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Biomedical subjects

Jason W Chin

Publications and source records attributed to Jason W Chin.

16 recordsLinked to original sources
PubMed2006

Expanding nucleic acid function in vitro and in vivo.

Nucleic Acids composed of the five natural bases and a phosphate backbone can be designed or evolved to have a wide variety of sequence-dependent functions. Recent in vitro work has addressed some outstanding issues in evolving nucleic acid catalysts, as well as the creation of prescribed shapes and arrays from oligonucleotides and long single-stranded nucleic acids. Nucleic acids have also been engineered in vivo, leading to new modes of gene regulation. It is likely that the improving ability to synthesize long DNA sequences will accelerate the creation of novel functions from nucleic acids.

Catalysis↗
PubMed2006

Programming and engineering biological networks.

Synthetic biology aims to build new functions in living organisms. Recent work has addressed the creation of synthetic epigenetic switches in mammalian cells and synthetic intracellular communication. Fundamentally new, and potentially scaleable, modes of gene regulation have been created that enable expansion of the scope of synthetic circuits. Increasingly sophisticated models of gene regulation that include stochastic effects are beginning to predict the behaviour of small synthetic networks. Overall, these advances suggest that a combination of molecular engineering and systems engineering should allow the creation of living matter capable of performing many useful and novel functions.

Biomedical Engineering↗
PubMed2006

A genetically encoded fluorescent amino acid.

The ability to introduce fluorophores selectively into proteins provides a powerful tool to study protein structure, dynamics, localization, and biomolecular interactions both in vitro and in vivo. Here, we report a strategy for the selective and efficient biosynthetic incorporation of a low-molecular-weight fluorophore into proteins at defined sites. The fluorescent amino acid 2-amino-3-(5-(dimethylamino)naphthalene-1-sulfonamide)propanoic acid (dansylalanine) was genetically encoded in Saccharomyces cerevisiae by using an amber nonsense codon and corresponding orthogonal tRNA/aminoacyl-tRNA synthetase pair. This environmentally sensitive fluorophore was selectively introduced into human superoxide dismutase and used to monitor unfolding of the protein in the presence of guanidinium chloride. The strategy described here should be applicable to a number of different fluorophores in both prokaryotic and eukaryotic organisms, and it should facilitate both biochemical and cellular studies of protein structure and function.

Alanine↗
PubMed2006

Functional epitopes at the ribosome subunit interface.

The ribosome is a 2.5-MDa molecular machine that synthesizes cellular proteins encoded in mRNAs. The 30S and 50S subunits of the ribosome associate through structurally defined intersubunit bridges burying 6,000 A(2), 80% of which is buried in conserved RNA-RNA interactions. Intersubunit bridges bind translation factors, may coordinate peptide bond formation and translocation and may be actively remodeled in the post-termination complex, but the functional importance of numerous 30S bridge nucleotides had been unknown. We carried out large-scale combinatorial mutagenesis and in vivo selections on 30S nucleotides that form RNA-RNA intersubunit bridges in the Escherichia coli ribosome. We determined the covariation and functional importance of bridge nucleotides, allowing comparison of the structural interface and phylogenetic data to the functional epitope. Our results reveal how information for ribosome function is partitioned across bridges, and suggest a subset of nucleotides that may have measurable effects on individual steps of the translational cycle.

Cloning, Molecular↗
PubMed2006

Modular approaches to expanding the functions of living matter.

The synthesis of increasingly complex unnatural networks embedded in living matter is an emerging theme in synthetic biology. Synthetic networks have allowed the creation of organisms endowed with toggle switches, logic gates, pattern-forming systems, oscillators, cellular sensors, new modes of gene regulation and expanded genetic codes. A common challenge of this work is the addition of specific new functions to complex living organisms. This requires spatial and temporal control of molecular interactions and fluxes to achieve the desired outcomes. Here we review recent successes in this emerging field and discuss strategies for addressing the challenges of increasing network complexity.

Animals↗
PubMed2005

Cellular logic with orthogonal ribosomes.

The creation and use of unnatural molecules to control cellular function is a long standing goal of the chemical community, but in general, these efforts have been directed at finding molecules to inhibit or activate a particular molecular target or function, or to elicit a particular phenotype. Here we show that multiple unnatural molecules (orthogonal ribosomes) can be used combinatorially, in a single cell, to program Boolean logic functions. These experiments show how attention to the molecular specificity of noncovalent interactions between unnatural macromolecules allows the synthesis of complex function from the "bottom-up" in living matter.

Base Sequence↗
PubMed2005

A network of orthogonal ribosome x mRNA pairs.

Synthetic biology promises the ability to program cells with new functions. Simple oscillators, switches, logic functions, cell-cell communication and pattern-forming circuits have been created by the connection of a small set of natural transcription factors and their binding sites in different ways to produce different networks of molecular interactions. However, the controlled synthesis of more complex synthetic networks and functions will require an expanded set of functional molecules with known molecular specificities. Here, we tailored the molecular specificity of duplicated Escherichia coli ribosome x mRNA pairs with respect to the wild-type ribosome and mRNAs to produce multiple orthogonal ribosome x orthogonal mRNA pairs that can process information in parallel with, but independent of, their wild-type progenitors. In these pairs, the ribosome exclusively translates the orthogonal mRNA, and the orthogonal mRNA is not a substrate for cellular ribosomes. We predicted and measured the network of interactions between orthogonal ribosomes and orthogonal mRNAs, and showed that they can be used to post-transcriptionally program the cell with Boolean logic.

