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Epithiodiketopiperazines (ETPs) are a structurally complex class of fungal natural products with potent anticancer activity. In ETPs, the diketopiperazine ring is spanned by a disulfide bond that is constrained in a high-energy eclipsed conformation. We employed computational, synthetic, and spectroscopic methods to investigate the physicochemical attributes of this atypical disulfide bond. We find that the disulfide bond is stabilized by two n→π* interactions, each with large energies (3-5 kcal/mol). The n→π* interactions in ETPs make disulfide reduction much more difficult, endowing stability in physiological environments in a manner that could impact their biological activity. These data reveal a previously unappreciated means to stabilize a disulfide bond and highlight the utility of the n→π* interaction in molecular design.

The glycyl radical enzyme (GRE) superfamily utilizes a glycyl radical cofactor to catalyze difficult chemical reactions in a variety of anaerobic microbial metabolic pathways. Recently, a GRE, trans-4-hydroxy-L-proline (Hyp) dehydratase (HypD), was discovered that catalyzes the dehydration of Hyp to (S)-Δ1-pyrroline-5-carboxylic acid (P5C). This enzyme is abundant in the human gut microbiome and also present in prominent bacterial pathogens. However, we lack an understanding of how HypD performs its unusual chemistry. Here, we have solved the crystal structure of HypD from the pathogen Clostridioides difficile with Hyp bound in the active site. Biochemical studies have led to the identification of key catalytic residues and have provided insight into the radical mechanism of Hyp dehydration.

The Diels-Alder reactivity of 4,4-difluoro-3,5-diphenyl-4H-pyrazole was investigated experimentally and computationally with endo-bicyclo[6.1.0]non-4-yne. The computationally predicted rate enhancement from hyperconjugative antiaromaticity induced by fluorination of cyclopentadienes at the 5-position extends to five-membered heterocyclic dienes containing a saturated center. 4,4-Difluoro-4H-pyrazoles are new electron-deficient dienes with rapid reactivities toward strained alkynes.

Lindner and Lemal showed that perfluorination of keto-enol systems significantly shifts the equilibrium toward the enol tautomer. Quantum mechanical calculations now reveal that the shift in equilibrium is the result of the stabilization of the enol tautomer by hyperconjugative π → σ*CF interactions and the destabilization of the keto tautomer by the electron withdrawal induced by the neighboring fluorine atoms. The preference for the enol tautomer further increases in smaller perfluorinated cyclic keto-enol systems. This trend is in contrast to the nonfluorinated compounds, where the enol is strongly disfavored in the smaller rings. The fluoro effect overrides the effect of the ring size that controls the equilibria in nonfluorinated compounds. The increased overlap of the enol π bond with the σ*CF orbitals of the allylic C-F bonds results in the increased preference for the enol tautomer in smaller perfluorinated keto-enol systems. We show here why the effect is much greater than in 3,3-difluorocyclooctyne.

Collagens are the foundational component of diverse tissues, including skin, bone, cartilage, and basement membranes, and are the most abundant protein class in animals. The fibrillar collagens are large, complex, multidomain proteins, all containing the characteristic triple helix motif. The most prevalent collagens are heterotrimeric, meaning that cells express at least two distinctive procollagen polypeptides that must assemble into specific heterotrimer compositions. The molecular mechanisms ensuring correct heterotrimeric assemblies are poorly understood - even for the most common collagen, type-I. The longstanding paradigm is that assembly is controlled entirely by the ~30 kDa globular C-propeptide (C-Pro) domain. Still, this dominating model for procollagen assembly has left many questions unanswered. Here, we show that the C-Pro paradigm is incomplete. In addition to the critical role of the C-Pro domain in templating assembly, we find that the amino acid sequence near the C terminus of procollagen's triple-helical domain plays an essential role in defining procollagen assembly outcomes. These sequences near the C terminus of the triple-helical domain encode conformationally stabilizing features that ensure only desirable C-Pro- mediated trimeric templates are committed to irreversible triple-helix folding. Incorrect C-Pro trimer assemblies avoid commitment to triple-helix formation thanks to destabilizing features in the amino acid sequences of their triple helix. Incorrect C-Pro assemblies are consequently able to dissociate and search for new binding partners. These findings provide a distinctive perspective on the mechanism of procollagen assembly, revealing the molecular basis by which incorrect homotrimer assemblies are avoided and setting the stage for a deeper understanding of the biogenesis of this ubiquitous protein. [ABSTRACT FROM AUTHOR]

