David A. Tirrell

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Name: Tirrell, David A.

Organization: California Institute of Technology , USA

Department: Division of Chemistry and Chemical Engineering

Title: (PhD)

Chemicals
References

Protein-Mediated Colloidal Assembly

Co-reporter:Maiko Obana, Bradley R. Silverman, and David A. Tirrell

Journal of the American Chemical Society October 11, 2017 Volume 139(Issue 40) pp:14251-14251

Publication Date(Web):September 12, 2017

DOI:10.1021/jacs.7b07798

Programmable colloidal assembly enables the creation of mesoscale materials in a bottom-up manner. Although DNA oligonucleotides have been used extensively as the programmable units in this paradigm, proteins, which exhibit more diverse modes of association and function, have not been widely used to direct colloidal assembly. Here we use protein–protein interactions to drive controlled aggregation of polystyrene microparticles, either through reversible coiled-coil interactions or through intermolecular isopeptide linkages. The sizes of the resulting aggregates are tunable and can be controlled by the concentration of immobilized surface proteins. Moreover, particles coated with different protein pairs undergo orthogonal assembly. We demonstrate that aggregates formed by association of coiled-coil proteins, in contrast to those linked by isopeptide bonds, are dispersed by treatment with chemical denaturants or soluble competing proteins. Finally, we show that protein–protein interactions can be used to assemble complex core–shell aggregates. This work illustrates a versatile strategy for engineering colloidal systems for use in materials science and biotechnology.

Analysis and Control of Chain Mobility in Protein Hydrogels

Co-reporter:Peter B. Rapp, Ahmad K. Omar, Jeff J. Shen, Maren E. Buck, Zhen-Gang Wang, and David A. Tirrell

Journal of the American Chemical Society March 15, 2017 Volume 139(Issue 10) pp:3796-3796

Publication Date(Web):February 22, 2017

DOI:10.1021/jacs.6b13146

Coiled-coil domains can direct the assembly of protein block copolymers into physically cross-linked, viscoelastic hydrogels. Here, we describe the use of fluorescence recovery after photobleaching (FRAP) to probe chain mobility in reversible hydrogels assembled from engineered proteins bearing terminal coiled-coil domains. We show that chain mobility can be related to the underlying dynamics of the coiled-coil domains by application of a three-state “hopping” model of chain migration. We further show that genetic programming allows the effective mobility of network chains to be varied 500-fold through modest changes in protein sequence. Destabilization of the coiled-coil domains by site-directed mutagenesis increases the effective diffusivity of probe chains. Conversely, probe mobility is reduced by expanding the hydrophobic surface area of the coiled-coil domains through introduction of the bulky leucine surrogate homoisoleucine. Predictions from the three-state model imply asymmetric sequential binding of the terminal domains. Brownian Dynamics simulations suggest that binding asymmetry is a general feature of reversible gels, arising from a loss in entropy as chains transition to a conformationally restricted bridged state.

4S-Hydroxylation of Insulin at ProB28 Accelerates Hexamer Dissociation and Delays Fibrillation

Co-reporter:Seth A. Lieblich, Katharine Y. Fang, Jackson K. B. Cahn, Jeffrey Rawson, Jeanne LeBon, H. Teresa Ku, and David A. Tirrell

Journal of the American Chemical Society June 28, 2017 Volume 139(Issue 25) pp:8384-8384

Publication Date(Web):June 9, 2017

DOI:10.1021/jacs.7b00794

Daily injections of insulin provide lifesaving benefits to millions of diabetics. But currently available prandial insulins are suboptimal: The onset of action is delayed by slow dissociation of the insulin hexamer in the subcutaneous space, and insulin forms amyloid fibrils upon storage in solution. Here we show, through the use of noncanonical amino acid mutagenesis, that replacement of the proline residue at position 28 of the insulin B-chain (ProB28) by (4S)-hydroxyproline (Hzp) yields an active form of insulin that dissociates more rapidly, and fibrillates more slowly, than the wild-type protein. Crystal structures of dimeric and hexameric insulin preparations suggest that a hydrogen bond between the hydroxyl group of Hzp and a backbone amide carbonyl positioned across the dimer interface may be responsible for the altered behavior. The effects of hydroxylation are stereospecific; replacement of ProB28 by (4R)-hydroxyproline (Hyp) causes little change in the rates of fibrillation and hexamer disassociation. These results demonstrate a new approach that fuses the concepts of medicinal chemistry and protein design, and paves the way to further engineering of insulin and other therapeutic proteins.

Programming Molecular Association and Viscoelastic Behavior in Protein Networks

Co-reporter:Lawrence J. Dooling;Maren E. Buck;Wen-Bin Zhang

Advanced Materials 2016 Volume 28( Issue 23) pp:4651-4657

Publication Date(Web):

DOI:10.1002/adma.201506216

Chemoenzymatic Labeling of Proteins for Imaging in Bacterial Cells

Co-reporter:Samuel H. Ho and David A. Tirrell

Journal of the American Chemical Society 2016 Volume 138(Issue 46) pp:15098-15101

Publication Date(Web):November 10, 2016

DOI:10.1021/jacs.6b07067

Reliable methods to determine the subcellular localization of bacterial proteins are needed for the study of prokaryotic cell biology. We describe here a simple and general technique for imaging of bacterial proteins in situ by fluorescence microscopy. The method uses the eukaryotic enzyme N-myristoyltransferase to modify the N-terminus of the protein of interest with an azido fatty acid. Subsequent strain-promoted azide–alkyne cycloaddition allows conjugation of dyes and imaging of tagged proteins by confocal fluorescence microscopy. We demonstrate the method by labeling the chemotaxis proteins Tar and CheA and the cell division proteins FtsZ and FtsA in Escherichia coli. We observe distinct spatial patterns for each of these proteins in both fixed and live cells. The method should prove broadly useful for protein imaging in bacteria.

