James R Swartz

Stanford University, Stanford, California, United States

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Publications (112)520.71 Total impact

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    ABSTRACT: Three maturase enzymes-HydE, HydF, and HydG-synthesize and insert the organometallic component of the [FeFe]-hydrogenase active site (the H cluster). HydG generates the first organometallic intermediates in this process, ultimately producing an [Fe(CO)2(CN)] complex. A limitation in understanding the mechanism by which this complex forms has been uncertainty regarding the precise metallocluster composition of HydG that comprises active enzyme. We herein show that the HydG auxiliary cluster must bind both l-cysteine and a dangler Fe in order to generate the [Fe(CO)2(CN)] product. These findings support a mechanistic framework in which a [(Cys)Fe(CO)2(CN)](-) species is a key synthon in H-cluster maturation.
    No preview · Article · Jan 2016 · Journal of the American Chemical Society
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    Full-text · Dataset · Jan 2016
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    Yuan Lu · James R. Swartz
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    ABSTRACT: We report the development of a well-defined flagellin-based nanoparticle stimulator and also provide a new mechanism of action model explaining how flagellin-triggered innate immunity has evolved to favor localized rather than potentially debilitating systemic immune stimulation. Cell-free protein synthesis (CFPS) was used to facilitate mutational analysis and precisely orientated display of flagellin on Hepatitis B core (HBc) protein virus-like particles (VLPs). The need for product stability and an understanding of mechanism of action motivated investigations indicating that the D0 domain of flagellin is sensitive to amino acid sequence independent hydrolysis – apparently due to the need for structural flexibility during natural flagellin polymerization. When D0-stabilized flagellin was attached to HBc VLPs with the D0 domain facing outward, flagellin’s tendency to polymerize caused the VLPs to precipitate. However, attaching the D0 domain to the VLP surface produced a stable nanoparticle adjuvant. Surprisingly, attaching only 2 flagellins per VLP provided the same 1 pM potency as did VLPs with about 33 attached flagellins suggesting that the TLR5 receptor is highly effective in delivering its intracellular signal. These observations suggest that flagellin’s protease sensitivity, tendency to aggregate, and very high affinity for TLR5 receptors limit its systemic distribution to favor localized immune stimulation.
    Preview · Article · Jan 2016 · Scientific Reports
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    ABSTRACT: Hydrogenase enzymes catalyze the rapid and reversible interconversion of H2 with protons and electrons. The active site of the [FeFe] hydrogenase is the H cluster, which consists of a [4Fe-4S]H subcluster linked to an organometallic [2Fe]H subcluster. Understanding the biosynthesis and catalytic mechanism of this structurally unusual active site will aid in the development of synthetic and biological hydrogenase catalysts for applications in solar fuel generation. The [2Fe]H subcluster is synthesized and inserted by three maturase enzymes-HydE, HydF, and HydG-in a complex process that involves inorganic, organometallic, and organic radical chemistry. HydG is a member of the radical S-adenosyl-l-methionine (SAM) family of enzymes and is thought to play a prominent role in [2Fe]H subcluster biosynthesis by converting inorganic Fe(2+), l-cysteine (Cys), and l-tyrosine (Tyr) into an organometallic [(Cys)Fe(CO)2(CN)](-) intermediate that is eventually incorporated into the [2Fe]H subcluster. In this Forum Article, the mechanism of [2Fe]H subcluster biosynthesis is discussed with a focus on how this key [(Cys)Fe(CO)2(CN)](-) species is formed. Particular attention is given to the initial metallocluster composition of HydG, the modes of substrate binding (Fe(2+), Cys, Tyr, and SAM), the mechanism of SAM-mediated Tyr cleavage to CO and CN(-), and the identification of the final organometallic products of the reaction.
