Stimulus-Induced Relief of Intentionally Incorporated Frustration Drives Refolding of a Water-Soluble Biomimetic Foldamer.

Frustrated, or nonoptimal, interactions have been proposed to be essential to a protein's ability to display responsive behavior such as allostery, conformational signaling, and signal transduction. However, the intentional incorporation of frustrated noncovalent interactions has not been explored as a design element in the field of dynamic foldamers. Here, we report the design, synthesis, characterization, and molecular dynamics simulations of the first dynamic water-soluble foldamer that, in response to a stimulus, exploits relief of frustration in its noncovalent network to structurally rearrange from a pleated to an intercalated columnar structure. Thus, relief of frustration provides the energetic driving force for structural rearrangement. This work represents a previously unexplored design element for the development of stimulus-responsive systems that has potential application to materials chemistry, synthetic biology, and molecular machines.

Design, Heterologous Expression, and Application of an Immobilized Protein Kinase.

Protein kinase A (PKA) is a biologically important enzyme for cell regulation, often referred to as the "central kinase". An immobilized PKA that retains substrate specificity and activity would be a useful tool for laboratory scientists, enabling targeted phosphorylation without interference from downstream kinase contamination or kinase autophosphorylation in sensitive assays. Moreover, it might also provide the benefits of robustness and reusability that are often associated with immobilized enzyme preparations. In this work, we describe the creation of a recombinant PKA fusion protein that incorporates the HaloTag covalent immobilization system. We demonstrate that protein fusion design, including affinity tag placement, is critical for optimal heterologous expression in Escherichia coli. Furthermore, we demonstrate various applications of our immobilized PKA, including the phosphorylation of recombinant PKA substrates, such as vasodilator-stimulated phosphoprotein, and endogenous PKA substrates in a cell lysate. This immobilized PKA also possesses robust activity and reusability over multiple trials. This work holds promise as a generalizable strategy for the production and application of immobilized protein kinases.

Covalent and non-covalent strategies for the immobilization of Tobacco Etch Virus protease (TEVp) on superparamagnetic nanoparticles.

Proteases with highly specific activities have numerous applications, including the cleavage of affinity tags (Flag; HA; His6X) and solubility promoting partners (GST; MBP) within the context of protein isolation and purification schemes. However, commercially sourced proteases such as Tobacco Etch Virus protease (TEVp) and Human Rhinovirus (HRV) 3C protease are typically applied as single use aliquots, which limits their cost-effectiveness. In addition, the presence of residual proteases in downstream applications can complicate analysis of the protein of interest. Thus, the creation of immobilized, reusable site-specific proteases would be of significant value to the life science community. In this work, we explore two strategies for the immobilization of TEV protease onto superparamagnetic iron oxide nanoparticles (SPIONs). In one strategy, a MBP-TEVp-Streptavidin fusion protein is immobilized on biotin-functionalized SPIONs. In a second strategy, TEV protease is covalently coupled onto SPIONs directly, via amine-mediated attachment, and indirectly, via HALO-tag mediated attachment. We demonstrate activity of our immobilized proteases in the presence of a MBP-GFP fusion protein containing the TEV protease target sequence (ENLYFQ|S). We then analyze time-dependent activity, longevity, and reuse of these immobilized protein preparations, comparing each approach. The protease immobilization strategies described in this work may be useful tools for simplifying challenging protein purification protocols, in addition to providing general methods for enzyme immobilization on SPIONs.

Phenolic Oxidation Using H2O2 via in Situ Generated para-Quinone Methides for the Preparation of para-Spiroepoxydienones.

Phenols are attractive starting materials for the preparation of highly substituted cyclohexane rings via dearomative processes. Herein we report an efficient preparation of dearomatized 1-oxaspiro[2.5]octa-4,7-dien-6-ones (para-spiroepoxydienones) via the nucleophilic epoxidation of in situ generated para-quinone methides from 4-(hydroxymethyl)phenols using aqueous H2O2. The developed protocol bypasses the need for stoichiometric bismuth reagents or diazomethane, which are frequently deployed for p-spiroepoxydienone preparation. The p-spiroepoxydienones are further elaborated in numerous downstream complexity-building transformations.

Wristwatch-associated ski injury.

Upper limb injuries are a common occurrence in snowboarders, and these may be reduced by wearing wrist guards that transmit the force of the fall across the forearm. We present a case of a 26-year-old man who experienced a displaced left radial and ulnar fracture related to wearing a wristwatch while snowboarding. The wristwatch acted as a fulcrum around which the arm was fractured, serving as a stress riser that focused the force of the fall. The fracture was treated using open reduction and internal fixation.