Engineered Aminoacyl-tRNA Synthetase for Cell-Selective Analysis of Mammalian Protein Synthesis

Co-reporter:Alborz Mahdavi; Graham D. Hamblin; Granton A. Jindal; John D. Bagert; Cathy Dong; Michael J. Sweredoski; Sonja Hess; Erin M. Schuman

Journal of the American Chemical Society 2016 Volume 138(Issue 13) pp:4278-4281

Publication Date(Web):March 18, 2016

DOI:10.1021/jacs.5b08980

Methods for cell-selective analysis of proteome dynamics will facilitate studies of biological processes in multicellular organisms. Here we describe a mutant murine methionyl-tRNA synthetase (designated L274GMmMetRS) that charges the noncanonical amino acid azidonorleucine (Anl) to elongator tRNAMet in hamster (CHO), monkey (COS7), and human (HeLa) cell lines. Proteins made in cells that express the synthetase can be labeled with Anl, tagged with dyes or affinity reagents, and enriched on affinity resin to facilitate identification by mass spectrometry. The method does not require expression of orthogonal tRNAs or depletion of canonical amino acids. Successful labeling of proteins with Anl in several mammalian cell lines demonstrates the utility of L274GMmMetRS as a tool for cell-selective analysis of mammalian protein synthesis.

Time-resolved proteomic analysis of quorum sensing in Vibrio harveyi

Co-reporter:John D. Bagert, Julia C. van Kessel, Michael J. Sweredoski, Lihui Feng, Sonja Hess, Bonnie L. Bassler and David A. Tirrell

Chemical Science 2016 vol. 7(Issue 3) pp:1797-1806

Publication Date(Web):23 Nov 2015

DOI:10.1039/C5SC03340C

Bacteria use a process of chemical communication called quorum sensing to assess their population density and to change their behavior in response to fluctuations in the cell number and species composition of the community. In this work, we identified the quorum-sensing-regulated proteome in the model organism Vibrio harveyi by bio-orthogonal non-canonical amino acid tagging (BONCAT). BONCAT enables measurement of proteome dynamics with temporal resolution on the order of minutes. We deployed BONCAT to characterize the time-dependent transition of V. harveyi from individual- to group-behaviors. We identified 176 quorum-sensing-regulated proteins at early, intermediate, and late stages of the transition, and we mapped the temporal changes in quorum-sensing proteins controlled by both transcriptional and post-transcriptional mechanisms. Analysis of the identified proteins revealed 86 known and 90 new quorum-sensing-regulated proteins with diverse functions, including transcription factors, chemotaxis proteins, transport proteins, and proteins involved in iron homeostasis.

SutA is a bacterial transcription factor expressed during slow growth in Pseudomonas aeruginosa

Co-reporter:Brett M. Babin;Megan Bergkessel;Michael J. Sweredoski;Annie Moradian;Sonja Hess;Dianne K. Newman

PNAS 2016 Volume 113 (Issue 5 ) pp:E597-E605

Publication Date(Web):2016-02-02

DOI:10.1073/pnas.1514412113

Microbial quiescence and slow growth are ubiquitous physiological states, but their study is complicated by low levels of metabolic activity. To address this issue, we used a time-selective proteome-labeling method [bioorthogonal noncanonical amino acid tagging (BONCAT)] to identify proteins synthesized preferentially, but at extremely low rates, under anaerobic survival conditions by the opportunistic pathogen Pseudomonas aeruginosa. One of these proteins is a transcriptional regulator that has no homology to any characterized protein domains and is posttranscriptionally up-regulated during survival and slow growth. This small, acidic protein associates with RNA polymerase, and chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing suggests that the protein associates with genomic DNA through this interaction. ChIP signal is found both in promoter regions and throughout the coding sequences of many genes and is particularly enriched at ribosomal protein genes and in the promoter regions of rRNA genes. Deletion of the gene encoding this protein affects expression of these and many other genes and impacts biofilm formation, secondary metabolite production, and fitness in fluctuating conditions. On the basis of these observations, we have designated the protein SutA (survival under transitions A).

Engineering the Dynamic Properties of Protein Networks through Sequence Variation

Co-reporter:Lawrence J. Dooling and David A. Tirrell

ACS Central Science 2016 Volume 2(Issue 11) pp:812

Publication Date(Web):October 18, 2016

DOI:10.1021/acscentsci.6b00205

The dynamic behavior of macromolecular networks dominates the mechanical properties of soft materials and influences biological processes at multiple length scales. In hydrogels prepared from self-assembling artificial proteins, stress relaxation and energy dissipation arise from the transient character of physical network junctions. Here we show that subtle changes in sequence can be used to program the relaxation behavior of end-linked networks of engineered coiled-coil proteins. Single-site substitutions in the coiled-coil domains caused shifts in relaxation time over 5 orders of magnitude as demonstrated by dynamic oscillatory shear rheometry and stress relaxation measurements. Networks with multiple relaxation time scales were also engineered. This work demonstrates how time-dependent mechanical responses of macromolecular materials can be encoded in genetic information.

Cell-specific proteomic analysis in Caenorhabditis elegans

Co-reporter:Kai P. Yuet;Meenakshi K. Doma;John T. Ngo;Michael J. Sweredoski;Robert L. J. Graham;Erin M. Schuman;Annie Moradian;Paul W. Sternberg;Sonja Hess

PNAS 2015 Volume 112 (Issue 9 ) pp:2705-2710

Publication Date(Web):2015-03-03

DOI:10.1073/pnas.1421567112

Proteomic analysis of rare cells in heterogeneous environments presents difficult challenges. Systematic methods are needed to enrich, identify, and quantify proteins expressed in specific cells in complex biological systems including multicellular plants and animals. Here, we have engineered a Caenorhabditis elegans phenylalanyl-tRNA synthetase capable of tagging proteins with the reactive noncanonical amino acid p-azido-l-phenylalanine. We achieved spatiotemporal selectivity in the labeling of C. elegans proteins by controlling expression of the mutant synthetase using cell-selective (body wall muscles, intestinal epithelial cells, neurons, and pharyngeal muscle) or state-selective (heat-shock) promoters in several transgenic lines. Tagged proteins are distinguished from the rest of the protein pool through bioorthogonal conjugation of the azide side chain to probes that permit visualization and isolation of labeled proteins. By coupling our methodology with stable-isotope labeling of amino acids in cell culture (SILAC), we successfully profiled proteins expressed in pharyngeal muscle cells, and in the process, identified proteins not previously known to be expressed in these cells. Our results show that tagging proteins with spatiotemporal selectivity can be achieved in C. elegans and illustrate a convenient and effective approach for unbiased discovery of proteins expressed in targeted subsets of cells.

