Development Of Chemical Probes Research Articles

Transitioning from the art to the science of thin films: 1964 to 2003

The origin of the modern era of thin film technology is closely linked to the development of vacuum technology. However, the more recent emergence of thin film science as an identifiable field of investigation parallels the development and adaptation of surface structural and chemical probes to provide in situ real-time atomic-scale data during film growth. The ready accessibility of high-resolution transmission electron microscopy also played an important role. The next step in this evolutionary process, which is occurring right now, is the addition of theory and modeling as standard approaches, used in combination with the above physical tools, to complement, design, and analyze experiments. The beautifully hierarchical complexity associated with thin film microstructural and surface morphological evolution, which arises from a diverse set of competitive kinetic instabilities operating on very different time and length scales, assures that thin film science and technology will remain a vital and exciting field long into the future. While the present focus of the field is on film growth from the vapor phase, there is an increasingly strong resurgent interest, driven by nanotechnology and the desire to extend self-organization to higher spatial orders, in film growth from solution.

Chemical Probes of Signal‐Transducing Proteins

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Fluorescent caged phosphoserine peptides as probes to investigate phosphorylation-dependent protein associations.

The development of chemical probes for the investigation of the complex phosphorylation signaling cascades that regulate biological events is crucial to understanding these processes. We describe herein a bifunctional probe that enables spatial and temporal release of a biologically active ligand while allowing simultaneous monitoring of its binding to the protein of interest. Substitution of Tyr(-2) for the enviromentally sensitive fluorescent amino acid DANA in the sequence RLYRpSLPA which is known to bind the 14-3-3 protein does not adversely affect binding affinity and allows monitoring of the binding process. The binding of the peptide to 14-3-3 places the fluorescent reporter unit into a hydrophobic pocket, which changes the fluorescent maximum emission intensity and wavelength. At the same time, the newly developed photolabile 1-(2-nitrophenyl)ethyl-caged phosphoserine allows control of the release of the biologically active ligand through unmasking of the key phosphoserine functionality upon UV irradiation.

Chemical probes of signal-transducing proteins.

Protein kinases are key participants in signal transduction pathways. A direct assessment of the relationship between the activity of any given protein kinase and the corresponding cellular phenotype has proven challenging. This is due to the large number of protein kinases encoded by the human genome coupled with intracellular temporal and spatial constraints that appear to further regulate the ultimate response of a cell to a stimulus. Our work has focused on the development of chemical probes to address the complexities associated with protein kinase-mediated cell signaling. These include the acquisition of highly selective substrates and inhibitors for specific members of the protein kinase family, the design and synthesis of light-activated signaling proteins and their corresponding inhibitors, and the preparation of fluorescent reporters of intracellular protein kinase action.

Activity-based probes for functional proteomics.

Achieving an understanding of the functional role of all proteins expressed by a complex organism will require the development of methods that enable the rapid monitoring of protein function on a global scale. Current genomics and proteomics technologies fall short of this goal since they measure only the relative abundance of transcripts and proteins, respectively. Recent efforts in several laboratories have led to the development of tagged chemical probes that selectively react with families of active enzymes based on shared mechanistic features. These activity-based probes (ABPs) permit the quantitation and comparison of multiple protein activities simultaneously in complex proteomes. In this paper, the general properties and design features of ABPs will be discussed with an emphasis on the use of ABPs for activity-based proteome analysis.

RNA Cleavage by C-1027 Chromophore, an Enediyne Antitumor Antibiotic: High Selectivity to an Anticodon Arm

This study demonstrates unique reactivity of the C-1027 chromophore toward a tRNA phe. In the presence of Mg 2+ ions where the tRNA phe attains a stable three-dimensional structure, the enediyne chromophore exhibits high cleavage selectivity to the anticodon arm. The present reactivity of the C-1027 chromophore is useful in development of new chemical probes for mapping of tertiary RNA structure. Considering the paucity of RNA repair mechanisms, RNA may be also an important biological target for certain enediyne antibiotics.

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Hundreds of genomes have been successfully sequenced to date, and the data are publicly available. At the same time, the advances in large-scale expression and purification of recombinant proteins have paved the way for structural genomics efforts. Frequently, however, little is known about newly expressed proteins calling for large-scale protein characterization to better understand their biochemical roles and to enable structure–function relationship studies. In the Structural Genomics Consortium (SGC), we have established a platform to characterize large numbers of purified proteins. This includes screening for ligands, enzyme assays, peptide arrays and peptide displacement in a 384-well format. In this review, we describe this platform in more detail and report on how our approach significantly increases the success rate for structure determination. Coupled with high-resolution X-ray crystallography and structure-guided methods, this platform can also be used toward the development of chemical probes through screening families of proteins against a variety of chemical series and focused chemical libraries.