Stability Of Collagen Triple Helix Research Articles

Investigating the effect of water on collagen triple helix stability

Collagen provides essential structural support to diverse body tissues through its unique triple helix structure. Collagen stability is crucial for proper tissue function and any disruptions in its stability can lead to various health issues. Prior research has identified multiple factors affecting collagen stability. However, despite its fundamental presence in biological systems, the influence of water remains inadequately explored. Here, we performed molecular dynamics simulations on collagen model peptides (CMPs) with varying experimental stability profiles to investigate the influence of water on collagen stability. Our simulations revealed variations in both the hydrogen bonding (H-bonding) patterns between the CMPs and surrounding water molecules, as well as the water networks formed around CMPs. We noted that water molecules form H-bonds with collagen residues which enhance its structural stability. Additionally, water molecules assemble around the collagen and form topological water networks (TWNs) through intermolecular H-bonding. These TWNs further stabilizes the collagen structure. Overall, this research provides insight into the role of water molecules in preserving the stability of the collagen triple helix.

Carbohydrate Co-Solutes Stabilize Collagen Triple Helices.

Carbohydrates are common co-solutes for the stabilization of proteins. The effect of carbohydrate solutions on the stability of collagen, the most abundant protein in mammals, is, however, underexplored. In this work, we studied the thermal stability of collagen triple helices derived from a molecularly defined collagen model peptide (CMP), Ac-(Pro-Hyp-Gly)7 -NH2 , in solutions of six common mono- and disaccharides. We show that the carbohydrates stabilize the collagen triple helix in a concentration-dependent manner, with an increase of the melting temperature of up to 17 °C. In addition, we show that the stabilizing effect is similar for all studied sugars, including trehalose, which is otherwise considered a privileged bioprotectant. The results provided insight into the effects of sugar co-solutes on collagen triple helices and can aid the selection of storage environments for collagen-based materials and probes.

Transcriptome analysis reveals the molecular basis of anti‐predation defence in Daphnia pulex simultaneously responding to Microcystis aeruginosa

Abstract1. The influence of cyanobacteria on interspecies interactions has received growing attention as a predictor of aquatic ecosystem function. It is well‐known that cyanobacteria greatly affect predator‐induced phenotypic plasticity in planktonic animals. However, the underlying molecular mechanism remains unclear.2. Here, we used Daphnia pulex as a model organism for inducible defence, examining its morphological and life‐history responses to fish kairomone while feeding on 100% Chlorella pyrenoides, or while feeding on 70% Chlorella and either microcystin‐producing or ‐free Microcystis aeruginosa, separately. Transcriptome profiles of kairomone‐exposed D. pulex fed different types of food were detected using RNA‐seq. How the differentially expressed genes and pathways identified are responsible for the altered adaptive traits under Microcystis stress was discussed.3. Daphnia pulex fed 100% Chlorella matured at a smaller body size, elongated tail spine length, and produced smaller offspring as adaptive morphological and reproductive responses to fish kairomone. These anti‐predation defences entailed a cost in decreased somatic growth rate. Microcystis stress caused defence trade‐offs in both morphology and reproduction: reduction of body size at maturity was stronger whereas tail spine elongation was unchanged or inhibited. Given that reproductive investment was reduced overall in Daphnia exposed to fish kairomones, there was a trade‐off for unchanged offspring size by reducing offspring number in Daphnia fed Microcystis. Defence‐induced costs to growth were increased by Microcystis exposure.4. Transcriptome analysis revealed that neuronal signals including acetylcholine and glutamate signalling implicated in kairomone reception and/or transmission exhibited stronger responses under Microcystis stress. Cuticle development associated with the biosynthesis of wax esters and chitin, modification of lipo‐chitin saccharides, and the stability of collagen triple helix apparently participated in the morphological changes. The arachidonic acid pathway and biosynthesis of cholesterol and steroid hormones mediated altered reproductive performance. In addition, metabolic processes such as detoxification, food assimilation, lysosome dysfunction and apoptosis activation were involved in the survival and growth of Daphnia, which may contribute to the increased defence‐induced cost in growth when fed Microcystis.5. Taken together, these findings advance our understanding of the molecular mechanisms underlying anti‐predation defensive responses by Daphnia to cyanobacterial stress. This work also provides a reference for further exploring the functional genes that mediate zooplankton–fish coevolution dynamics under eutrophic conditions.

The Yin and Yang of How N-Terminal Acyl Caps Affect Collagen Triple Helices.

