein complex to ionizing radiation has never been studied, despite its fundamental importance. In this work, we probe the direct effects of VUV and soft X-ray photons on isolated models of the collagen triple helix, by coupling a tandem mass spectrometer to a synchrotron beamline. Single-photon absorption is found to induce electronic excitation, ionization and conversion into internal energy leading to inter- and intra-molecular fragmentation, mainly due to Gly-Pro peptide bond cleavages. Our results indicate that a range of molecular orbitals is probed, and that a smooth transition from excitation to ionization occurs with increasing photon energy. Moreover, they support a stabilization of the triple helix models by proline hydroxylation via stereoelectronic effects.
A strategy inspired by tweezer receptors has been employed to develop a new host-guest system. The Hybridization into a collagen-like triple helix is the driving force for the recognition that occurs with high affinity and selectivity. Several systems have been screened to find the best host-guest pair and this strategy may be implemented for tag fused protein recongnition.
Proline is unique among proteinogenic amino acids because a pyrrolidine ring links its amino group to its side chain. This heterocycle constrains the conformations of the main chain and thus templates particular secondary structures. Proline residues undergo posttranslational modification at the 4-position to yield 4-hydroxyproline, which is especially prevalent in collagen. Interest in characterizing the effects of this modification led to the use of 4-fluoroprolines to enhance inductive properties relative to the hydroxyl group of 4-hydroxyproline and to eliminate contributions from hydrogen bonding. The strong inductive effect of the fluoro group has three main consequences: enforcing a particular pucker upon the pyrrolidine ring, biasing the conformation of the preceding peptide bond, and accelerating cis–trans prolyl peptide bond isomerization. These subtle yet reliable modulations make 4-fluoroproline incorporation a complement to traditional genetic approaches for exploring structure–function relationships in peptides and proteins, as well as for endowing peptides and proteins with conformational stability.
Connective tissues, the supporting structures of the body include cartilage, ligaments, tendons, fascia, joint capsules, the subepidermal portions (corium) of the skin, important elements of the heart valves, aorta and small blood vessels and, finally, bone. In general, these tissues comprise cellular and fibrous constituents embedded in the extracellular matrix of so-called ground substance. The major components of such tissues are the fibrous proteins, collagen and elastin, and the interfibrillar mucopolysaccharides or proteoglycans; and the relative proportions of these components govern the properties of the different connective tissues. For example, tendon, a tissue requiring high tensile strength and little elasticity, is comprised of over 80 per cent collagen (Table I), whereas cartilage, which is required to be more resilient to pressure than other tissues, has a high content of mucopolysaccharides and contains approximately 50 per cent collagen (Table I).
Collagen is the oldest extracellular matrix component in the animal kingdom. Currently, 29 homo- and hetero-trimmers have been identified, all of which have in common the amino acid repeat [Gly-X-Y]n that distinguishes their primary structure from other proteins and enables the signature quaternary structure, the triple helix. Depending on the aggregate structure, collagens have been classified as fibrous, non-fibrous, filamentous and fibril associated collagens with interrupted triple-helices. Among them, collagen type I is the most abundant collagen in mammalian tissues (70–90% of the collagen found in the body). Collagen type I fibrils are the primary structural elements of all connective tissues, providing a structural scaffold for other components primarily due to native cross-linking pathway of lysyl oxidase. Collagen type I is also associated with cell interaction, migration, attachment, differentiation and organisation. For these reasons, collagen type I is extensively used for tissue engineering applications. Mammalian extracted collagen (acid or pepsin derived) is almost exclusively used for industrial applications in leather, food, biomaterial, cosmetic and pharmaceutical industry. Fear, however, of interspecies transmission of disease has encouraged the development of synthetic and recombinant collagen technologies, which may hold the future in biomaterials applications. Numerous fabrication, stabilisation and functionalisation strategies have been developed over the years in order to produce tissue facsimiles that will promote functional regeneration. A number of collagen-specific assays have also been developed to ensure reproducibility and full characterisation of collagen preparations and collagen-based devices.
The proper handling of hydroxyamino acids is an important aspect of effective peptide synthesis. However, syntheses of peptides containing these amino acids often lead to problems. Some of these problems arise from uncertainty as to whether a protecting group is actually needed for the hydroxyl function. Many peptides have been synthesized successfully using amino acid derivatives containing free hydroxyl groups, whereas in other situations such attempts have led to serious side reactions. The chapter presents the survey of the field of synthesis of peptides containing serine, threonine, and tyrosine, as well as related hydroxyl-containing amino acids, and discusses suggestions as to the need for and use of protecting groups. It also discusses all the aspects of peptide synthesis, including protection of the hydroxyl group, and presents representative laboratory procedures for synthesis of derivatives and peptides, as well as tables of appropriate amino acid derivatives.
The integrin-mediated interaction of cells with laminins has recently been reviewed by Aumailley et al.1 In this chapter, the structural aspects of laminin integrin interaction will be emphasized. At least seven integrins have been identified which bind to laminin (Fig. 4.3.1). They were determined by using inhibiting antibodies, affinity chromatography and more recently, adherence of cells transfected with individual integrin α-subunits. The recognition sites within laminin-1 for the integrins were mapped to distinct regions and domains using defined proteolytic laminin fragments or more recently, using fragments expressed recombinantly. The most detailed investigations were carried out with laminin-1 from the murine EHS tumor.
