Noncanonical Amino Acids in the Interrogation of Cellular Protein Synthesis

Proteins in living cells can be made receptive to bioorthogonal chemistries through metabolic labeling with appropriately designed noncanonical amino acids (ncAAs). In the simplest approach to metabolic labeling, an amino acid analog replaces one of the natural amino acids specified by the protein’s gene (or genes) of interest. Through manipulation of experimental conditions, the extent of the replacement can be adjusted. This approach, often termed residue-specific incorporation, allows the ncAA to be incorporated in controlled proportions into positions normally occupied by the natural amino acid residue. For a protein to be labeled in this way with an ncAA, it must fulfill just two requirements: (i) the corresponding natural amino acid must be encoded within the sequence of the protein at the genetic level, and (ii) the protein must be expressed while the ncAA is in the cell. Because this approach permits labeling of proteins throughout the cell, it has enabled us to develop strategies to track cellular protein synthesis by tagging proteins with reactive ncAAs. In procedures similar to isotopic labeling, translationally active ncAAs are incorporated into proteins during a “pulse” in which newly synthesized proteins are tagged. The set of tagged proteins can be distinguished from those made before the pulse by bioorthogonally ligating the ncAA side chain to probes that permit detection, isolation, and visualization of the labeled proteins. Noncanonical amino acids with side chains containing azide, alkyne, or alkene groups have been especially useful in experiments of this kind. They have been incorporated into proteins in the form of methionine analogs that are substrates for the natural translational machinery. The selectivity of the method can be enhanced through the use of mutant aminoacyl tRNA synthetases (aaRSs) that permit incorporation of ncAAs not used by the endogenous biomachinery. Through expression of mutant aaRSs, proteins can be tagged with other useful ncAAs, including analogs that contain ketones or aryl halides. High-throughput screening strategies can identify aaRS variants that activate a wide range of ncAAs. Controlled expression of mutant synthetases has been combined with ncAA tagging to permit cell-selective metabolic labeling of proteins. Expression of a mutant synthetase in a portion of cells within a complex cellular mixture restricts labeling to that subset of cells. Proteins synthesized in cells not expressing the synthetase are neither labeled nor detected. In multicellular environments, this approach permits the identification of the cellular origins of labeled proteins. In this Account, we summarize the tools and strategies that have been developed for interrogating cellular protein synthesis through residue-specific tagging with ncAAs. We describe the chemical and genetic components of ncAA-tagging strategies and discuss how these methods are being used in chemical biology

Mutant methionyl-tRNA synthetase from bacteria enables site-selective N-terminal labeling of proteins expressed in mammalian cells

Newly synthesized cellular proteins can be tagged with a variety of metabolic labels that distinguish them from preexisting proteins and allow them to be identified and tracked. Many such labels are incorporated into proteins via the endogenous cellular machinery and can be used in numerous cell types and organisms. Though broad applicability has advantages, we aimed to develop a strategy to restrict protein labeling to specified mammalian cells that express a transgene. Here we report that heterologous expression of a mutant methionyl-tRNA synthetase from Escherichia coli permits incorporation of azidonorleucine (Anl) into proteins made in mammalian (HEK293) cells. Anl is incorporated site-selectively at N-terminal positions (in competition with initiator methionines) and is not found at internal sites. Site selectivity is enabled by the fact that the bacterial synthetase aminoacylates mammalian initiator tRNA, but not elongator tRNA. N-terminally labeled proteins can be selectively conjugated to a variety of useful probes; here we demonstrate use of this system in enrichment and visualization of proteins made during various stages of the cell cycle. N-terminal incorporation of Anl may also be used to engineer modified proteins for therapeutic and other applications

Cleavable Biotin Probes for Labeling of Biomolecules via Azide−Alkyne Cycloaddition

The azide−alkyne cycloaddition provides a powerful tool for bio-orthogonal labeling of proteins, nucleic acids, glycans, and lipids. In some labeling experiments, e.g., in proteomic studies involving affinity purification and mass spectrometry, it is convenient to use cleavable probes that allow release of labeled biomolecules under mild conditions. Five cleavable biotin probes are described for use in labeling of proteins and other biomolecules via azide−alkyne cycloaddition. Subsequent to conjugation with metabolically labeled protein, these probes are subject to cleavage with either 50 mM Na_2S_2O_4, 2% HOCH_2CH_2SH, 10% HCO_2H, 95% CF_3CO_2H, or irradiation at 365 nm. Most strikingly, a probe constructed around a dialkoxydiphenylsilane (DADPS) linker was found to be cleaved efficiently when treated with 10% HCO_2H for 0.5 h. A model green fluorescent protein was used to demonstrate that the DADPS probe undergoes highly selective conjugation and leaves a small (143 Da) mass tag on the labeled protein after cleavage. These features make the DADPS probe especially attractive for use in biomolecular labeling and proteomic studies