    No preview · Article · Dec 2015 · Inorganic Chemistry
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    Kunal K. Mehta · Niklaus H. Evitt · James R. Swartz
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    ABSTRACT: We have developed a mixture of enzymes and chemicals that completely lyse cyanobacteria. Since the treatment involves only readily-available chemicals and simple proteins that degrade the components of the cyanobacterial cell wall, it can easily be used in high-throughput applications requiring lysis for subsequent intracellular measurements. Our lysis technique consistently enables complete lysis of several different cyanobacterial strains, and we demonstrated that DNA, mRNA, and proteins are preserved in the lysates. Chemical lysis can be superior to existing techniques because of its convenience, reliability, and amenability to a variety of downstream applications. Electronic supplementary material The online version of this article (doi:10.1186/s13036-015-0007-y) contains supplementary material, which is available to authorized users.
    Full-text · Article · Dec 2015 · Journal of Biological Engineering
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    ABSTRACT: Virus-like particles (VLPs) have been extensively explored as nanoparticle vehicles for many applications in biotechnology (e.g., vaccines, drug delivery, imaging agents, biocatalysts). However, amino acid sequence plasticity relative to subunit expression and nanoparticle assembly has not been explored. Whereas the hepatitis B core protein (HBc) VLP appears to be the most promising model for fundamental and applied studies; particle instability, antigen fusion limitations, and intrinsic immunogenicity have limited its development. Here, we apply Escherichia coli-based cell-free protein synthesis (CFPS) to rapidly produce and screen HBc protein variants that still self-assemble into VLPs. To improve nanoparticle stability, artificial covalent disulfide bridges were introduced throughout the VLP. Negative charges on the HBc VLP surface were then reduced to improve surface conjugation. However, removal of surface negative charges caused low subunit solubility and poor VLP assembly. Solubility and assembly as well as surface conjugation were greatly improved by transplanting a rare spike region onto the common shell structure. The newly stabilized and extensively modified HBc VLP had almost no immunogenicity in mice, demonstrating great promise for medical applications. This study introduces a general paradigm for functional improvement of complex protein assemblies such as VLPs. This is the first study, to our knowledge, to systematically explore the sequence plasticity of viral capsids as an approach to defining structure function relationships for viral capsid proteins. Our observations on the unexpected importance of the HBc spike tip charged state may also suggest new mechanistic routes toward viral therapeutics that block capsid assembly.
    No preview · Article · Oct 2015 · Proceedings of the National Academy of Sciences
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    ABSTRACT: Hydrogenases catalyze the redox interconversion of protons and H2, an important reaction for a number of metabolic processes and for solar fuel production. In FeFe hydrogenases, catalysis occurs at the H cluster, a metallocofactor comprising a [4Fe-4S]H subcluster coupled to a [2Fe]H subcluster bound by CO, CN(-), and azadithiolate ligands. The [2Fe]H subcluster is assembled by the maturases HydE, HydF, and HydG. HydG is a member of the radical S-adenosyl-l-methionine family of enzymes that transforms Fe and l-tyrosine into an [Fe(CO)2(CN)] synthon that is incorporated into the H cluster. Although it is thought that the site of synthon formation in HydG is the "dangler" Fe of a [5Fe] cluster, many mechanistic aspects of this chemistry remain unresolved including the full ligand set of the synthon, how the dangler Fe initially binds to HydG, and how the synthon is released at the end of the reaction. To address these questions, we herein show that l-cysteine (Cys) binds the auxiliary [4Fe-4S] cluster of HydG and further chelates the dangler Fe. We also demonstrate that a [4Fe-4S]aux[CN] species is generated during HydG catalysis, a process that entails the loss of Cys and the [Fe(CO)2(CN)] fragment; on this basis, we suggest that Cys likely completes the coordination sphere of the synthon. Thus, through spectroscopic analysis of HydG before and after the synthon is formed, we conclude that Cys serves as the ligand platform on which the synthon is built and plays a role in both Fe(2+) binding and synthon release.