(Pro-Pro-Gly)(10) [(PPG(10))], a collagen-like polypeptide, forms a triple-helical, polyproline-II structure in aqueous solution at temperatures somewhat lower than physiological, with a melting temperature of 24.5 degrees C. In this article, we present circular dichroism spectra that demonstrate an increase of the melting temperature with the addition of increasing amounts of D2O to an H2O solution of (PPG)(10), with the melting temperature reaching 40 degrees C in pure D2O. A thermodynamic analysis of the data demonstrates that this result is due to an increasing enthalphy of unfolding in D2O vs. H2O. To provide a theoretical explanation for this result, we have used a model for hydration of (PPG)(10) that we developed previously, in which inter-chain water bridges are formed between sterically crowded waters and peptide bond carbonyls. Energy minimizations were performed upon this model using hydrogen bond parameters for water, and altered hydrogen bond parameters that reproduced the differences in carbonyl oxygen-water oxygen distances found in small-molecule crystal structures containing oxygen-oxygen hydrogen bonds between organic molecules and H2O or D2O. It was found that using hydrogen bond parameters that reproduced the distance typical of hydrogen bonds to D2O resulted in a significant lowering of the potential energy of hydrated (PPG)(10). This lowering of the energy involved energetic terms that were only indirectly related to the altered hydrogen bond parameters, and were therefore not artifactual; the intra-(PPG(10)) energy, plus the water-(PPG(10)) van der Waals energy (not including hydrogen bond interactions), were lowered enough to qualitatively account for the lower enthalpy of the triple-helical conformation, relative to the unfolded state, in D2O vs. H2O. This result indicates that the geometry of the carbonyl-D2O hydrogen bonds allows formation of good hydrogen bonds without making as much of an energetic sacrifice from other factors as in the case of hydration by H2O.
Previous reports demonstrated that synthetic polytripeptides of the structure (Pro-Pro-Gly)10, prepared with a specific modification of the Merrifield technique, formed triple helical structures in aqueous solution which were similar to the triple helical structures of tropocollagen. The studies presented here were undertaken in order to answer the question of whether the individual polytripeptide chains within the triple helices were packed in a parallel fashion similar to vertebrate collagen or in an antiparallel fashion similar to the structure which has been suggested for collagen from the cuticle of the round worm, Ascaris lumbricoides. It was found that electrostatic interactions between the COOH-terminal and NH-terminal groups of (Pro-Pro-Gly)10 could be used to probe the detailed structure of triple helices formed by the polytripeptide.
Titration of (Pro-Pro-Gly)10 in triple helical conformation indicated that the pK of the NH-terminal group was 0.59 units lower than the pK for the same group in nonhelical (Pro-Pro-Gly)5. The titration of the NH-terminal group of helical (Pro-Pro-Gly)10 was significantly broader than the theoretically predicted curve, and the pK increased after the polytripeptide was heated and quenched.
Furthermore, the melting curve for the helix coil transition of (Pro-Pro-Gly)10 was changed by protonation of the NH-terminal or COOH-terminal groups. The Tm for the dipolar ion form was about 10° lower than for either the fully protonated or unprotonated forms of the helical polytripeptide.
The results indicate that in the triple helical structure formed by (Pro-Pro-Gly)10 the three chains are in register and parallel.
Sequenced polytripeptides, (Pro-Pro-Gly)n, (n = 10, 15, 20), with defined molecular weights were synthesized by the solid-phase method. Conformational changes of these sample as a function of temperature were studied by measurements of optical rotation and sedimentation equilibrium. The temperature dependence of optical rotation was shown similar to thermal transition of collagen molecule. Each of these polymers existed as a timer at lower temperature. (Pro-Pro-Gly)10 existed as a monomer at higher temperature, and the others were expected to behave analogously.
Previous studies demonstrated that (Pro-Pro-Gly)10 and (Pro-Pro-Gly)20 prepared by a specific modification of the Merrifield technique form triple-helical structures in aqueous solution similar to tropocollagen. In the present studies we used electron microscopy to demonstrate that at about 4 °C the same polytripeptides also form larger structures which we consider to be micro-crystalline segments.Examination of the micro-crystalline segments by negative staining with sodium silicotungstate demonstrated that one lateral dimension was relatively constant and was approximately equal to the theoretical length of the polytripeptides in a collagen-like triple helix. Also, when (Pro-Pro-Gly)20 was negatively stained with uranyl acetate, smaller structures were seen which were of the same length as the “regular dimension” of the micro-crystalline segments and which probably were individual triple helices. Selective positive staining of either the amino-terminal or the carboxy-terminal groups indicated that the ends of the polytripeptide chains were packed in an anti-parallel fashion within the segments.The results obtained by both negative and positive staining are best explained by a scheme in which (a) the polytripeptide chains within triple helices are in register and parallel, and (b) the micro-crystalline segments are formed by the lateral aggregation of triple helices in an anti-parallel fashion.
The properties of anhydrous hydrogen fluoride (HF) as a reagent for the acidolysis of various protective groups have been studied. Amino acids with various protective groups were each treated with HF at 0 or 20°C in the presence of anisole, and the reaction products were tested. Thus, HF was found to be much better than the other known reagents, such as hydrogen bromide and trifluoroacetic acid. In the present study, the following groups were shown, for the first time, to be removed safely: the nitro group in nitroarginine, the diphenylmethyl group in diphenylmethylamide, the t-butyl group in S-t-butylcysteine, the isopropyloxycarbonyl group, and the isopropyl ester group. A new and convenient apparatus was designed for the safe handling of HF for peptide synthesis.