N-terminal acylation is a common tool for the installation of functional moieties (e.g., sensors or bioactive molecules) on collagen model peptides (CMPs). The N-acyl group and its length are generally assumed to have little or no influence on the properties of the collagen triple helix formed by the CMP. Here, we show that the length of short (C1-C4) acyl capping groups has different effects on the thermal stability of collagen triple helices in POG, OGP, and GPO frames. While the effect of different capping groups on the stability of triple helices in the GPO frame is negligible, longer acyl chains stabilize OGP triple helices but destabilize POG analogues. The observed trends arise from a combination of steric repulsion, the hydrophobic effect, and n → π* interactions. Our study provides a basis for the design of N-terminally functionalized CMPs with predictable effects on triple helix stability.

Concentration-mediated Folding and Unfolding of Collagen Triple Helix.

Collagen has been widely utilized in tissue engineering, regenerative medicine and cosmetics. Collagen of low concentrations is frequently applied to reduce the production cost, while it may result in the loss of triple helical structure and bioactivity. CD and NMR techniques have enhanced our understanding of collagen triple helix, while they require high concentrations of collagen samples. We have systematically investigated the folding and unfolding features of collagen mimetic peptides at a broad variety of concentrations in order to decipher the role of the concentration in the triple helical stability. Peptide FAM-G(POG)10 was synthesized by the solid phase synthesis method. Fluorescence spectra of peptide FAM-G(POG)10 at different concentrations were recorded. The unfolding and folding profiles of peptide FAM-G(POG)10 with concentrations varying from 1 nM to 100 μM were examined. The effect of concentration on the folding and unfolding capability of peptide FAMG( POG)10 was investigated. Fluorescence characterization of peptide FAM-G(POG)10 under widely varying concentrations from 1 nM to 100 μM has revealed that concentration played a critical role in the stability of collagen peptides. The two-phase pattern of the concentration-dependent folding and unfolding curves has for the first time demonstrated the presence of a critical concentration for the collagen peptide to trigger the complete folding of the triple helix and to maintain the triple helix structure. It is noteworthy that the triple helix structure of collagen peptides was very stable at μM-level concentrations from both the folding and unfolding perspectives. It has significantly contributed to our understanding of collagen triple helix stability at low and ultra-low concentrations, and provided valuable and practical guidelines for the preparation of collagen-based products.

Predicting Collagen Triple Helix Stability through Additive Effects of Terminal Residues and Caps.

Collagen model peptides (CMPs) consisting of proline-(2S,4R)-hydroxyproline-glycine (POG) repeats have provided a breadth of knowledge of the triple helical structure of collagen, the most abundant protein in mammals. Predictive tools for triple helix stability have, however, lagged behind since the effect of CMPs with different frames ([POG]n , [OGP]n , or [GPO]n ) and capped or uncapped termini have so far been underestimated. Here, we elucidated the impact of the frame, terminal functional group and its charge on the stability of collagen triple helices. Combined experimental and theoretical studies with frame-shifted, capped and uncapped CMPs revealed that electrostatic interactions, strand preorganization, interstrand H-bonding, and steric repulsion at the termini contribute to triple helix stability. We show that these individual contributions are additive and allow for the prediction of the melting temperatures of CMP trimers.

Predicting Collagen Triple Helix Stability through Additive Effects of Terminal Residues and Caps

AbstractCollagen model peptides (CMPs) consisting of proline‐(2S,4R)‐hydroxyproline‐glycine (POG) repeats have provided a breadth of knowledge of the triple helical structure of collagen, the most abundant protein in mammals. Predictive tools for triple helix stability have, however, lagged behind since the effect of CMPs with different frames ([POG]n, [OGP]n, or [GPO]n) and capped or uncapped termini have so far been underestimated. Here, we elucidated the impact of the frame, terminal functional group and its charge on the stability of collagen triple helices. Combined experimental and theoretical studies with frame‐shifted, capped and uncapped CMPs revealed that electrostatic interactions, strand preorganization, interstrand H‐bonding, and steric repulsion at the termini contribute to triple helix stability. We show that these individual contributions are additive and allow for the prediction of the melting temperatures of CMP trimers.

Frame Shifts Affect the Stability of Collagen Triple Helices.