Prometastatic GPCR CD97 Is a Direct Target of Tumor Suppressor microRNA-126

Co-reporter:Ying Y. Lu, Michael J. Sweredoski, David Huss, Rusty Lansford, Sonja Hess, and David A. Tirrell

ACS Chemical Biology 2014 Volume 9(Issue 2) pp:334

Publication Date(Web):November 25, 2013

DOI:10.1021/cb400704n

Tumor suppressor microRNA-126 (miR-126) is often down-regulated in cancer cells, and its overexpression is found to inhibit cancer metastasis. To elucidate the mechanism of tumor suppression by miR-126, we analyzed the proteomic response to miR-126 overexpression in the human metastatic breast cancer cell line MDA-MB-231. To acquire quantitative, time-resolved information, we combined two complementary proteomic methods, BONCAT and SILAC. We discovered a new direct target of miR-126: CD97, a pro-metastatic G-protein-coupled receptor (GPCR) that has been reported to promote tumor cell invasion, endothelial cell migration, and tumor angiogenesis. This discovery establishes a link between down-regulation of miR-126 and overexpression of CD97 in cancer and provides new mechanistic insight into the role of miR-126 in inhibiting both cell-autonomous and non-cell-autonomous cancer progression.

Cell Surface Display Yields Evolvable, Clickable Antibody Fragments

Co-reporter:Dr. James A. Van Deventer;Kai P. Yuet; Tae Hyeon Yoo; David A. Tirrell

ChemBioChem 2014 Volume 15( Issue 12) pp:1777-1781

Publication Date(Web):

DOI:10.1002/cbic.201402184

Abstract

Non-canonical amino acids (ncAAs) provide powerful tools for engineering the chemical and physical properties of proteins. However, introducing ncAAs into proteins can affect protein properties in unpredictable ways, thus necessitating screening efforts to identify mutants with desirable properties. In this work, we describe an Escherichia coli cell surface display platform for the directed evolution of clickable antibody fragments. This platform enabled isolation of antibody fragments with improved digoxigenin binding and modest affinity maturation in several different ncAA contexts. Azide-functionalized fragments exhibited improved binding kinetics relative to their methionine counterparts, facile chemical modification through azide–alkyne cycloaddition, and retention of binding properties after modification. The results described here suggest new possibilities for protein engineering, including modulation of molecular recognition events by ncAAs and direct screening of libraries of chemically modified proteins.

Chemical Tools for Temporally and Spatially Resolved Mass Spectrometry-Based Proteomics

Co-reporter:Kai P. Yuet

Annals of Biomedical Engineering 2014 Volume 42( Issue 2) pp:299-311

Publication Date(Web):2014 February

DOI:10.1007/s10439-013-0878-3

Accurate measurements of the abundances, synthesis rates and degradation rates of cellular proteins are critical for understanding how cells and organisms respond to changes in their environments. Over the past two decades, there has been increasing interest in the use of mass spectrometry for proteomic analysis. In many systems, however, protein diversity as well as cell and tissue heterogeneity limit the usefulness of mass spectrometry-based proteomics. As a result, researchers have had difficulty in systematically identifying proteins expressed within specified time intervals, or low abundance proteins expressed in specific tissues or in a few cells in complex microbial systems. In this review, we present recently-developed tools and strategies that probe these two subsets of the proteome: proteins synthesized during well-defined time intervals—temporally resolved proteomics—and proteins expressed in predetermined cell types, cells or cellular compartments—spatially resolved proteomics—with a focus on chemical and biological mass spectrometry-based methodologies.

Synthesis of bioactive protein hydrogels by genetically encoded SpyTag-SpyCatcher chemistry

Co-reporter:Fei Sun;Wen-Bin Zhang;Alborz Mahdavi;Frances H. Arnold

PNAS 2014 Volume 111 (Issue 31 ) pp:11269-11274

Publication Date(Web):2014-08-05

DOI:10.1073/pnas.1401291111

Protein-based hydrogels have emerged as promising alternatives to synthetic hydrogels for biomedical applications, owing to the precise control of structure and function enabled by protein engineering. Nevertheless, strategies for assembling 3D molecular networks that carry the biological information encoded in full-length proteins remain underdeveloped. Here we present a robust protein gelation strategy based on a pair of genetically encoded reactive partners, SpyTag and SpyCatcher, that spontaneously form covalent isopeptide linkages under physiological conditions. The resulting “network of Spies” may be designed to include cell-adhesion ligands, matrix metalloproteinase-1 cleavage sites, and full-length globular proteins [mCherry and leukemia inhibitory factor (LIF)]. The LIF network was used to encapsulate mouse embryonic stem cells; the encapsulated cells remained pluripotent in the absence of added LIF. These results illustrate a versatile strategy for the creation of information-rich biomaterials.

Identification of secreted bacterial proteins by noncanonical amino acid tagging

Co-reporter:Alborz Mahdavi;Janek Szychowski;John T. Ngo;Michael J. Sweredoski;Robert L. J. Graham;Sonja Hess;Olaf Schneewind;Sarkis K. Mazmanian;

Proceedings of the National Academy of Sciences 2014 111(1) pp:433-438

Publication Date(Web):December 17, 2013

DOI:10.1073/pnas.1301740111

Pathogenic microbes have evolved complex secretion systems to deliver virulence factors into host cells. Identification of these factors is critical for understanding the infection process. We report a powerful and versatile approach to the selective labeling and identification of secreted pathogen proteins. Selective labeling of microbial proteins is accomplished via translational incorporation of azidonorleucine (Anl), a methionine surrogate that requires a mutant form of the methionyl-tRNA synthetase for activation. Secreted pathogen proteins containing Anl can be tagged by azide-alkyne cycloaddition and enriched by affinity purification. Application of the method to analysis of the type III secretion system of the human pathogen Yersinia enterocolitica enabled efficient identification of secreted proteins, identification of distinct secretion profiles for intracellular and extracellular bacteria, and determination of the order of substrate injection into host cells. This approach should be widely useful for the identification of virulence factors in microbial pathogens and the development of potential new targets for antimicrobial therapy.

