ABSTRACT Protein engineering often combines multiple mutations, yet predicting how these combinations affect stability remains a fundamental challenge. The double‐mutant effect often departs from the sum of its constituent single‐mutant contributions, a phenomenon known as mutation epistasis or non‐additivity. Anticipating this is critical, as the number of candidate pairs grows combinatorially and each non‐additive pair can either strengthen or cancel an intended design. Despite growing interest, current approaches to predicting epistasis remain limited, and systematically identifying which mutation pairs will behave non‐additively remains an open problem. Here, we present a Dynamic Residue Contact Network Clique (DynRCNC) model to capture the non‐additive mutation pairs. DynRCNC provides a protein native network with residue‐residue dynamic coupling from molecular dynamics (MD) simulations and forms a three‐tier prediction hierarchy with six criteria. The model was applied to 265 double‐mutation pairs from seven proteins and achieved a weighted Matthews correlation coefficient of 0.422 under leave‐one‐protein‐out cross‐validation (weighted LOPOCV MCC) on the full benchmark. It transfers across proteins better than random‐forest, gradient‐boosting, and logistic‐regression models trained on the same structural and dynamic features. Needing no training data from the target protein and reporting the criterion behind each prediction, DynRCNC gives an interpretable, mechanistic route to prioritizing candidate mutation pairs.
ABSTRACT Collagen plays a pivotal role in animal body structure, but hydrogels of reconstituted collagens often have poor mechanical performance. Chemical crosslinking or the addition of other biopolymers are often used as a remedy. Inspired by the many natural collagen‐interacting proteins that modify the mechanics of collagen hydrogels, we explore protein‐based physical crosslinking of collagen as a route to modulate the mechanics of collagen hydrogels. A protein‐based crosslinker for collagen was designed based on an adhesion domain from Staphylococcus aureus . A tetramerization domain was coupled to an elastin‐like polypeptide as a spacer, followed by the collagen adhesion domain, yielding a tetrafunctional crosslinking. Circular Dichroism spectroscopy shows that the crosslinker has the expected secondary structure. Using quartz crystal microbalance, we demonstrate the tetrameric crosslinker binds rat tail Collagen I and fish collagen, even though the latter is largely denatured as evidenced by circular dichroism spectroscopy. Rheometry shows strong enhancement of elastic moduli and strain‐stiffening for rat tail collagen hydrogels, but smaller effects on the reduced fish collagen. Because CNA35 recognizes the collagen triple helix, this contrast indicates that the crosslinker acts specifically on triple‐helical collagen, and points to the importance of improved extraction protocols for alternative collagens that preserve native triple‐helical conformations.
ABSTRACT The collagen triple helix is the most abundant protein structural motif in animals. It tolerates any amino acid in its Xaa and Yaa positions but demands glycine (Gly) at every third position, where the residue packs into the sterically forbidding core of the triple helix and donates the recurring interstrand N–H⋯OC hydrogen bond that stitches the three strands together. (2 S )‐Proline (Pro) and (2 S ,4 R )‐4‐hydroxyproline (Hyp) are enriched far above their proteome‐wide abundance because they preorganize individual strands into the polyproline II conformation, lowering the entropic cost of folding. We asked whether the triple helix is intrinsically stable enough to form from strands that carry no such preorganization, that is, from polyglycine. To offset the conformational entropy of a Gly‐rich tract, we embedded Gly 6 within flanking triple‐helical (Pro‐Hyp‐Gly) n clamps in Ac‐(Pro‐Hyp‐Gly) 6 ‐Gly 6 ‐(Pro‐Hyp‐Gly) 5 ‐Pro‐Hyp‐NH 2 and compared it with an otherwise identical peptide in which Gly 6 was replaced by two [2‐(2‐aminoethoxy)ethoxy]acetic acid units, a flexible, length‐matched spacer that lacks most of the regularly spaced backbone amides of Gly 6 . Circular dichroism spectroscopy revealed the diagnostic positive band at 225 nm. The molar ellipticity of the spacer‐containing control was 86% of that of the Gly 6 ‐containing peptide, matching the 35/41 = 85% ratio of residues capable of triple‐helix formation and consistent with the Gly 6 segment contributing to the triple‐helical signal. Thermal denaturation yielded a T m of 46.8°C for the Gly 6 ‐containing peptide, 6.3°C higher than that of the matched spacer control. This added stability indicates that the polyglycine tract is not merely accommodated within the helix but contributes stabilizing interstrand hydrogen bonds. Together, these data indicate that three polyglycine strands can form a collagen triple helix when flanked by triple‐helical clamps. This result is consistent with the idea that the triple‐helical architecture predates the recruitment of Pro and Hyp, informs the evolutionary origin of collagen, and offers a blunt‐ended, symmetric scaffold for designing peptidic triple helices.
This study investigates how pre-esterification dry heat treatment (DHT) of corn starch at 170°C, 180°C, and 200°C influences the functional properties of subsequently synthesized starch citrates. Structural and morphological changes induced by DHT and esterification were analyzed using scanning electron microscopy, X-ray diffractometry, and Fourier-Transform Infrared spectroscopy. Starch citrates were characterized for their resistance to pancreatic α-amylase and amyloglucosidase, as well as their water absorption capacity (WAC) at 37°C. Results demonstrated a strong correlation between DHT temperature and the functional properties of the final citrate. Increasing the DHT temperature significantly increased the resistant starch fraction and WAC. Specifically, citrates from native starch had a resistant starch (RS) fraction of 91%, while citrates from starch pre-treated at 200°C exhibited a markedly increased RS fraction of 98% and a 64% increase in WAC. These findings indicate that DHT is an effective pre-treatment for enhancing the dietary fiber potential and hydration properties of starch citrates.
Biogenic silver nanoparticles (AgNPs) are emerging as valuable candidates in biomedical research, offering advantages such as cost-effectiveness, eco-friendly preparation methods, and superior biocompatibility. Owing to the challenges in the treatment of chronic skin injury, this study utilizes biogenic AgNPs (synthesized using Shilajit as a reducing and stabilizing agent) to develop gel and spray formulations (constituting fish collagen and l-lysine) for wound-healing applications. FTIR analysis indicated the involvement of functional groups from the Shilajit extract in synthesizing crystalline spherical AgNPs of 140-160 nm diameter. EDAX analysis reported 0.6% (by weight) silver in both formulations, with major elemental peaks for nitrogen, carbon, and oxygen, indicating the presence of polysaccharides. The MTT assay on HDFa cells confirmed that both preparations were nontoxic, as validated by FDA staining, which signified intact cell morphology. In vivo findings demonstrated that ShilAgNPs-Fcol-Lys formulations promote rapid wound closure, effective extracellular matrix (ECM) deposition, advanced neovascularization, and improved tissue remodeling compared with controls and standard treatments. Well-organized collagen deposition and efficient remodeling were confirmed by histology and immunohistochemistry, with the spray formulation performing better than the gel. Thus, ShilAgNPs-Fcol-Lys formulations offer a biocompatible, safe, and cost-effective alternative for treating deep excisional wounds.