Binding Sites↗
PubMed2004

Substrate recognition by the AAA+ chaperone ClpB.

The AAA+ protein ClpB cooperates with the DnaK chaperone system to solubilize and refold proteins from an aggregated state. The substrate-binding site of ClpB and the mechanism of ClpB-dependent protein disaggregation are largely unknown. Here we identified a substrate-binding site of ClpB that is located at the central pore of the first AAA domain. The conserved Tyr251 residue that lines the central pore contributes to substrate binding and its crucial role was confirmed by mutational analysis and direct crosslinking to substrates. Because the positioning of an aromatic residue at the central pore is conserved in many AAA+ proteins, a central substrate-binding site involving this residue may be a common feature of this protein family. The location of the identified binding site also suggests a possible translocation mechanism as an integral part of the ClpB-dependent disaggregation reaction.

Adenosine Triphosphatases↗
PubMed2003

Adding amino acids with novel reactivity to the genetic code of Saccharomyces cerevisiae.

Using a novel genetic selection, we have identified a series of mutants of the E. coli tyrosyl-tRNA synthetase that selectively charge an amber suppressor tRNA with p-(propargyloxy)phenylalanine and p-azidophenylalanine in yeast. These evolved tRNA-synthetase pairs can be used to site-specifically label proteins with functional groups orthogonal to normal biological chemistries. As an example, we have shown that proteins containing these amino acids can be efficiently bioconjugated with small organic molecules by a [3 + 2] cycloaddition reaction that is mild enough for the manipulation of biological samples.

Acetylene↗
PubMed2003

An expanded eukaryotic genetic code.

We describe a general and rapid route for the addition of unnatural amino acids to the genetic code of Saccharomyces cerevisiae. Five amino acids have been incorporated into proteins efficiently and with high fidelity in response to the nonsense codon TAG. The side chains of these amino acids contain a keto group, which can be uniquely modified in vitro and in vivo with a wide range of chemical probes and reagents; a heavy atom-containing amino acid for structural studies; and photocrosslinkers for cellular studies of protein interactions. This methodology not only removes the constraints imposed by the genetic code on our ability to manipulate protein structure and function in yeast, it provides a gateway to the systematic expansion of the genetic codes of multicellular eukaryotes.

Amino Acids↗
PubMed2003

Progress toward an expanded eukaryotic genetic code.

Expanding the eukaryotic genetic code to include unnatural amino acids with novel properties would provide powerful tools for manipulating protein function in eukaryotic cells. Toward this goal, a general approach with potential for isolating aminoacyl-tRNA synthetases that incorporate unnatural amino acids with high fidelity into proteins in Saccharomyces cerevisiae is described. The method is based on activation of GAL4-responsive HIS3, URA3, or lacZ reporter genes by suppression of amber codons in GAL4. The optimization of GAL4 reporters is described, and the positive and negative selection of active Escherichia coli tyrosyl-tRNA synthetase (EcTyrRS)/tRNA(CUA) is demonstrated. Importantly, both selections can be performed on a single cell and with a range of stringencies. This method will facilitate the isolation of a range of aminoacyl-tRNA synthetase (aaRS)/tRNA(CUA) activities from large libraries of mutant synthetases.

Amino Acids↗
PubMed2002

Addition of p-azido-L-phenylalanine to the genetic code of Escherichia coli.

We report the selection of a new orthogonal aminoacyl tRNA synthetase/tRNA pair for the in vivo incorporation of a photocrosslinker, p-azido-l-phenylalanine, into proteins in response to the amber codon, TAG. The amino acid is incorporated in good yield with high fidelity and can be used to crosslink interacting proteins.

Azides↗
PubMed2002

Addition of a photocrosslinking amino acid to the genetic code of Escherichiacoli.

Benzophenones are among the most useful photocrosslinking agents in biology. We have evolved an orthogonal aminoacyl-tRNA synthetase/tRNA pair that makes possible the in vivo incorporation of p-benzoyl-l-phenylalanine into proteins in Escherichia coli in response to the amber codon, TAG. This unnatural amino acid was incorporated with high translational efficiency and fidelity into the dimeric protein glutathione S-transferase. Irradiation resulted in efficient crosslinking (>50%) of the protein subunits. This methodology may prove useful for discovering and defining protein interactions in vitro and in vivo.

Amino Acids↗
PubMed2002

A view to a kill: ligands for Bcl-2 family proteins.

Apoptosis is the essential process of programmed cell death that, in multicellular organisms, regulates development and maintains homeostasis. Defects in the apoptotic molecular machinery that result in either excessive or insufficient apoptosis are observed in a remarkably wide range of human disease, prompting intense interest in pro- and anti-apoptotic proteins as therapeutic targets. A number of recent reports have described the discovery of ligands for anti-apoptotic Bcl-2 family proteins by a variety of approaches, including computational, combinatorial and evolutionary strategies. Both the design of ligands and the exploration of their mechanisms of action have been greatly enhanced by recent high-resolution structure determinations of proteins from this family. Several of the newly discovered ligands promote apoptosis, and some do so even in the face of overexpressed anti-apoptotic Bcl-2 proteins. Ligands that overcome the protective effects associated with up-regulation of anti-apoptotic Bcl-2 proteins represent especially promising therapeutic leads.

Apoptosis↗