Designing collagens to shed light on the multi-scale structure-function mapping of matrix disorders.

Collagens are the most abundant structural proteins in the extracellular matrix of animals and play crucial roles in maintaining the structural integrity and mechanical properties of tissues and organs while mediating important biological processes. Fibrillar collagens have a unique triple helix structure with a characteristic repeating sequence of (Gly-X-Y)n. Variations within the repetitive sequence can cause misfolding of the triple helix, resulting in heritable connective tissue disorders. The most common variations are single-point missense mutations that lead to the substitution of a glycine residue with a bulkier amino acid (Gly â†’ X). In this review, we will first discuss the importance of collagen's triple helix structure and how single Gly substitutions can impact its folding, structure, secretion, assembly into higher-order structures, and biological functions. We will review the role of "designer collagens," i.e., synthetic collagen-mimetic peptides and recombinant bacterial collagen as model systems to include Gly â†’ X substitutions observed in collagen disorders and investigate their impact on structure and function utilizing in vitro studies. Lastly, we will explore how computational modeling of collagen peptides, especially molecular and steered molecular dynamics, has been instrumental in probing the effects of Gly substitutions on structure, receptor binding, and mechanical stability across multiple length scales.

Purification of recombinant bacterial collagens containing structural perturbations.

Streptococcus pyogenes-derived recombinant bacterial collagen-like proteins (CLPs) are emerging as a potential biomaterial for biomedical research and applications. Bacterial CLPs form stable triple helices and lack specific interactions with human cell surface receptors, thus enabling the design of novel biomaterials with specific functional attributes. Bacterial collagens have been instrumental in understanding collagen structure and function in normal and pathological conditions. These proteins can be readily produced in E. coli, purified using affinity chromatography, and subsequently isolated after cleavage of the affinity tag. Trypsin is a widely used protease during this purification step since the triple helix structure is resistant to trypsin digestion. However, the introduction of Gly→X mutations or natural interruptions within CLPs can perturb the triple helix structure, making them susceptible to trypsin digestion. Consequently, removing the affinity tag and isolating collagen-like (CL) domains containing mutations is impossible without degradation of the product. We present an alternative method to isolate CL domains containing Gly→X mutations utilizing a TEV protease cleavage site. Protein expression and purification conditions were optimized for designed protein constructs to achieve high yield and purity. Enzymatic digestion assays demonstrated that CL domains from wild-type CLPs could be isolated by digestion with either trypsin or TEV protease. In contrast, CLPs containing Gly→Arg mutations are readily digested by trypsin while digestion with TEV protease cleaved the His6-tag, enabling the isolation of mutant CL domains. The developed method can be adapted to CLPs containing various new biological sequences to develop multifunctional biomaterials for tissue engineering applications.

Molecular underpinnings of integrin binding to collagen-mimetic peptides containing vascular Ehlers-Danlos syndrome-associated substitutions.

Collagens carry out critical extracellular matrix (ECM) functions by interacting with numerous cell receptors and ECM components. Single glycine substitutions in collagen III, which predominates in vascular walls, result in vascular Ehlers-Danlos syndrome (vEDS), leading to arterial, uterine, and intestinal rupture and an average life expectancy of <50 years. Collagen interactions with integrin α2ß1 are vital for platelet adhesion and activation; however, how these interactions are impacted by vEDS-associated mutations and by specific amino acid substitutions is unclear. Here, we designed collagen-mimetic peptides (CMPs) with previously reported Gly → Xaa (Xaa = Ala, Arg, or Val) vEDS substitutions within a high-affinity integrin α2ß1-binding motif, GROGER. We used these peptides to investigate, at atomic-level resolution, how these amino acid substitutions affect the collagen III-integrin α2ß1 interaction. Using a multitiered approach combining biological adhesion assays, CD, NMR, and molecular dynamics (MD) simulations, we found that these substitutions differentially impede human mesenchymal stem cell spreading and integrin α2-inserted (α2I) domain binding to the CMPs and were associated with triple-helix destabilization. Although an Ala substitution locally destabilized hydrogen bonding and enhanced mobility, it did not significantly reduce the CMP-integrin interactions. MD simulations suggested that bulkier Gly → Xaa substitutions differentially disrupt the CMP-α2I interaction. The Gly → Arg substitution destabilized CMP-α2I side-chain interactions, and the Gly → Val change broke the essential Mg2+ coordination. The relationship between the loss of functional binding and the type of vEDS substitution provides a foundation for developing potential therapies for managing collagen disorders.

Phytoremediation of aspirin and tetracycline by Brassica juncea.

With the increasing release of pharmaceutical drugs in the environment, research is in progress for investigating alternative methods for their remediation. Various studies have shown the phytoremediation potential of Brassica juncea for metals. The current study was aimed at evaluating the phytoremediation potential of B. juncea for two different pharmaceutical drugs i.e. aspirin and tetracycline in in-vitro conditions. The seeds of B. juncea were germinated and grown for a period of 28 and 24 days for aspirin and tetracycline, respectively. The study analyzed the remediation rate of B. juncea for the selected drugs in three different sets of varying concentration along with any phytotoxic effects exerted by the drugs on the seeds. Preliminary results showed that the average remediation rate of aspirin and tetracycline at the end of experiment was approximately 90% and 71%, respectively. As initial drug concentrations were increased in the media, the remediation rate also improved. However, at higher concentrations, the plants showed phytotoxicity as depicted by the decrease in shoot length of the germinated seeds. These preliminary results indicated that B. juncea could tolerate and remediate pharmaceutical drugs such as analgesics and antibiotics.