Motivation Post-translational modifications (PTMs) increase the diversity of the proteome and are vital to organismal life and therapeutic strategies. Deep learning has been used to predict PTM locations. Still, limitations in datasets and their analyses compromise success. Results We evaluated the use of known PTM sites in prediction via sequence-based deep learning algorithms. For each PTM, known locations of that PTM were encoded as a separate amino acid before sequences were encoded via word embedding and passed into a convolutional neural network that predicts the probability of that PTM at a given site. Without labeling known PTMs, our models are on par with others. With labeling, however, we improved significantly upon extant models. Moreover, knowing PTM locations can increase the predictability of a different PTM. Our findings highlight the importance of PTMs for the installation of additional PTMs. We anticipate that including known PTM locations will enhance the performance of other proteomic machine learning algorithms. Availability and implementation Sitetack is available as a web tool at https://sitetack.net ; the source code, representative datasets, instructions for local use, and select models are available at https://github.com/clair-gutierrez/sitetack. [ABSTRACT FROM AUTHOR]

Wound healing is a complex process that requires the intervention of cytoactive factors. The one-time application of soluble factors to a wound bed does not maintain a steady, sufficient concentration. Here we investigated the benefits of anchoring a factor in a wound bed via a tether to endogenous collagen. We used a collagen-mimetic peptide (CMP) as a pylon. The CMP binds to damaged but not intact collagen and thus localizes a pendant cytoactive factor in the regions of a wound bed that require intervention. As a model factor, we chose substance P, a peptide of the tachykinin family that promotes wound healing. Using splinted wounds in db/db mice, we found that the one-time application of a CMP-substance P conjugate enhances wound healing compared to unconjugated substance P and other controls. Specifically, all 16 wounds treated with the conjugate closed more thoroughly and, did so with extensive re-epithelialization and mitigated inflammatory activity. These data validate a simple and general strategy for re-engineering wound beds by the integration of beneficial cytoactive factors. Copyright © 2014 John Wiley & Sons, Ltd.

Alongside vaccines and antiviral therapeutics, diagnostic tools are a crucial aid in combating the COVID‐19 pandemic caused by the etiological agent SARS‐CoV‐2. All common assays for infection rely on the detection of viral sub‐components, including structural proteins of the virion or fragments of the viral genome. Selective pressure imposed by human intervention of COVID‐19 can, however, induce viral mutations that decrease the sensitivity of diagnostic assays based on biomolecular structure, leading to an increase in false‐negative results. In comparison, mutations are unlikely to alter the function of viral proteins, and viral machinery is under less selective pressure from vaccines and therapeutics. Accordingly, diagnostic assays that rely on biomolecular function can be more robust than ones that rely on biopolymer structure. Toward this end, we used a split intein to create a circular ribonuclease zymogen that is activated by the SARS‐CoV‐2 main protease, 3CLpro. Zymogen activation by 3CLpro leads to a >300‐fold increase in ribonucleolytic activity, which can be detected with a highly sensitive fluorogenic substrate. This coupled assay can detect low nanomolar concentrations of 3CLpro within a timeframe comparable to that of common antigen‐detection protocols. More generally, the concept of detecting a protease by activating a ribonuclease could be the basis of diagnostic tools for other indications. [ABSTRACT FROM AUTHOR]

Animal‐sourced hydrogels, such as collagen, are widely used as extracellular‐matrix (ECM) mimics in tissue engineering but are plagued with problems of reproducibility, immunogenicity, and contamination. Synthetic, chemically defined hydrogels can avoid such issues. Despite the abundance of collagen in the ECM, synthetic collagen hydrogels are extremely rare due to design challenges brought on by the triple‐helical structure of collagen. Sticky‐ended symmetric self‐assembly (SESSA) overcomes these challenges by maximizing interactions between the strands of the triple helix, allowing the assembly of collagen‐mimetic peptides (CMPs) into robust synthetic collagen nanofibers. This optimization, however, also minimizes interfiber contacts. In this work, symmetric association states for the SESSA of short CMPs to probe their increased propensity for interfiber association are modelled. It is found that 33‐residue CMPs not only self‐assemble through sticky ends, but also form hydrogels. These self‐assemblies behave with remarkable consistency across multiple scales and present a clear link between their triple‐helical architecture and the properties of their hydrogels. The results show that SESSA is an effective and robust design methodology that enables the rational design of synthetic collagen hydrogels. [ABSTRACT FROM AUTHOR]