Collagen model peptides (CMPs), composed of proline-(2S,4R)-hydroxyproline-glycine (POG) repeat units, have been extensively used to study the structure and stability of triple-helical collagen─the dominant structural protein in mammals─at the molecular level. Despite the more than 50-year history of CMPs and numerous studies on the relationship between the composition of single-stranded CMPs and the thermal stability of the assembled triple helices, little attention has been paid to the effects arising from their terminal residues. Here, we show that frame-shifted CMPs, which share POG repeat units but terminate with P, O, or G, form triple helices with vastly different thermal stabilities. A melting temperature difference as high as 16°C was found for triple helices from 20-mers Ac-OG[POG]6-NH2 and Ac-[POG]6PO-NH2, and triple helices of the constitutional isomers Ac-[POG]7-NH2 and Ac-[GPO]7-NH2 melt 10 °C apart. A combination of thermal denaturation, circular dichroism and NMR spectroscopic studies, and molecular dynamics simulations revealed that the stability differences originate from the propensity of the peptide termini to preorganize into a polyproline-II helical structure. Our results advise that care must be taken when designing peptide mimics of structural proteins, as subtle changes in the terminal residues can significantly affect their properties. Our findings also provide a general and straightforward tool for tuning the stability of CMPs for applications as synthetic materials and biological probes.

CollagenTransformer: End-to-End Transformer Model to Predict Thermal Stability of Collagen Triple Helices Using an NLP Approach.

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.

Terminal repeats impact collagen triple-helix stability through hydrogen bonding.

Nearly 30% of human proteins have tandem repeating sequences. Structural understanding of the terminal repeats is well-established for many repeat proteins with the common α-helix and β-sheet foldings. By contrast, the sequence-structure interplay of the terminal repeats of the collagen triple-helix remains to be fully explored. As the most abundant human repeat protein and the most prevalent structural component of the extracellular matrix, collagen features a hallmark triple-helix formed by three supercoiled polypeptide chains of long repeating sequences of the Gly-X-Y triplets. Here, with CD characterization of 28 collagen-mimetic peptides (CMPs) featuring various terminal motifs, as well as DSC measurements, crystal structure analysis, and computational simulations, we show that CMPs only differing in terminal repeat may have distinct end structures and stabilities. We reveal that the cross-chain hydrogen bonding mediated by the terminal repeat is key to maintaining the triple-helix's end structure, and that disruption of it with a single amide to carboxylate substitution can lead to destabilization as drastic as 19 °C. We further demonstrate that the terminal repeat also impacts how strong the CMP strands form hybrid triple-helices with unfolded natural collagen chains in tissue. Our findings provide a spatial profile of hydrogen bonding within the CMP triple-helix, marking a critical guideline for future crystallographic or NMR studies of collagen, and algorithms for predicting triple-helix stability, as well as peptide-based collagen assemblies and materials. This study will also inspire new understanding of the sequence-structure relationship of many other complex structural proteins with repeating sequences.

ColGen: An end-to-end deep learning model to predict thermal stability of de novo collagen sequences

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.

Modulating the Stability of Collagen Triple Helices by Terminal Charged Residues.

Cationic or anionic residues are frequently located at the termini of proteins because their charged side chain can form electrostatic interactions with a terminal carboxylate or ammonium group to stabilize the structure under physiological conditions. Here, we used collagen-mimetic peptides (CMPs) to examine how the terminal charge-charge interactions affect the collagen triple helix stability. We designed a series of CMPs with either a Lys or Glu incorporated into the terminus and measured their pH-dependent stability. The results showed that the terminal electrostatic attractions stabilized the triple helix, while the terminal electrostatic repulsions destabilized the trimer. The data also revealed that the repulsions imposed a greater effect than did the attractions on the triple helix. An amino acid with a shorter side chain, such as aspartate and ornithine, was also installed to investigate the length effect on electrostatic interactions, which was found to be insignificant. Meanwhile, simultaneously incorporating cationic and anionic residues into the termini showed slight additive stabilization effects but pronounced additive destabilization consequences. We have demonstrated that the collagen triple helix stability can be modulated by introducing a cationic or anionic residue into the terminus of a peptide, giving useful information for the design of collagen-associated materials.

Influence of Lipidation on the Folding and Stability of Collagen Triple Helices-An Experimental and Theoretical Study.