    Full-text · Article · Aug 2015 · Proceedings of the National Academy of Sciences
  • Franklin Lu · Phillip R. Smith · Kunal Mehta · James R. Swartz
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    ABSTRACT: Sustainable production of biochemicals from cellulosic biomass is an attractive alternative to chemical production from fossil fuels. We describe the further development of a three protein synthetic pathway to convert NADPH and H+ to hydrogen using a ferredoxin-NADP+ reductase, a ferredoxin, and the [FeFe] hydrogenase from Clostridium pasteurianum at a rate greater than 14 mmol H2 L−1 hr−1 using natural enzymes. We also demonstrate the feasibility of coupling this pathway to a cell-free extract to convert glucose to hydrogen in a potentially cost effective manner. Both of these accomplishments serve as the basis for further engineering to optimize both the yield and productivity of a low-cost cell-free process for the production of highly reduced biochemicals.
    No preview · Article · Aug 2015 · International Journal of Hydrogen Energy
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    ABSTRACT: Hydrogenases use complex metal cofactors to catalyze the reversible formation of hydrogen. In [FeFe]-hydrogenases, the H-cluster cofactor includes a diiron subcluster containing azadithiolate, three CO, and two CN(-) ligands. During the assembly of the H cluster, the radical S-adenosyl methionine (SAM) enzyme HydG lyses the substrate tyrosine to yield the diatomic ligands. These diatomic products form an enzyme-bound Fe(CO)x(CN)y synthon that serves as a precursor for eventual H-cluster assembly. To further elucidate the mechanism of this complex reaction, we report the crystal structure and EPR analysis of HydG. At one end of the HydG (βα)8 triosephosphate isomerase (TIM) barrel, a canonical [4Fe-4S] cluster binds SAM in close proximity to the proposed tyrosine binding site. At the opposite end of the active-site cavity, the structure reveals the auxiliary Fe-S cluster in two states: one monomer contains a [4Fe-5S] cluster, and the other monomer contains a [5Fe-5S] cluster consisting of a [4Fe-4S] cubane bridged by a μ2-sulfide ion to a mononuclear Fe(2+) center. This fifth iron is held in place by a single highly conserved protein-derived ligand: histidine 265. EPR analysis confirms the presence of the [5Fe-5S] cluster, which on incubation with cyanide, undergoes loss of the labile iron to yield a [4Fe-4S] cluster. We hypothesize that the labile iron of the [5Fe-5S] cluster is the site of Fe(CO)x(CN)y synthon formation and that the limited bonding between this iron and HydG may facilitate transfer of the intact synthon to its cognate acceptor for subsequent H-cluster assembly.
    Preview · Article · Jan 2015 · Proceedings of the National Academy of Sciences
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    ABSTRACT: The two cyanide ligands in the assembled cluster of [FeFe] hydrogenase originate from exogenous L-tyrosine. Using selectively-labeled tyrosine substrates, the cyanides were isotopically-labeled via a recently developed in vitro maturation procedure allowing. advanced electron par-amagnetic resonance techniques to probe the electronic structure of the catalytic core of the enzyme. The ratio of the isotropic 13C hyperfine interactions for the two CN- ligands-a reporter of spin density on their respective coordinating iron ions-collapses from ≈5.8 for the Hox form of hydrogenase to <2 for the CO-inhibited form. Additionally, when the maturation was carried out using [15N]-tyrosine, no features previously ascribed to the nitrogen of the bridging dithiolate ligand were observed suggesting that this bridge is not sourced from tyrosine.