A Genetically Encoded AND Gate for Cell-Targeted Metabolic Labeling of Proteins

Co-reporter:Alborz Mahdavi ; Thomas H. Segall-Shapiro ; Songzi Kou ; Granton A. Jindal ; Kevin G. Hoff ; Shirley Liu ; Mohsen Chitsaz ; Rustem F. Ismagilov ; Jonathan J. Silberg

Journal of the American Chemical Society 2013 Volume 135(Issue 8) pp:2979-2982

Publication Date(Web):February 13, 2013

DOI:10.1021/ja400448f

We describe a genetic AND gate for cell-targeted metabolic labeling and proteomic analysis in complex cellular systems. The centerpiece of the AND gate is a bisected methionyl-tRNA synthetase (MetRS) that charges the Met surrogate azidonorleucine (Anl) to tRNAMet. Cellular protein labeling occurs only upon activation of two different promoters that drive expression of the N- and C-terminal fragments of the bisected MetRS. Anl-labeled proteins can be tagged with fluorescent dyes or affinity reagents via either copper-catalyzed or strain-promoted azide–alkyne cycloaddition. Protein labeling is apparent within 5 min after addition of Anl to bacterial cells in which the AND gate has been activated. This method allows spatial and temporal control of proteomic labeling and identification of proteins made in specific cellular subpopulations. The approach is demonstrated by selective labeling of proteins in bacterial cells immobilized in the center of a laminar-flow microfluidic channel, where they are exposed to overlapping, opposed gradients of inducers of the N- and C-terminal MetRS fragments. The observed labeling profile is predicted accurately from the strengths of the individual input signals.

Controlling Macromolecular Topology with Genetically Encoded SpyTag–SpyCatcher Chemistry

Co-reporter:Wen-Bin Zhang ; Fei Sun ; David A. Tirrell ;Frances H. Arnold

Journal of the American Chemical Society 2013 Volume 135(Issue 37) pp:13988-13997

Publication Date(Web):August 21, 2013

DOI:10.1021/ja4076452

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.

Selective Functionalization of the Protein N Terminus with N-Myristoyl Transferase for Bioconjugation in Cell Lysate

Co-reporter:Chethana Kulkarni;Dr. Tamara L. Kinzer-Ursem; David A. Tirrell

ChemBioChem 2013 Volume 14( Issue 15) pp:1958-1962

Publication Date(Web):

DOI:10.1002/cbic.201300453

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

Co-reporter:Erin M. Schuman;John T. Ngo

PNAS 2013 Volume 110 (Issue 13 ) pp:4992-4997

Publication Date(Web):2013-03-26

DOI:10.1073/pnas.1216375110

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.

Two-Strain, Cell-Selective Protein Labeling in Mixed Bacterial Cultures

Co-reporter:Frank Truong ; Tae Hyeon Yoo ; Thomas J. Lampo

Journal of the American Chemical Society 2012 Volume 134(Issue 20) pp:8551-8556

Publication Date(Web):May 10, 2012

DOI:10.1021/ja3004667

Cell-selective metabolic labeling of proteins with noncanonical amino acids enables the study of proteomic changes in specified subpopulations of complex multicellular systems. For example, azidonorleucine (Anl) and 2-aminooctynoic acid, both of which are activated by an engineered methionyl-tRNA synthetase (designated NLL-MetRS), are excluded from proteins made in wild-type cells but incorporated readily into proteins made in cells that carry NLL-MetRS. To expand the set of tools available for cell-selective metabolic labeling, we sought a MetRS variant capable of activating propargylglycine (Pra). Pra was chosen as the target amino acid because its alkynyl side chain can be selectively and efficiently conjugated to azide-functionalized fluorescence probes and affinity tags. Directed evolution, using active-site randomization and error-prone PCR, yielded a MetRS variant (designated PraRS) capable of incorporating Pra at near-quantitative levels into proteins made in a Met-auxotrophic strain of Escherichia coli cultured in Met-depleted media. Proteins made in E. coli strains expressing PraRS were labeled with Pra in Met-supplemented media as shown by in-gel fluorescence after conjugation to Cy5-azide. The combined use of NLL-MetRS and PraRS enabled differential, cell-selective labeling of marker proteins derived from two bacterial strains cocultured in media supplemented with Met, Anl, and Pra. Treatment of the mixed marker proteins by sequential strain-promoted and copper(I)-catalyzed cycloadditions allowed straightforward identification of the cellular origin of each protein.

State-Selective Metabolic Labeling of Cellular Proteins

Co-reporter:John T. Ngo, Brett M. Babin, Julie A. Champion, Erin M. Schuman, and David A. Tirrell

ACS Chemical Biology 2012 Volume 7(Issue 8) pp:1326

Publication Date(Web):June 12, 2012

DOI:10.1021/cb300238w

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 PBAD promoter and the SoxRS regulon and provides a basis for analysis of more complex systems including spatially heterogeneous microbial cultures and biofilms.

Noncanonical Amino Acids in the Interrogation of Cellular Protein Synthesis

Co-reporter:John T. Ngo and David A. Tirrell

Accounts of Chemical Research 2011 Volume 44(Issue 9) pp:677

Publication Date(Web):August 4, 2011

DOI:10.1021/ar200144y

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.

A BODIPY-Cyclooctyne for Protein Imaging in Live Cells

Co-reporter:Dr. Kimberly E. Beatty;Dr. Janek Szychowski;Dr. John D. Fisk ; David A. Tirrell

ChemBioChem 2011 Volume 12( Issue 14) pp:2137-2139

Publication Date(Web):

DOI:10.1002/cbic.201100277

Homoisoleucine: A Translationally Active Leucine Surrogate of Expanded Hydrophobic Surface Area

Co-reporter:James A. Van Deventer; John D. Fisk; David A. Tirrell

ChemBioChem 2011 Volume 12( Issue 5) pp:700-702

Publication Date(Web):

DOI:10.1002/cbic.201000731

Collective Cell Migration on Artificial Extracellular Matrix Proteins Containing Full-Length Fibronectin Domains

Co-reporter:Eileen Fong

Advanced Materials 2010 Volume 22( Issue 46) pp:5271-5275

Publication Date(Web):

DOI:10.1002/adma.201002448

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

Co-reporter:Jeremiah A. Johnson ; Ying Y. Lu ; Alan O. Burts ; Yeon-Hee Lim ; M. G. Finn ; Jeffrey T. Koberstein ; Nicholas J. Turro ; David A. Tirrell ;Robert H. Grubbs

Journal of the American Chemical Society 2010 Volume 133(Issue 3) pp:559-566

Publication Date(Web):December 13, 2010

DOI:10.1021/ja108441d

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.