The folding of triple-helical collagen, the most abundant protein in nature, relies on the nucleation and propagation along the strands. Hydrophobic moieties are crucial for the folding and stability of numerous proteins. Instead, nature uses for collagen a trimerization domain and cis-trans prolyl isomerases to facilitate and accelerate triple helix formation. Yet, pendant hydrophobic moieties endow triple-helical collagen with hyperstability and accelerate the cis-trans isomerization to an extent that thermally induced unfolding and folding of collagen triple helices take place at the same speed. Here, we systematically explored the effect of pendant fatty acids on the folding and stability of collagen triple helices. Thermal denaturation and kinetic studies with a series of collagen mimetic peptides (CMPs) bearing saturated and unsaturated fatty acids with different lengths revealed that longer and more flexible fatty acid appendages increase the stability and the folding rate of collagen triple helices. Molecular dynamics simulations combined with experimental data indicate that the hydrophobic appendages stabilize the triple helix by interaction with the grooves of the collagen triple helix and accelerate the folding and unfolding process by creating a molten globule-like intermediate.

Quantitative Analysis of the Positional Distribution of Hydroxyproline in Collagenous Gly-Xaa-Yaa Sequences by LC-MS with Partial Acid Hydrolysis and Precolumn Derivatization.

Collagen is extensively modified by various enzymes, including prolyl hydroxylases. Pro residues at the Yaa position of repeating Gly-Xaa-Yaa amino acid sequences are mostly hydroxylated to 4-hydroxyproline (4Hyp), which is essential for the thermal stability of collagen triple helix. In contrast, Pro residues at the Xaa position are rarely modified to 3Hyp and 4Hyp, the biological function of which is poorly understood. Overall estimation of prolyl hydroxylation with discrimination of the position (Xaa or Yaa) and hydroxylation type (4Hyp or 3Hyp) has been difficult to perform using traditional methods. In the present study, we developed a novel position-specific analytical method featuring LC-MS detection of collagenous Gly-containing dipeptides, including Gly-Pro, Pro-Gly, Gly-4Hyp, Gly-3Hyp, and 4Hyp-Gly, after partial acid hydrolysis and precolumn derivatization using 3-aminopyridyl-N-hydroxysuccinimidyl carbamate (APDS). We performed acid hydrolysis at 55 °C with HCl/trifluoroacetic acid/water (2:1:1, v/v) to avoid peptide inversion and imbalanced peptide generation observed for collagenous model peptides. The positional distribution of Pro, 4Hyp, and 3Hyp can be calculated from the relative concentrations of the APDS-derivatized dipeptides, and in combination with amino acid analysis, we can determine their absolute contents at the Xaa and Yaa positions. Bovine type I, III, and V collagens were analyzed by the established method, and the amount of 4Hyp was higher than that of 3Hyp at the Xaa position in type I and III collagens. In addition, we clearly showed that collagen extracted from earthworm cuticles has an extremely high content of Xaa position 4Hyp, reaching over 10% of the total amino acids.

Triple-Helix-Stabilizing Effects in Collagen Model Peptides Containing PPII-Helix-Preorganized Diproline Modules.

Collagen model peptides (CMPs) serve as tools for understanding stability and function of the collagen triple helix and have a potential for biomedical applications. In the past, interstrand cross‐linking or conformational preconditioning of proline units through stereoelectronic effects have been utilized in the design of stabilized CMPs. To further study the effects determining collagen triple helix stability we investigated a series of CMPs containing synthetic diproline‐mimicking modules (ProMs), which were preorganized in a PPII‐helix‐type conformation by a functionalizable intrastrand C2 bridge. Results of CD‐based denaturation studies were correlated with calculated (DFT) conformational preferences of the ProM units, revealing that the relative helix stability is mainly governed by an interplay of main‐chain preorganization, ring‐flip preference, adaptability, and steric effects. Triple helix integrity was proven by crystal structure analysis and binding to HSP47.

Triple‐Helix‐Stabilizing Effects in Collagen Model Peptides Containing PPII‐Helix‐Preorganized Diproline Modules

AbstractCollagen model peptides (CMPs) serve as tools for understanding stability and function of the collagen triple helix and have a potential for biomedical applications. In the past, interstrand cross‐linking or conformational preconditioning of proline units through stereoelectronic effects have been utilized in the design of stabilized CMPs. To further study the effects determining collagen triple helix stability we investigated a series of CMPs containing synthetic diproline‐mimicking modules (ProMs), which were preorganized in a PPII‐helix‐type conformation by a functionalizable intrastrand C2 bridge. Results of CD‐based denaturation studies were correlated with calculated (DFT) conformational preferences of the ProM units, revealing that the relative helix stability is mainly governed by an interplay of main‐chain preorganization, ring‐flip preference, adaptability, and steric effects. Triple helix integrity was proven by crystal structure analysis and binding to HSP47.