    Preview · Article · Aug 2014 · Journal of the American Chemical Society
  • Jon M Kuchenreuther · Stacey A Shiigi · James R Swartz
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    ABSTRACT: Many organometallic cofactors are highly complex and require multiple accessory proteins for both their assembly and transfer to a target protein. A cell-free system in which the biosynthetic pathway for a prosthetic group has been fully or even partially reconstructed enables investigations of the reaction sequence as well as the cofactor itself. As a model for the in vitro assembly of protein-bound metal centers, we describe a procedure for the cell-free synthesis of the H-cluster in the context of producing purified and active [FeFe] hydrogenase samples for spectroscopic studies. In general terms, this in vitro system is a combination of non-purified accessory proteins, exogenous substrates, and purified hydrogenase apoprotein. We also describe methods for making the required components used in the cell-free system. Specifically, these procedures include anaerobic expression of heterologous metalloproteins in Escherichia coli, anaerobic cell lysate production, and anaerobic metalloprotein purification using Strep-Tactin(®) chromatography.
    No preview · Article · Mar 2014 · Methods in molecular biology (Clifton, N.J.)
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    ABSTRACT: Three iron-sulfur proteins--HydE, HydF, and HydG--play a key role in the synthesis of the [2Fe](H) component of the catalytic H-cluster of FeFe hydrogenase. The radical S-adenosyl-L-methionine enzyme HydG lyses free tyrosine to produce p-cresol and the CO and CN(-) ligands of the [2Fe](H) cluster. Here, we applied stopped-flow Fourier transform infrared and electron-nuclear double resonance spectroscopies to probe the formation of HydG-bound Fe-containing species bearing CO and CN(-) ligands with spectroscopic signatures that evolve on the 1- to 1000-second time scale. Through study of the (13)C, (15)N, and (57)Fe isotopologs of these intermediates and products, we identify the final HydG-bound species as an organometallic Fe(CO)2(CN) synthon that is ultimately transferred to apohydrogenase to form the [2Fe](H) component of the H-cluster.
    No preview · Article · Jan 2014 · Science
  • Yuan Lu · John P Welsh · James R Swartz
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    ABSTRACT: The rapid dissemination of the 2009 pandemic H1N1 influenza virus emphasizes the need for universal influenza vaccines that would broadly protect against multiple mutated strains. Recent efforts have focused on the highly conserved hemagglutinin (HA) stem domain, which must undergo a significant conformational change for effective viral infection. Although the production of isolated domains of multimeric ectodomain proteins has proven difficult, we report a method to rapidly produce the properly folded HA stem domain protein from influenza virus A/California/05/2009 (H1N1) by using Escherichia coli-based cell-free protein synthesis and a simple refolding protocol. The T4 bacteriophage fibritin foldon placed at the C terminus of the HA stem domain induces trimer formation. Placing emphasis on newly exposed protein surfaces, several hydrophobic residues were mutated, two polypeptide segments were deleted, and the number of disulfide bonds in each monomer was reduced from four to two. High pH and Brij 35 detergent emerged as the most beneficial factors for improving the refolding yield. To stabilize the trimer of the HA stem-foldon fusion, new intermolecular disulfide bonds were finally introduced between foldon monomers and between stem domain monomers. The correct immunogenic conformation of the stabilized HA stem domain trimer was confirmed by using antibodies CR6261, C179, and FI6 that block influenza infection by binding to the HA stem domain trimer. These results suggest great promise for a broadly protective vaccine and also demonstrate a unique approach for producing individual domains of complex multimeric proteins.
    No preview · Article · Jan 2014 · Proceedings of the National Academy of Sciences
  • Cem Albayrak · James R Swartz
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    ABSTRACT: We report the synthesis of active polymers of super-folder green fluorescent protein (sfGFP) in one step using Click chemistry. Up to six copies of the non-natural amino acids (nnAAs) p¬-azido-L-phenylalanine (pAzF) or p-propargyloxy-L-phenylalanine (pPaF) were site-specifically inserted into sfGFP by cell-free protein synthesis (CFPS). sfGFP containing two or three copies of these nnAAs were coupled by copper-catalyzed azide-alkyne cycloaddition to synthesize linear or branched protein polymers, respectively. The protein polymers retained ≥63% of their specific activity (i.e. fluorescence) after coupling. Polymerization of a concentrated solution of triply-substituted sfGFP resulted in fluorescent macromolecular particles. Our method can be generalized to synthesize polymers of a protein or co-polymers of any two or more proteins, and the conjugation sites can be determined exactly by standard genetic manipulation. Polymers of proteins and small molecules can also be created with this technology to make a new class of scaffolds or biomaterials.
