High-throughput virtual search of small molecules for controlling the mechanical stability of human CD4.

Protein mechanical stability determines the function of a myriad of proteins, especially proteins from the extracellular matrix. Failure to maintain protein mechanical stability may result in diseases and disorders such as cancer, cardiomyopathies, or muscular dystrophy. Thus, developing mutation-free approaches to enhance and control the mechanical stability of proteins using pharmacology-based methods may have important implications in drug development and discovery. Here, we present the first approach that employs computational high-throughput virtual screening and molecular docking to search for small molecules in chemical libraries that function as mechano-regulators of the stability of human cluster of differentiation 4, receptor of HIV-1. Using single-molecule force spectroscopy, we prove that these small molecules can increase the mechanical stability of CD4D1D2 domains over 4-fold in addition to modifying the mechanical unfolding pathways. Our experiments demonstrate that chemical libraries are a source of mechanoactive molecules and that drug discovery approaches provide the foundation of a new type of molecular function, that is, mechano-regulation, paving the way toward mechanopharmacology.

Evolution of CRISPR-associated endonucleases as inferred from resurrected proteins.

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas9 is an effector protein that targets invading DNA and plays a major role in the prokaryotic adaptive immune system. Although Streptococcus pyogenes CRISPR-Cas9 has been widely studied and repurposed for applications including genome editing, its origin and evolution are poorly understood. Here, we investigate the evolution of Cas9 from resurrected ancient nucleases (anCas) in extinct firmicutes species that last lived 2.6 billion years before the present. We demonstrate that these ancient forms were much more flexible in their guide RNA and protospacer-adjacent motif requirements compared with modern-day Cas9 enzymes. Furthermore, anCas portrays a gradual palaeoenzymatic adaptation from nickase to double-strand break activity, exhibits high levels of activity with both single-stranded DNA and single-stranded RNA targets and is capable of editing activity in human cells. Prediction and characterization of anCas with a resurrected protein approach uncovers an evolutionary trajectory leading to functionally flexible ancient enzymes.

Enzymatically produced cellulose nanocrystals as reinforcement for waterborne polyurethane and its applications.

Waterborne polyurethanes (WBPUs) have been proposed as ecofriendly elastomers with several applications in coatings and adhesives. WBPU's physicochemical properties can be enhanced by the addition of cellulose nanocrystals (CNCs). The way CNCs are isolated has a strong effect on their properties and can determine their role as reinforcement. In this work, CNCs produced using ancestral endoglucanase (EnCNCs) were used as reinforcement for WBPU and compared with CNC produced by sulfuric acid hydrolysis (AcCNC). The enzymatic method produced highly thermostable and crystalline CNCs. The addition of small contents of EnCNCs improved the thermomechanical stability and mechanical properties of WBPUs, even better than commercial AcCNCs. Besides, WBPU reinforced by adding EnCNCs was studied as a coating for paper materials, increasing its abrasion resistance and as electrospun nanocomposite mats where EnCNCs helped maintaining the morphology of the fibers.