Decoding Collagen Triple Helix Stability by Means of Hybrid DFT Simulations.

Collagen is a protein family defined by a triple helix motif, which comprises roughly one-third of the total human protein content. Decoding the reasons underlying the stability of the collagen triple helix is of both fundamental and applicative relevance, for instance, to guide collagen protein engineering. In principle, full quantum mechanical approaches based on density functional theory (DFT) are ideal to study the subtle physicochemical features of collagen. Unfortunately, the huge size of the protein prevents the straightforward application of DFT to realistic collagen protein models. In this paper, we propose a new realistic model of the collagen protein based on a periodic approach. The protein model exploits the intrinsic symmetry of the collagen triple helix, dramatically lowering the cost of the simulations. This allows using accurate hybrid DFT simulations (B3LYP-D/TZP) for systematic studies of the collagen protein features. We have tested the proposed model/level-of-theory combination to analyze the well-known proline-conformation/collagen-stability relationship. For this purpose, we have performed an extensive conformational analysis of the proline ring within the protein, clarifying some of the reasons linking specific ring conformations to helix positions. Throughout our data analysis, we have also obtained "for free" the collagen interstrand binding energy. Simulation results demonstrate that London dispersion interactions play a dominant role in the whole helix stability. The good agreement with the experimental data validates the use of the proposed model/level of theory to assist the active field of collagen-like peptide synthesis.

Hydrophobic Moieties Bestow Fast-Folding and Hyperstability on Collagen Triple Helices.

Trans amide bonds and fast cis- trans isomerization of Xaa-Pro bonds are crucial for the stability and folding rate of collagen, the most abundant protein in mammals. Here, we explored the effect of pendant hydrophobic moieties on the folding and stability of collagen triple helices. Kinetic studies with a series of collagen model peptides showed that a local hydrophobic environment accelerates cis- trans isomerization to an extent that thermally induced unfolding and folding of the collagen triple helix take place at the same speed. Thermal denaturation studies revealed that the hydrophobic appendages provide hyperstable collagen triple helices ( Tm = 70 °C).

Cross-Linked Collagen Triple Helices by Oxime Ligation.

Covalent cross-links are crucial for the folding and stability of triple-helical collagen, the most abundant protein in nature. Cross-linking is also an attractive strategy for the development of synthetic collagen-based biocompatible materials. Nature uses interchain disulfide bridges to stabilize collagen trimers. However, their implementation into synthetic collagen is difficult and requires the replacement of the canonical amino acids (4R)-hydroxyproline and proline by cysteine or homocysteine, which reduces the preorganization and thereby stability of collagen triple helices. We therefore explored alternative covalent cross-links that allow for connecting triple-helical collagen via proline residues. Here, we present collagen model peptides that are cross-linked by oxime bonds between 4-aminooxyproline (Aop) and 4-oxoacetamidoproline placed in coplanar Xaa and Yaa positions of neighboring strands. The covalently connected strands folded into hyperstable collagen triple helices (Tm ≈ 80 °C). The design of the cross-links was guided by an analysis of the conformational properties of Aop, studies on the stability and functionalization of Aop-containing collagen triple helices, and molecular dynamics simulations. The studies also show that the aminooxy group exerts a stereoelectronic effect comparable to fluorine and introduce oxime ligation as a tool for the functionalization of synthetic collagen.

PH‐Responsive Aminoproline‐Containing Collagen Triple Helices

(4S)- and (4R)-configured aminoproline (Amp) residues were used as pH-responsive probes to tune the thermal stability of collagen triple helices in acidic and basic environments. The different steric and stereoelectronic properties of amino versus ammonium groups lead to a switch of the ring pucker of Amp upon changing the pH. The choice of the position of Amp within collagen model peptides (CMPs) as well as the absolute configuration at C(4) of the pH-responsive probe allows for tuning of the stability of Amp-containing collagen triple helices over a broad range. Comparative quantum chemical calculations on the steric and stereoelectronic effects of amino and ammonium groups versus fluorine, hydroxy, chlorine, and methyl substituents support the experimental findings. The research also shows that substitution of the naturally occurring hydroxy group in collagen by electron-withdrawing groups with a larger hydration shell than that of the hydroxy group is not tolerated.