Automatic Radiated Susceptibility Test System for Payload Equipment

An automatic radiated susceptibility test system (ARSTS) was developed for NASA Lewis Research Center's Electro-magnetic Interference laboratory. According to MSFC-SPEC 521B, any electrical or electronic equipment that will be transported by the spacelab and space shuttle must be tested for susceptibility to electromagnetic interference. This state-of-the-art automatic test system performs necessary calculations; analyzes, processes, and records a great quantity of measured data; and monitors the equipment being tested in real-time and with minimal user intervention. ARSTS reduces costly test time, increases test accuracy, and provides reliable test results

A Nuclear Fragmentation Energy Deposition Model

A formalism for target fragment transport is presented with application to energy loss spectra in thin silicon devices. A nuclear database is recommended that agrees well with the measurements of P.J. McNulty et al. (1980) using surface barrier detectors. High-energy events observed by P.J. McNulty et al. (1981), which are not predicted by intranuclear cascade models, are well represented by this wor

Passive Tracking System and Method

System and methods are disclosed for passively determining the location of a moveable transmitter utilizing a pair of phase shifts at a receiver for extracting a direction vector from a receiver to the transmitter. In a preferred embodiment, a phase difference between the transmitter and receiver is extracted utilizing a noncoherent demodulator in the receiver. The receiver includes antenna array with three antenna elements, which preferably are patch antenna elements placed apart by one-half wavelength. Three receiver channels are preferably utilized for simultaneously processing the received signal from each of the three antenna elements. Multipath transmission paths for each of the three receiver channels are indexed so that comparisons of the same multipath component are made for each of the three receiver channels. The phase difference for each received signal is determined by comparing only the magnitudes of received and stored modulation signals to determine a winning modulation symbol

Properties of traditional bamboo carrying poles have implications for user interactions

Compliant bamboo poles have long been used for load carriage in Asian cultures. Although this custom differs from Western conventions of rigid body attachments (e.g. backpack), potential benefits include reduced peak shoulder forces as well as metabolic transport cost savings. Evidence that carrying a flexible pole benefits locomotion remains mixed, perhaps in part because the properties of pole design (e.g. bamboo material, structural geometry, etc.) have largely been neglected. These properties influence vibrational forces and consequently, the energy required by the user to manage the oscillations. We collected authentic bamboo poles from northern Vietnam and characterized their design parameters. Four poles were extensively studied in the lab (load-deflection testing, resonance testing, and computed tomography scans of three-dimensional geometry), and 10 others were tested at a rural Vietnamese farm site (basic measures of form and resonance). A mass-spring-damper model was used to characterize a relationship between resonant frequency (which affects the energetics of the pole-carrier system) and pole properties concerning stiffness, damping, etc. Model predictions of resonant frequencies agreed well with empirical data. Although measured properties suggest the poles are not optimally designed to reduce peak oscillation forces, resonant frequencies are within range of a typical human walking cadence, and this is likely to have a consequence on locomotion energetics

State-Selective Metabolic Labeling of Cellular Proteins

Transcriptional activity from a specified promoter can provide a useful marker for the physiological state of a cell. Here we introduce a method for selective tagging of proteins made in cells in which specified promoters are active. Tagged proteins can be modified with affinity reagents for enrichment or with fluorescent dyes for visualization. The method allows state-selective analysis of the proteome, whereby proteins synthesized in predetermined physiological states can be identified. The approach is demonstrated by proteome-wide labeling of bacterial proteins upon activation of the P_(BAD) promoter and the SoxRS regulon and provides a basis for analysis of more complex systems including spatially heterogeneous microbial cultures and biofilms

Ultra-Wideband Angle-of-Arrival Tracking Systems

Systems that measure the angles of arrival of ultra-wideband (UWB) radio signals and perform triangulation by use of those angles in order to locate the sources of those signals are undergoing development. These systems were originally intended for use in tracking UWB-transmitter-equipped astronauts and mobile robots on the surfaces of remote planets during early stages of exploration, before satellite-based navigation systems become operational. On Earth, these systems could be adapted to such uses as tracking UWB-transmitter-equipped firefighters inside buildings or in outdoor wildfire areas obscured by smoke. The same characteristics that have made UWB radio advantageous for fine resolution ranging, covert communication, and ground-penetrating radar applications in military and law-enforcement settings also contribute to its attractiveness for the present tracking applications. In particular, the waveform shape and the short duration of UWB pulses make it possible to attain the high temporal resolution (of the order of picoseconds) needed to measure angles of arrival with sufficient precision, and the low power spectral density of UWB pulses enables UWB radio communication systems to operate in proximity to other radio communication systems with little or no perceptible mutual interference