The present work describes a general method for the selective attachment of proteins to solid surfaces through their C-termini that can be used for the efficient creation of protein chips. Our method is based in the chemoselective reaction between a protein C-terminal alpha-thioester and a modified surface containing N-terminal Cys residues. alpha-Thioester proteins can be obtained using standard recombinant techniques by using expression vectors containing engineered inteins. This new method was used to immobilize two fluorescent proteins and a functional SH3 domain using a protein microarrayer.

This paper describes a physical organic study of the relationship between the rate for an interfacial Diels-Alder reaction and the steric environment around the reacting molecules. The study used as a model reaction the cycloaddition of cyclopentadiene with a self-assembled monolayer (SAM) presenting benzoquinone groups surrounded by hydroxyl-terminated alkanethiolates. The accessibility of the quinone was varied by preparing monolayers from hydroquinone-terminated alkanethiols of different lengths [HS(CH(2))(n)-HQ, n = 6-14] and a hydroxyl-terminated alkanethiol [HS(CH(2))(11)-OH] of constant length. Cyclic voltammetry was used to measure the rate of the reaction by monitoring the decay of the redox-active quinone. The second-order rate constant showed a modest change as the position of quinone was varied relative to the hydroxyl groups of the monolayer. For monolayers wherein the quinone groups were extended from the interface, the rate constants oscillated near 0.20 M(-1) s(-1) with an even-odd dependence on the length of the alkanethiol. For monolayers that positioned the quinone groups below the surrounding hydroxyl groups, the rate constants decreased by approximately 8-fold. Examination of the activation parameters revealed that the quinone groups that were positioned below the interface (and in a crowded environment) reacted with an enthalpy of activation that was 4 kcal/mol greater than did the quinones that were accessible at the interface. The reaction of the buried quinone, however, proceeded with an entropy of activation that was more favorable by 13 eu, and therefore with a similar free energy of activation. The combination of SAMs for preparing model interfaces and cyclic voltammetry for measuring rates provides a new opportunity for physical organic studies of interfacial reactions.

Without a trace: The immobilization of biologically active proteins onto a solid support through split‐intein‐mediated protein trans‐splicing reactions requires only very dilute samples. Not only is this process traceless but it does not require purification of the protein, thereby making possible the direct immobilization of proteins from complex mixtures such as cell lysates.

Antibody arrays hold considerable potential in a variety of applications including proteomics research, drug discovery, and diagnostics. Many of the schemes used to fabricate the arrays fail to immobilize the antibodies at a uniform density or in a single orientation; consequently, the immobilized antibodies recognize their antigens with variable efficiency. This paper describes a strategy to immobilize antibodies in a single orientation, with a controlled density, using the covalent interaction between cutinase and its suicide substrate. Protein fusions between cutinase and five antibodies of three different types (scFv, V(HH), and FN3) were prepared and immobilized upon self-assembled monolayers (SAMs) presenting a phosphonate capture ligand. The immobilized antibodies exhibit high affinity and selectivity for their target antigens, as monitored by surface plasmon resonance and fluorescence scanning. Furthermore, by changing the density of capture ligand on the SAM the density of the immobilized antibody could be controlled. The monolayers, which also present a tri(ethylene glycol) group, are inert to nonspecific adsorption of proteins and allow the detection of a specific antigen in a complex mixture. The demonstration of cutinase-directed antibody immobilization with insert SAMs provides a straightforward and robust method for preparing antibody chips.

Electromagnetic fields (EMF) are physical energy fields generated by electrically charged objects, and specific ranges of EMF can influence numerous biological processes, which include the control of cell fate and plasticity. In this study, we show that electromagnetized gold nanoparticles (AuNPs) in the presence of specific EMF conditions facilitate an efficient direct lineage reprogramming to induced dopamine neurons in vitro and in vivo. Remarkably, electromagnetic stimulation leads to a specific activation of the histone acetyltransferase Brd2, which results in histone H3K27 acetylation and a robust activation of neuron-specific genes. In vivo dopaminergic neuron reprogramming by EMF stimulation of AuNPs efficiently and non-invasively alleviated symptoms in mouse Parkinson's disease models. This study provides a proof of principle for EMF-based in vivo lineage conversion as a potentially viable and safe therapeutic strategy for the treatment of neurodegenerative disorders.

We present a novel approach to examine cell migration using dynamically adhesive substrates consisting of three spatially and functionally distinct regions: the first is permanently nonadhesive to cells, the second is permanently adhesive, and the final region is electrochemically switched from nonadhesive to adhesive. We applied a double microcontact printing approach to pattern gold surfaces with carboxylic acid-terminated self-assembled monolayers (SAMs) that permit initial cell adhesion, with methyl-terminated SAMs that permit adsorption of a nonadhesive, and with tri(ethylene glycol)-terminated SAMs that can be electrochemically "switched" to permit cell migration from a prespecified pattern onto a new pattern. Using these substrates, we investigated the migration of epithelial cells from monolayers onto narrow, branching tracks of extracellular matrix in order to characterize how lead cells influence the direction of movement of followers. Time-lapse imaging revealed that, on average, five cells consistently chose one branch before other cells entered the second branch, providing evidence to suggest that intercellular communication plays an important role in guiding the cohesive movement of epithelial sheets. This platform may be of use in furthering our understanding of the mechanisms underlying cellular decision-making during migration in both individual and multicellular contexts.

Recently, significant advances have been made in live cell imaging owing to the rapid development of selective labeling of proteins in vivo. Green fluorescent protein (GFP) was the first example of fluorescent reporters genetically introduced to protein of interest (POI). While GFP and various types of engineered fluorescent proteins (FPs) have been actively used for live cell imaging for many years, the size and the limited windows of fluorescent spectra of GFP and its variants set limits on possible applications. In order to complement FP-based labeling methods, alternative approaches that allow incorporation of synthetic fluorescent probes to target POIs were developed. Synthetic fluorescent probes are smaller than fluorescent proteins, often have improved photochemical properties, and offer a larger variety of colors. These synthetic probes can be introduced to POIs selectively by numerous approaches that can be largely categorized into chemical recognition-based labeling, which utilizes metal-chelating peptide tags and fluorophore-carrying metal complexes, and biological recognition-based labeling, such as (1) specific non-covalent binding between an enzyme tag and its fluorophore-carrying substrate, (2) self-modification of protein tags using substrate variants conjugated to fluorophores, (3) enzymatic reaction to generate a covalent binding between a small molecule substrate and a peptide tag, and (4) split-intein-based C-terminal labeling of target proteins. The chemical recognition-based labeling reaction often suffers from compromised selectivity of metal-ligand interaction in the cytosolic environment, consequently producing high background signals. Use of protein-substrate interactions or enzyme-mediated reactions generally shows improved specificity but each method has its limitations. Some examples are the presence of large linker protein, restriction on the choice of introducible probes due to the substrate specificity of enzymes, and competitive reaction mediated by an endogenous analogue of the introduced protein tag. These limitations have been addressed, in part, by the split-intein-based labeling approach, which introduces fluorescent probes with a minimal size (~4 amino acids) peptide tag. In this review, the advantages and the limitations of each labeling method are discussed.

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