David A. Tirrell

In situ visualization and dynamics of newly synthesized proteins in rat hippocampal neurons

Protein translation has been implicated in different forms of synaptic plasticity, but direct in situ visualization of new proteins is limited to one or two proteins at a time. Here we describe a metabolic labeling approach based on incorporation of noncanonical amino acids into proteins followed by chemoselective fluorescence tagging by means of click chemistry. After a brief incubation with azidohomoalanine or homopropargylglycine, a robust fluorescent signal was detected in somata and dendrites. Pulse-chase application of azidohomoalanine and homopropargylglycine allowed visualization of proteins synthesized in two sequential time periods. This technique can be used to detect changes in protein synthesis and to evaluate the fate of proteins synthesized in different cellular compartments. Moreover, using strain-promoted cycloaddition, we explored the dynamics of newly synthesized membrane proteins using single-particle tracking and quantum dots. The newly synthesized proteins showed a broad range of diffusive behaviors, as would be expected for a pool of labeled proteins that is heterogeneous.

Core-Clickable PEG-Branch-Azide Bivalent-Bottle-Brush Polymers by ROMP: Grafting-Through and Clicking-To

The combination of highly efficient polymerizations with modular click coupling reactions has enabled the synthesis of a wide variety of novel nanoscopic structures. Here we demonstrate the facile synthesis of a new class of clickable, branched nanostructures, polyethylene glycol (PEG)-branch-azide bivalent-brush polymers, facilitated by graft-through ring-opening metathesis polymerization of a branched norbornene-PEG-chloride macromonomer followed by halide-azide exchange. The resulting bivalent-brush polymers possess azide groups at the core near a polynorbornene backbone with PEG chains extended into solution; the structure resembles a unimolecular micelle. We demonstrate copper-catalyzed azide-alkyne cycloaddition (CuAAC) click-to coupling of a photocleavable doxorubicin (DOX)-alkyne derivative to the azide core. The CuAAC coupling was quantitative across a wide range of nanoscopic sizes (∼6-∼50 nm); UV photolysis of the resulting DOX-loaded materials yielded free DOX that was therapeutically effective against human cancer cells.

Residue-specific incorporation of non-canonical amino acids into proteins: recent developments and applications

Residue-specific incorporation of non-canonical amino acids into proteins allows facile alteration and enhancement of protein properties. In this review, we describe recent technical developments and applications of residue-specific incorporation to problems ranging from elucidation of biochemical mechanisms to engineering of protein-based biomaterials. We hope to inform the reader of the ease and broad utility of residue-specific non-canonical amino acid incorporation with the goal of inspiring investigators outside the field to consider applying this tool to their own research.

A photoreversible protein-patterning approach for guiding stem cell fate in three-dimensional gels

Although biochemically patterned hydrogels are capable of recapitulating many critical aspects of the heterogeneous cellular niche, exercising spatial and temporal control of the presentation and removal of biomolecular signalling cues in such systems has proved difficult. Here, we demonstrate a synthetic strategy that exploits two bioorthogonal photochemistries to achieve reversible immobilization of bioactive full-length proteins with good spatial and temporal control within synthetic, cell-laden biomimetic scaffolds. A photodeprotection-oxime-ligation sequence permits user-defined quantities of proteins to be anchored within distinct subvolumes of a three-dimensional matrix, and an ortho-nitrobenzyl ester photoscission reaction facilitates subsequent protein removal. By using this approach to pattern the presentation of the extracellular matrix protein vitronectin, we accomplished reversible differentiation of human mesenchymal stem cells to osteoblasts in a spatially defined manner. Our protein-patterning approach should provide further avenues to probe and direct changes in cell physiology in response to dynamic biochemical signalling.

Three-Dimensional Traction Force Microscopy: A New Tool for Quantifying Cell-Matrix Interactions

The interactions between biochemical processes and mechanical signaling play important roles during various cellular processes such as wound healing, embryogenesis, metastasis, and cell migration. While traditional traction force measurements have provided quantitative information about cell matrix interactions in two dimensions, recent studies have shown significant differences in the behavior and morphology of cells when placed in three-dimensional environments. Hence new quantitative experimental techniques are needed to accurately determine cell traction forces in three dimensions. Recently, two approaches both based on laser scanning confocal microscopy have emerged to address this need. This study highlights the details, implementation and advantages of such a three-dimensional imaging methodology with the capability to compute cellular traction forces dynamically during cell migration and locomotion. An application of this newly developed three-dimensional traction force microscopy (3D TFM) technique to single cell migration studies of 3T3 fibroblasts is presented to show that this methodology offers a new quantitative vantage point to investigate the three-dimensional nature of cell-ECM interactions.

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 proteins 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.

Live‐Cell Imaging of Cellular Proteins by a Strain‐Promoted Azide–Alkyne Cycloaddition

Live and let dye: Three coumarin-cyclooctyne conjugates have been used to label proteins tagged with azidohomoalanine in Rat-1 fibroblasts. All three fluorophores labeled intracellular proteins with fluorescence enhancements ranging from eight- to 20-fold. These conjugates are powerful tools for visualizing biomolecule dynamics in living cells.

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(2)S(2)O(4), 2% HOCH(2)CH(2)SH, 10% HCO(2)H, 95% CF(3)CO(2)H, 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(2)H 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.

Controlling Macromolecular Topology with Genetically Encoded SpyTag–SpyCatcher Chemistry

Control of molecular topology constitutes a fundamental challenge in macromolecular chemistry. Here we describe the synthesis and characterization of artificial elastin-like proteins (ELPs) with unconventional nonlinear topologies including circular, tadpole, star, and H-shaped proteins using genetically encoded SpyTag-SpyCatcher chemistry. SpyTag is a short polypeptide that binds its protein partner SpyCatcher and forms isopeptide bonds under physiological conditions. Sequences encoding SpyTag and SpyCatcher can be strategically placed into ELP genes to direct post-translational topological modification in situ. Placement of SpyTag at the N-terminus and SpyCatcher at the C-terminus directs formation of circular ELPs. Induction of expression at 16 °C with 10 μM IPTG yields 80% monomeric cyclic protein. When SpyTag is placed in the middle of the chain, it exhibits an even stronger tendency toward cyclization, yielding up to 94% monomeric tadpole proteins. Telechelic ELPs containing either SpyTag or SpyCatcher can be expressed, purified, and then coupled spontaneously upon mixing in vitro. Block proteins, 3-arm or 4-arm star proteins, and H-shaped proteins have been prepared, with the folded CnaB2 domain that results from the SpyTag-SpyCatcher reaction as the molecular core or branch junction. The modular character of the SpyTag-SpyCatcher strategy should make it useful for preparing nonlinear macromolecules of diverse sequence and structure.

Direct visualization of newly synthesized target proteins in situ

Protein synthesis is a dynamic process that tunes the cellular proteome in response to internal and external demands. Metabolic labeling approaches identify the general proteomic response but cannot visualize specific newly synthesized proteins within cells. Here we describe a technique that couples noncanonical amino acid tagging or puromycylation with the proximity ligation assay to visualize specific newly synthesized proteins and monitor their origin, redistribution and turnover in situ.