    No preview · Article · Nov 2013 · ACS Synthetic Biology
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    ABSTRACT: [FeFe] hydrogenases rapidly produce H2, which has stimulated interest in their application for renewable energy technologies. In addition, their catalytic iron center termed the H-cluster has inspired the design of many synthetic catalysts, albeit few with ability to evolve H2, and a better understanding of how the H-cluster is made could further the development of H2-based technologies. HydE, HydF, and HydG are three enzymes that assemble the H-cluster and activate [FeFe] hydrogenases, and recent work has shed light on H-cluster biosynthesis such as the formation of the cofactor’s CO and CN ligands from free tyrosine. Nonetheless, challenges with experimentally probing the reaction pathway have left several aspects unresolved due in part to the O2 sensitive nature of both the maturation proteins as well as the H-cluster. In this work, we combine multiple techniques to study the HydG maturase and its radical SAM chemistry. We use rapid freeze quench methods along with EPR spectroscopy in order to examine the radical SAM reaction and determine the kinetics of its products. In doing so, we identify a novel benzylomethyl-4-olate (BM4O) radical species that derives from free tyrosine radical, which presumably forms during the generation of CO and CN– molecules. We also use stopped-flow infrared (SF-FTIR) spectroscopy to examine the HydG chemistry in situ. Specifically, we observe the concurrent formation of CO and CN– species during the radical SAM reaction, which we attribute to a HydG-specific iron compound coordinated by at least two CO and one CN–. We then utilize our cell-free hydrogenase maturation platform in conjunction with ENDOR spectroscopy to trace 57Fe nuclei from HydG to the [FeFe] hydrogenase, providing conclusive evidence that the HydG maturase synthesizes an iron compound precursor of the H-cluster. From this work, and in contrast to the current paradigm, we present a new reaction sequence for the assembly of the H-cluster and the maturation of [FeFe] hydrogenases.
    No preview · Conference Paper · Nov 2013
  • Jamin Koo · Tim Schnabel · Stacey Shiigi · James R. Swartz
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    ABSTRACT: Hydrogen (H2) has the potential to become a sustainable fuel depending on its production process. One possible process involves biological H2 evolution using enzymes known as hydrogenases. The maximum catalytic activity of [FeFe] hydrogenases can produce H2 at a turn over frequency (TOF) in the order of 10,000 s-1. However, these hydrogenases are highly sensitive to oxygen (O2), and it is not currently possible to combine [FeFe] hydrogenases with solar water splitting where O2 is a byproduct. For these reasons, there is great interest in using protein engineering to evolve O2-tolerance in these enzymes. Previous work has discovered a mutant of the Clostridium pasteurianum [FeFe] hydrogenase (CpI) with enhanced O2-tolerance during H2 consumption. However, tolerance was not observed when the mutant was producing hydrogen. We therefore sought to develop a high-throughput assay to screen for oxygen tolerance during H2 production. It has been previously shown that hydrogen molecules can undergo a series of reactions in thin films of Pd-Pt/WO3 to reduce the WO3 layer. The reduction is accompanied by a change in color. Our screen uses such a sensor to monitor hydrogen production in the individual wells of a 96-wells plate. A custom fabricated elastomeric gasket provides a seal between the sensor plate and a 96-well plate. A standard CCD camera provides time-lapse images and image analysis software gives a quantitative assessment of the rate of color formation. The device is also designed so that gases of known H2 partial pressure can be injected below the 96-wells plate for calibration purposes. We will describe the device and show results from initial evaluation of mutated hydrogenases.