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

Co-reporter:Janek Szychowski ; Alborz Mahdavi ; Jennifer J. L. Hodas ; John D. Bagert ; John T. Ngo ; Peter Landgraf ; Daniela C. Dieterich ; Erin M. Schuman

Journal of the American Chemical Society 2010 Volume 132(Issue 51) pp:18351-18360

Publication Date(Web):December 8, 2010

DOI:10.1021/ja1083909

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 Na2S2O4, 2% HOCH2CH2SH, 10% HCO2H, 95% CF3CO2H, 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% HCO2H 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.

Yielding Behavior in Injectable Hydrogels from Telechelic Proteins

Co-reporter:Bradley D. Olsen, Julia A. Kornfield, and David A. Tirrell

Macromolecules 2010 Volume 43(Issue 21) pp:9094-9099

Publication Date(Web):October 13, 2010

DOI:10.1021/ma101434a

Injectable hydrogels show substantial promise for use in minimally invasive tissue engineering and drug delivery procedures. A new injectable hydrogel material, developed from recombinant telechelic proteins expressed in E. coli, demonstrates shear thinning by 3 orders of magnitude at large strains. Large-amplitude oscillatory shear illustrates that shear thinning is due to yielding within the bulk of the gel, and the rheological response and flow profiles are consistent with a shear-banding mechanism for yielding. The sharp yielding transition and large magnitude of the apparent shear thinning allow gels to be injected through narrow gauge needles with only gentle hand pressure. After injection the gels reset to full elastic strength in seconds due to rapid re-formation of the physical network junctions, allowing self-supporting structures to be formed. The shear thinning and recovery behavior is largely independent of the midblock length, enabling genetic engineering to be used to control the equilibrium modulus of the gel without loss of the characteristic yielding behavior. The shear-banding mechanism localizes deformation during flow into narrow regions of the gels, allowing more than 95% of seeded cells to survive the injection process.

Live-Cell Imaging of Cellular Proteins by a Strain-Promoted AzideAlkyne Cycloaddition

Co-reporter:Dr. Kimberly E. Beatty;Dr. John D. Fisk;Dr. Brian P. Smart;Ying Ying Lu;Dr. Janek Szychowski;Dr. Matthew J. Hangauer;Dr. Jeremy M. Baskin; Carolyn R. Bertozzi; David A. Tirrell

ChemBioChem 2010 Volume 11( Issue 15) pp:2092-2095

Publication Date(Web):

DOI:10.1002/cbic.201000419

Hydration dynamics at fluorinated protein surfaces

Co-reporter:Oh-Hoon Kwon;Tae Hyeon Yoo;Christina M. Othon;James A. Van Deventer;Ahmed H. Zewail

PNAS 2010 Volume 107 (Issue 40 ) pp:17101-17106

Publication Date(Web):2010-10-05

DOI:10.1073/pnas.1011569107

Water-protein interactions dictate many processes crucial to protein function including folding, dynamics, interactions with other biomolecules, and enzymatic catalysis. Here we examine the effect of surface fluorination on water-protein interactions. Modification of designed coiled-coil proteins by incorporation of 5,5,5-trifluoroleucine or (4S)-2-amino-4-methylhexanoic acid enables systematic examination of the effects of side-chain volume and fluorination on solvation dynamics. Using ultrafast fluorescence spectroscopy, we find that fluorinated side chains exert electrostatic drag on neighboring water molecules, slowing water motion at the protein surface.

Boundary crossing in epithelial wound healing

Co-reporter:David A. Tirrell;Shelly Tzlil;Eileen Fong

PNAS 2010 Volume 107 (Issue 45 ) pp:19302-19307

Publication Date(Web):2010-11-09

DOI:10.1073/pnas.1008291107

The processes of wound healing and collective cell migration have been studied for decades. Intensive research has been devoted to understanding the mechanisms involved in wound healing, but the role of cell-substrate interactions is still not thoroughly understood. Here we probe the role of cell-substrate interactions by examining in vitro the healing of monolayers of human corneal epithelial (HCE) cells cultured on artificial extracellular matrix (aECM) proteins. We find that the rate of wound healing is dependent on the concentration of fibronectin-derived (RGD) cell-adhesion ligands in the aECM substrate. The wound closure rate varies nearly sixfold on the substrates examined, despite the fact that the rates of migration and proliferation of individual cells show little sensitivity to the RGD concentration (which varies 40-fold). To explain this apparent contradiction, we study collective migration by means of a dynamic Monte Carlo simulation. The cells in the simulation spread, retract, and proliferate with probabilities obtained from a simple phenomenological model. The results indicate that the overall wound closure rate is determined primarily by the rate at which cells cross the boundary between the aECM protein and the matrix deposited under the cell sheet.

Generation of Surface-Bound Multicomponent Protein Gradients

Co-reporter:Kechun Zhang Dr.;Ayae Sugawara Dr.

ChemBioChem 2009 Volume 10( Issue 16) pp:2617-2619

Publication Date(Web):

DOI:10.1002/cbic.200900542

Biosynthesis and Stability of Coiled-Coil Peptides Containing (2S,4R)-5,5,5-Trifluoroleucine and (2S,4S)-5,5,5-Trifluoroleucine

Co-reporter:Jin Kim Montclare ;Soojin Son Dr.;Ginevra A. Clark;Krishna Kumar

ChemBioChem 2009 Volume 10( Issue 1) pp:84-86

Publication Date(Web):

DOI:10.1002/cbic.200800164

Introduction of an Aliphatic Ketone into Recombinant Proteins in a Bacterial Strain that Overexpresses an Editing-Impaired Leucyl-tRNA Synthetase

Co-reporter:Yi Tang ;Pin Wang ;James A. Van Deventer;A. James Link

ChemBioChem 2009 Volume 10( Issue 13) pp:2188-2190

Publication Date(Web):

