Modular functionalization of crystalline graphene by recombinant proteins: a nanoplatform for probing biomolecules.

Graphene, as well as other two-dimensional materials, is a promising candidate for use in bioimaging, therapeutic drug delivery, and bio-sensing applications. Here, we developed a protocol to functionalize graphene with recombinant proteins using genetically encoded SpyTag-SpyCatcher chemistry. SpyTag forms a covalent isopeptide bond with its genetically encoded partner SpyCatcher through spontaneous amidation under physiological conditions. The functionalization protocol developed is based on the use of short proteins as a linker, where two graphene-binding-peptides (GBPs) are attached to both ends of SpyTag (referred to as GStG), followed by the covalent conjugation with SpyCatcher-fusion proteins. The proposed method enables the decoration of crystalline graphene with various proteins, such as fluorescent proteins and affibody molecules that bind to cancerous cells. This scheme, which takes advantage of the cleanness of single-crystal graphene and the robustness of SpyTag-SpyCatcher chemistry, provides a versatile platform on which to study the biomolecule-surface and cell-substrate interactions and, indeed, may lead to a new way of designing biomedical devices. The interaction between peptides and graphene was clearly shown using molecular dynamics simulation and proven using specially designed experiments.

Controlling the Integration of Polyvinylpyrrolidone onto Substrate by Quartz Crystal Microbalance with Dissipation To Achieve Excellent Protein Resistance and Detoxification.

Blood purification systems, in which the adsorbent removes exogenous and endogenous toxins from the blood, are widely used in clinical practice. To improve the protein resistance of and detoxification by the adsorbent, researchers can modify the adsorbent with functional molecules, such as polyvinylpyrrolidone (PVP). However, achieving precise control of the functional molecular density, which is crucial to the activity of the adsorbent, remains a significant challenge. In the present study, we prepared a model system for blood purification adsorbents in which we controlled the integration density of PVP molecules of different molecular weights on an Au substrate by quartz crystal microbalance with dissipation (QCM-D). We characterized the samples with atomic force microscopy, X-ray photoelectron spectroscopy, and QCM-D and found that the molecular density and the chain length of the PVP molecules played important roles in determining the properties of the sample. At the optimal condition, the modified sample demonstrated strong resistance to plasma proteins, decreasing the adsorption of human serum albumin (HSA) and fibrinogen (Fg) by 92.5% and 79.2%, respectively. In addition, the modified sample exhibited excellent detoxification, and the adsorption of bilirubin increased 2.6-fold. Interestingly, subsequent atomistic molecular dynamics simulations indicated that the favorable interactions between PVP and bilirubin were dominated by hydrophobic interactions. An in vitro platelet adhesion assay showed that the adhesion of platelets on the sample decreased and that the platelets were maintained in an inactivated state. The CCK-8 assay indicated that the modified sample exhibited negligible cytotoxicity to L929 cells. These results demonstrated that our method holds great potential for the modification of adsorbents in blood purification systems.

Stapling of unprotected helical peptides via photo-induced intramolecular thiol-yne hydrothiolation.

Peptide stapling emerged as a versatile strategy to recapitulate the bioactive helical conformation of unstructured short peptides in water to improve their therapeutic properties in targeting intracellular "undruggable" targets. Here, we describe the development of photo-induced intramolecular thiol-yne macrocyclization for rapid access to short stapled peptides with enhanced biophysical properties. This new peptide stapling technique provides rapid access to conformationally constrained helices with satisfying functional group tolerance. Notably, the vinyl sulfide linkage shows distinct lipophilicity with reduced membrane toxicity compared to the corresponding all-hydrocarbon analogue. As a proof of principle, we constructed stabilized helices modulating intracellular estrogen receptor (ER)-coactivator interactions with a nanomolar binding affinity, enhanced serum stability, a diffuse cellular distribution and selective cytotoxicity towards ER-positive MCF-7 cells.