    No preview · Conference Paper · Nov 2013
  • James R. Swartz

    No preview · Article · Nov 2013 · Chemical Engineering Progress
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    ABSTRACT: The radical S-adenosylmethionine (SAM) enzyme HydG lyses free l-tyrosine to produce CO and CN− for the assembly of the catalytic H cluster of FeFe hydrogenase. We used electron paramagnetic resonance spectroscopy to detect and characterize HydG reaction intermediates generated with a set of 2H, 13C, and 15N nuclear spin-labeled tyrosine substrates. We propose a detailed reaction mechanism in which the radical SAM reaction, initiated at an N-terminal 4Fe-4S cluster, generates a tyrosine radical bound to a C-terminal 4Fe-4S cluster. Heterolytic cleavage of this tyrosine radical at the Cα-Cβ bond forms a transient 4-oxidobenzyl (4OB•) radical and a dehydroglycine bound to the C-terminal 4Fe-4S cluster. Electron and proton transfer to this 4OB• radical forms p-cresol, with the conversion of this dehydroglycine ligand to Fe-bound CO and CN−, a key intermediate in the assembly of the 2Fe subunit of the H cluster.
    No preview · Article · Oct 2013 · Science
  • Yuan Lu · John P Welsh · Wei Chan · James R Swartz
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    ABSTRACT: Bacterial flagellin has been explored as a potential vaccine adjuvant for enhancing immune responses. In this paper, we describe Escherichia coli-based cell-free protein synthesis (CFPS) as a method to rapidly produce soluble phase 1 flagellin (FliC) protein from Salmonella typhimurium. The yield was about 300 µg/ml and the product had high affinity for the TLR5 receptor (EC50 = 2.4 ± 1.4 pM). The flagellin coding sequence was first optimized for cell-free expression. We then found that the D0 domain at the C-terminus of flagellin was susceptible to proteolytic degradation in the CFPS system. Proteolysis was reduced by protease inhibitors, the use of protease-deficient cell extracts or deletion of the flagellin D0 domain. A human Toll-Like Receptor 5 (hTLR5)-specific bioactivity analysis of purified flagellin demonstrated that, although the D0 domain is far from the TLR5 recognition region, it is important for flagellin bioactivity. We next incorporated a non-natural amino acid displaying an alkyne moiety into flagellin using the CFPS system and attached flagellin to hepatitis B core virus-like particle (VLP) using bioorthogonal azide-alkyne cycloaddition reaction. The ordered and oriented VLP display of flagellin increased its specific TLR5 stimulation activity by approximately 10-fold. Biotechnol. Bioeng. © 2013 Wiley Periodicals, Inc.
    No preview · Article · Aug 2013 · Biotechnology and Bioengineering
  • Cem Albayrak · K. C. Jones · James R. Swartz
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    ABSTRACT: Cell-free protein synthesis (CFPS) has emerged as a practical method for producing a broad variety of proteins. In addition, the direct accessibility to the reaction environment makes CFPS particularly suitable as a learning vehicle for fundamental biological concepts. Here, we describe its implementation as a teaching tool for a high school laboratory course. Ninety students in a biotechnology class used CFPS to study the effects of the concentrations of amino acids, cell extract, DNA, and the energy source on accumulation of active super-folder green fluorescent protein. Students estimated product concentrations simply by comparing solution colors to a printed green color gradient. This simple and inexpensive method allows for immediate measurements, and 26 of the 30 groups observed measurable product concentrations within 60 min. These student-generated data were then discussed to illustrate concepts of data analysis such as outliers and standard deviation. We also combined the laboratory experience with a visit to a university campus that included a laboratory tour and a college-style lecture. Our overall objective was to excite the students about the scientific enterprise and to instill a sense of personal relevance and attainability so that these students could realistically consider technical careers.
    No preview · Article · Jul 2013 · Journal of Science Education and Technology