DOI:10.1002/cbic.200900407

Discovery of Escherichia coli methionyl-tRNA synthetase mutants for efficient labeling of proteins with azidonorleucine in vivo

Co-reporter:I. Caglar Tanrikulu;Emmanuelle Schmitt;Yves Mechulam;William A. Goddard III

PNAS 2009 Volume 106 (Issue 36 ) pp:15285-15290

Publication Date(Web):2009-09-08

DOI:10.1073/pnas.0905735106

Incorporation of noncanonical amino acids into cellular proteins often requires engineering new aminoacyl-tRNA synthetase activity into the cell. A screening strategy that relies on cell-surface display of reactive amino acid side-chains was used to identify a diverse set of methionyl-tRNA synthetase (MetRS) mutants that allow efficient incorporation of the methionine (Met) analog azidonorleucine (Anl). We demonstrate that the extent of cell-surface labeling in vivo is a good indicator of the rate of Anl activation by the MetRS variant harbored by the cell. By screening at low Anl concentrations in Met-supplemented media, MetRS variants with improved activities toward Anl and better discrimination against Met were identified.

Enzymatic N-terminal Addition of Noncanonical Amino Acids to Peptides and Proteins

Co-reporter:Rebecca E. Connor;Konstantin Piatkov;Alexer Varshavsky

ChemBioChem 2008 Volume 9( Issue 3) pp:366-369

Publication Date(Web):

DOI:10.1002/cbic.200700605

Cell Response to RGD Density in Cross-Linked Artificial Extracellular Matrix Protein Films

Co-reporter:Julie C. Liu and David A. Tirrell

Biomacromolecules 2008 Volume 9(Issue 11) pp:

Publication Date(Web):October 1, 2008

DOI:10.1021/bm800469j

This study examines the adhesion, spreading, and migration of human umbilical vein endothelial cells on cross-linked films of artificial extracellular matrix (aECM) proteins. The aECM proteins described here were designed for application in small-diameter grafts and are composed of elastin-like structural repeats and fibronectin cell-binding domains. aECM-RGD contains the RGD sequence derived from fibronectin; the negative control protein aECM-RDG contains a scrambled cell-binding domain. The covalent attachment of poly(ethylene glycol) (PEG) to aECM substrates reduced nonspecific cell adhesion to aECM-RDG-PEG but did not preclude sequence-specific adhesion of endothelial cells to aECM-RGD-PEG. Variation in ligand density was accomplished by the mixing of aECM-RGD-PEG and aECM-RDG-PEG prior to cross-linking. Increasing the density of RGD domains in cross-linked films resulted in more robust cell adhesion and spreading but did not affect cell migration speed. Control of cell-binding domain density in aECM proteins can thus be used to modulate cell adhesion and spreading and will serve as an important design tool as these materials are further developed for use in surgery, tissue engineering, and regenerative medicine.

Structure and mechanical properties of artificial protein hydrogels assembled through aggregation of leucine zipper peptide domains

Co-reporter:Wei Shen, Julia A. Kornfield and David A. Tirrell

Soft Matter 2007 vol. 3(Issue 1) pp:99-107

Publication Date(Web):09 Nov 2006

DOI:10.1039/B610986A

Artificial protein hydrogels made from a triblock protein (designated AC10A, where A is an acidic zipper domain and C10 comprises 10 repeats of the nonapeptide sequence exhibit normalized plateau storage moduli (G′∞/nkT) less than 0.13 at all concentrations, pH values, and ionic strengths examined. These gels are surprisingly soft due to loop formation at the expense of bridges between physical junctions. Molecular-level evidence of loop formation is provided by strong fluorescence energy transfer (FRET) between distinct chromophores placed at the C- and N-termini of labelled chains diluted in an excess of unlabelled chains. The tendency to form loops originates from the compact size of the random coil midblock (mean RH(C10) ≈ 20 Å, determined from quasi-elastic light scattering of C10), and is facilitated by the ability of the leucine zipper domains to form antiparallel aggregates. Although the aggregation number of the leucine zipper domains is small (tetrameric, determined from multi-angle static light scattering of AC10 diblock), the average center-to-center distance between aggregates is roughly 1.5 times the average end-to-end distance of the C10 domain in a 7% w/v network. To avoid stretching the C10 domain, the chains tend to form loops. Changes in pH or ionic strength that expand the polyelectrolyte midblock favor bridging, leading to greater G′∞ as long as leucine zipper endblocks do not dissociate. Understanding of the network structure provided successful design strategies to increase the rigidity of these hydrogels. In contrast to intuitive design concepts for rubber and gel materials, it was shown that increasing either the length or the charge density of the midblock increases rigidity, because fewer chains are wasted in loop formation.

High-Throughput Screening for Methionyl-tRNA Synthetases That Enable Residue-Specific Incorporation of Noncanonical Amino Acids into Recombinant Proteins in Bacterial Cells

Co-reporter:Tae Hyeon Yoo;David A. Tirrell

Angewandte Chemie 2007 Volume 119(Issue 28) pp:

Publication Date(Web):14 JUN 2007

DOI:10.1002/ange.200700779

Einfacher Einbau: Eine effiziente Methode zur Identifizierung von Mutanten-Methionyl-tRNA-Synthetasen (MetRS) wurde entwickelt, mit der nichtkanonische Aminosäuren global in rekombinante Proteine eingebaut werden können. Mithilfe dieser Methode wurde eine MetRS-Variante identifiziert, die den nahezu quantitativen Ersatz von Methionin durch 6,6,6-Trifluornorleucin ermöglicht (siehe Schema).

High-Throughput Screening for Methionyl-tRNA Synthetases That Enable Residue-Specific Incorporation of Noncanonical Amino Acids into Recombinant Proteins in Bacterial Cells

Co-reporter:Tae Hyeon Yoo;David A. Tirrell

Angewandte Chemie International Edition 2007 Volume 46(Issue 28) pp:

Publication Date(Web):14 JUN 2007

DOI:10.1002/anie.200700779

A change for the better: An efficient method for the identification of mutant methionyl-tRNA synthetases (MetRS) has been developed that enables global incorporation of noncanonical amino acids into recombinant proteins. By using the method, an MetRS variant has been identified that enables near-quantitative replacement of methionine by 6,6,6-trifluoronorleucine (see scheme).

Evolution of a fluorinated green fluorescent protein

Co-reporter:Tae Hyeon Yoo;A. James Link

PNAS 2007 104 (35 ) pp:13887-13890

Publication Date(Web):2007-08-28

DOI:10.1073/pnas.0701904104

The fluorescence of bacterial cells expressing a variant (GFPm) of the green fluorescent protein (GFP) was reduced to background levels by global replacement of the leucine residues of GFPm by 5,5,5-trifluoroleucine. Eleven rounds of random mutagenesis and screening via fluorescence-activated cell sorting yielded a GFP mutant containing 20 amino acid substitutions. The mutant protein in fluorinated form showed improved folding efficiency both in vivo and in vitro, and the median fluorescence of cells expressing the fluorinated protein was improved ≈650-fold in comparison to that of cells expressing fluorinated GFPm. The success of this approach demonstrates the feasibility of engineering functional proteins containing many copies of abiological amino acid constituents.

Evolving Proteins of Novel Composition

Co-reporter:Jin Kim Montclare

Angewandte Chemie International Edition 2006 Volume 45(Issue 27) pp:

Publication Date(Web):8 JUN 2006

DOI:10.1002/anie.200600088

Changing its nature: Global incorporation of noncanonical amino acids can alter the behavior of proteins in useful ways. In some cases, however, replacement of natural amino acids by noncanonical analogues (blue bars in picture) can cause loss of protein stability. After several generations, functional proteins of non-natural composition were prepared through residue-specific incorporation combined with directed evolution.

Stabilization of bzip Peptides through Incorporation of Fluorinated Aliphatic Residues

Co-reporter:Soojin Son;I. Caglar Tanrikulu Dr.

ChemBioChem 2006 Volume 7(Issue 8) pp:

Publication Date(Web):7 JUN 2006

DOI:10.1002/cbic.200500420

Two fluorinated amino acids, 5,5,5-trifluoroisoleucine (5TFI) and (2S,3R)-4,4,4-trifluorovaline (4TFV), which have been shown to serve as isoleucine surrogates in protein synthesis in Escherichia coli, have been incorporated in vivo into basic leucine zipper (bzip) peptides derived from GCN4. The extents of residue-specific incorporation of 5TFI and 4TFV were 90 and 88 %, respectively, of the encoded isoleucine residues, as evidenced by MALDI mass spectrometry and amino acid analysis. Both circular dichroism and equilibrium sedimentation studies of the fluorinated bzip peptides indicated preservation of secondary and higher-order protein structure. Thermal-denaturation experiments showed an increase of 27 °C in melting temperature when isoleucine was replaced by 5TFI. However, the T_m of the peptide containing 4TFV was increased by only 4 °C over that of the peptide containing valine. Similar trends were observed in chemical denaturation studies in which ΔΔG_unfold in water was determined to be 2.1 or 0.3 kcal mol⁻¹ upon incorporation of 5TFI or 4TFV, respectively. When the fluorinated peptides were tested for DNA binding, both their affinity and specificity were similar to those of the respective hydrogenated peptides. These results suggest that fluorinated amino acids, even when introduced into the same positions, can have markedly different effects on the physical properties of proteins, while having little impact on secondary and higher-order structure.

Fluorescence Visualization of Newly Synthesized Proteins in Mammalian Cells

Co-reporter:Kimberly E. Beatty;Julie C. Liu Dr.;Fang Xie;Daniela C. Dieterich Dr.;Erin M. Schuman ;Qian Wang

Angewandte Chemie International Edition 2006 Volume 45(Issue 44) pp:

Publication Date(Web):11 OCT 2006

DOI:10.1002/anie.200602114

Playing tag: Noncanonical amino acid tagging enables the selective fluorescent visualization of newly synthesized proteins in mammalian cells (see the picture). Susceptibility to tagging is determined by the spatial and temporal character of the protein synthesis, thus providing a complement to methods which identify relevant members of the proteome.

Stereoselective Incorporation of an Unsaturated Isoleucine Analogue into a Protein Expressed in E. coli

Co-reporter:Marissa L. Mock;Thierry Michon ;Jan C. M. van Hest

ChemBioChem 2006 Volume 7(Issue 1) pp:

Publication Date(Web):5 JAN 2006

DOI:10.1002/cbic.200500201

The unsaturated amino acid 2-amino-3-methyl-4-pentenoic acid (E-Ile) was prepared in the form of its (2S,3S),(2R,3R) and (2S,3R),(2R,3S) stereoisomeric pairs. The translational activities of SS-E-Ile and SR-E-Ile were assessed in an E. coli strain rendered auxotrophic for isoleucine. SS-E-Ile was incorporated into the test protein mouse dihydrofolate reductase (mDHFR) in place of isoleucine at a rate of up to 72 %; SR-E-Ile yielded no conclusive evidence for incorporation. ATP/PP_iexchange assays indicated that SS-E-Ile was activated by the isoleucyl-tRNA synthetase at a rate comparable to that characteristic of isoleucine; SR-E-Ile was activated approximately 100-times more slowly than SS-E-Ile.

Discovery of aminoacyl-tRNA synthetase activity through cell-surface display of noncanonical amino acids

Co-reporter:A. James Link;Mandy K. S. Vink;Nicholas J. Agard;Jennifer A. Prescher;Carolyn R. Bertozzi;

Proceedings of the National Academy of Sciences 2006 103(27) pp:10180-10185

Publication Date(Web):June 26, 2006

DOI:10.1073/pnas.0601167103

The incorporation of noncanonical amino acids into recombinant proteins in Escherichia coli can be facilitated by the introduction of new aminoacyl-tRNA synthetase activity into the expression host. We describe here a screening procedure for the identification of new aminoacyl-tRNA synthetase activity based on the cell surface display of noncanonical amino acids. Screening of a saturation mutagenesis library of the E. coli methionyl-tRNA synthetase (MetRS) led to the discovery of three MetRS mutants capable of incorporating the long-chain amino acid azidonorleucine into recombinant proteins with modest efficiency. The Leu-13 → Gly (L13G) mutation is found in each of the three MetRS mutants, and the MetRS variant containing this single mutation is highly efficient in producing recombinant proteins that contain azidonorleucine.

Alternative Translations of a Single RNA Message: An Identity Switch of (2S,3R)-4,4,4-Trifluorovaline between Valine and Isoleucine Codons

Co-reporter:Pin Wang;Alfio Fichera;Krishna Kumar

Angewandte Chemie 2004 Volume 116(Issue 28) pp:

Publication Date(Web):21 JUN 2004

DOI:10.1002/ange.200454036

Bei der Übersetzung verändert: Ein bakterieller Expressionswirt wurde so konzipiert, dass eine einzige RNA-Botschaft abhängig von den relativen Geschwindigkeiten konkurrierender Aminoacylierungsreaktionen unterschiedlich gelesen wird. (2S,3R)-4,4,4-Trifluorvalin kann Isoleucin- oder Valincodons zugeordnet werden, je nachdem ob der bakterielle Wirt die Isoleucyl- oder die Valyl-tRNA-Synthetase (IleRS bzw. ValRS; siehe Schema) überexprimiert.

Alternative Translations of a Single RNA Message: An Identity Switch of (2S,3R)-4,4,4-Trifluorovaline between Valine and Isoleucine Codons

Co-reporter:Pin Wang;Alfio Fichera;Krishna Kumar

Angewandte Chemie International Edition 2004 Volume 43(Issue 28) pp:

Publication Date(Web):21 JUN 2004

DOI:10.1002/anie.200454036

Changed in translation: Bacterial expression hosts can be engineered so that a single RNA message can be read in different ways depending on the relative rates of competing aminoacylation reactions. The (2S,3R)-4,4,4-trifluorovaline can be assigned either to isoleucine or to valine codons according to whether the bacterial host overexpresses the isoleucyl- or the valyl-tRNA synthetase (IleRS and ValRS, respectively; see scheme).

Endothelial cell adhesion to the fibronectin CS5 domain in artificial extracellular matrix proteins

Co-reporter:Sarah C. Heilshorn, Kathleen A. DiZio, Eric R. Welsh, David A. Tirrell

Biomaterials 2003 Volume 24(Issue 23) pp:4245-4252

Publication Date(Web):October 2003

DOI:10.1016/S0142-9612(03)00294-1

This study examines the spreading and adhesion of human umbilical vein endothelial cells (HUVEC) on artificial extracellular matrix (aECM) proteins containing sequences derived from elastin and fibronectin. Three aECM variants were studied: aECM 1 contains lysine residues periodically spaced within the protein sequence and three repeats of the CS5 domain of fibronectin, aECM 2 contains periodically spaced lysines and three repeats of a scrambled CS5 sequence, and aECM 3 contains lysines at the protein termini and five CS5 repeats. Comparative cell binding and peptide inhibition assays confirm that the tetrapeptide sequence REDV is responsible for HUVEC adhesion to aECM proteins that contain the CS5 domain. Furthermore, more than 60% of adherent HUVEC were retained on aECM 1 after exposure to physiologically relevant shear stresses (⩽100 dynes/cm2). Finally, the levels of thrombogenic markers (tissue plasminogen activator and plasminogen activator inhibitor–1) secreted by HUVEC monolayers on aECM 1 were found to be similar to those secreted by HUVEC monolayers cultured on fibronectin. These characteristics, along with the physical strength and elasticity of crosslinked films prepared from these materials, make aECM proteins promising candidates for application in small-diameter vascular grafts.

Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation

Co-reporter:Kristi L. Kiick;Eliana Saxon;Carolyn R. Bertozzi

PNAS 2002 Volume 99 (Issue 1 ) pp:19-24

Publication Date(Web):2002-01-08

DOI:10.1073/pnas.012583299

The introduction of chemically unique groups into proteins by means of non-natural amino acids has numerous applications in protein engineering and functional studies. One method to achieve this involves the utilization of a non-natural amino acid by the cell's native translational apparatus. Here we demonstrate that a methionine surrogate, azidohomoalanine, is activated by the methionyl-tRNA synthetase of Escherichia coli and replaces methionine in proteins expressed in methionine-depleted bacterial cultures. We further show that proteins containing azidohomoalanine can be selectively modified in the presence of other cellular proteins by means of Staudinger ligation with triarylphosphine reagents. Incorporation of azide-functionalized amino acids into proteins in vivo provides opportunities for protein modification under native conditions and selective labeling of proteins in the intracellular environment.

Biosynthesis of Proteins Incorporating a Versatile Set of Phenylalanine Analogues

Co-reporter:Kent Kirshenbaum Dr.;Isaac S. Carrico

ChemBioChem 2002 Volume 3(Issue 2-3) pp:

Publication Date(Web):7 MAR 2002

DOI:10.1002/1439-7633(20020301)3:2/3<235::AID-CBIC235>3.0.CO;2-7

Unnatural amino acids with useful chemical functionality can replace phenylalanine in bacterial proteins. Coexpression of a promiscuous phenylalanine-tRNA synthetase mutant enables the synthesis of target proteins bearing iodophenyl, cyanophenyl, ethynylphenyl, azidophenyl, and pyridyl groups (see general structures). Proteins incorporating the analogues have a range of potential applications, including Pd-mediated conjugation (R=CCH), photoaffinity labeling (R=N₃), X-ray phasing (R=I), and novel metal coordination (R=pyridyl).

Chemistry at the crossroads

Co-reporter:M Reza Ghadiri, David A Tirrell

Current Opinion in Chemical Biology 2000 Volume 4(Issue 6) pp:661-662

Publication Date(Web):1 December 2000

DOI:10.1016/S1367-5931(00)00156-3

Expanding the Scope of Protein Biosynthesis by Altering the Methionyl-tRNA Synthetase Activity of a Bacterial Expression Host

Co-reporter:Kristi L. Kiick;Jan C. M. van Hest;David A. Tirrell

Angewandte Chemie 2000 Volume 112(Issue 12) pp:

Publication Date(Web):14 JUN 2000

DOI:10.1002/1521-3757(20000616)112:12<2232::AID-ANGE2232>3.0.CO;2-0

Cell-selective proteomics for biological discovery

Co-reporter:Shannon E Stone, Weslee S Glenn, Graham D Hamblin, David A Tirrell