Recursos de colección

Caltech Authors (171.365 recursos)

Repository of works by Caltech published authors.

Group = Caltech Center for Environmental Microbial Interactions (CEMI)

Mostrando recursos 1 - 20 de 72

  1. Catalytic iron-carbene intermediate revealed in a cytochrome c carbene transferase

    Lewis, Russell D.; Garcia-Borràs, Marc; Chalkley, Matthew J.; Buller, Andrew R.; Houk, K. N.; Kan, S. B. Jennifer; Arnold, Frances H.
    Recently, heme proteins have been discovered and engineered by directed evolution to catalyze chemical transformations that are biochemically unprecedented. Many of these nonnatural enzyme-catalyzed reactions are assumed to proceed through a catalytic iron porphyrin carbene (IPC) intermediate, although this intermediate has never been observed in a protein. Using crystallographic, spectroscopic, and computational methods, we have captured and studied a catalytic IPC intermediate in the active site of an enzyme derived from thermostable Rhodothermus marinus (Rma) cytochrome c. High-resolution crystal structures and computational methods reveal how directed evolution created an active site for carbene transfer in an electron transfer protein and...

  2. Metabolic interactions between dynamic bacterial subpopulations

    Rosenthal, Adam Z.; Qi, Yutao; Hormoz, Sahand; Park, Jin; Hsin-Jung Li, Sophia; Elowitz, Michael
    Individual microbial species are known to occupy distinct metabolic niches within multi-species communities. However, it has remained largely unclear whether metabolic specialization can similarly occur within a clonal bacterial population. More specifically, it is not clear what functions such specialization could provide and how specialization could be coordinated dynamically. Here, we show that exponentially growing Bacillus subtilis cultures divide into distinct interacting metabolic subpopulations, including one population that produces acetate, and another population that differentially expresses metabolic genes for the production of acetoin, a pH-neutral storage molecule. These subpopulations exhibit distinct growth rates and dynamic interconversion between states. Furthermore, acetate...

  3. Fe-Mediated Nitrogen Fixation with a Metallocene Mediator: Exploring pK_a Effects and Demonstrating Electrocatalysis

    Chalkley, Matthew J.; Del Castillo, Trevor J.; Matson, Benjamin D.; Peters, Jonas C.
    Substrate selectivity in reductive multi-electron/proton catalysis with small molecules such as N_2, CO_2, and O_2 is a major challenge for catalyst design, especially where the competing hydrogen evolution reaction (HER) is thermodynamically and kinetically competent. In this study, we investigate how the selectivity of a tris(phosphine)borane iron(I) catalyst, P_3^BFe^+, for catalyzing the nitrogen reduction reaction (N_2RR, N_2-to-NH_3 conversion) versus HER changes as a function of acid pK_a. We find that there is a strong correlation between pKa and N_2RR efficiency. Stoichiometric studies indicate that the anilinium triflate acids employed are only compatible with the formation of early stage intermediates of...

  4. Biomolecular Ultrasound and Sonogenetics

    Maresca, David; Lakshmanan, Anupama; Abedi, Mohamed; Bar-Zion, Avinoam; Farhadi, Arash; Lu, George J.; Szablowski, Jerzy O.; Wu, Di; Yoo, Sangjin; Shapiro, Mikhail G.
    Visualizing and modulating molecular and cellular processes occurring deep within living organisms is fundamental to our study of basic biology and disease. Currently, the most sophisticated tools available to dynamically monitor and control cellular events rely on light-responsive proteins, which are difficult to use outside of optically transparent model systems, cultured cells, or surgically accessed regions owing to strong scattering of light by biological tissue. In contrast, ultrasound is a widely used medical imaging and therapeutic modality that enables the observation and perturbation of internal anatomy and physiology but has historically had limited ability to monitor and control specific cellular...

  5. A Thermodynamic Model for Redox-Dependent Binding of Carbon Monoxide at Site-Differentiated, High Spin Iron Clusters

    Arnett, Charles H.; Chalkley, Matthew J.; Agapie, Theodor
    Binding of N_2 and CO by the FeMo-cofactor of nitrogenase depends on the redox level of the cluster, but the extent to which pure redox chemistry perturbs the affinity of high spin iron clusters for π-acids is not well understood. Here, we report a series of site-differentiated iron clusters which reversibly bind CO in redox states Fe^(II)_4 through Fe^(II)Fe^(III)_3. One electron redox events result in small changes in the affinity for (at most ~400-fold) and activation of CO (at most 28 cm^(-1) for ν_(CO)). The small influence of redox chemistry on the affinity of these high spin, valence-localized clusters for...

  6. Proteins, air and water: reporter genes for ultrasound and magnetic resonance imaging

    Lu, George J.; Farhadi, Arash; Mukherjee, Arnab; Shapiro, Mikhail G.
    A long-standing goal of molecular imaging is to visualize cellular function within the context of living animals, necessitating the development of reporter genes compatible with deeply penetrant imaging modalities such as ultrasound and magnetic resonance imaging (MRI). Until recently, no reporter genes for ultrasound were available, and most genetically encoded reporters for MRI were limited by metal availability or relatively low sensitivity. Here we review how these limitations are being addressed by recently introduced reporter genes based on air-filled and water-transporting biomolecules. We focus on gas-filled protein nanostructures adapted from buoyant microbes, which scatter sound waves, perturb magnetic fields and...

  7. Ultrastructure of Shewanella oneidensis MR-1 nanowires revealed by electron cryotomography

    Subramanian, Poorna; Pirbadian, Sahand; El-Naggar, Mohamed Y.; Jensen, Grant J.
    Bacterial nanowires have garnered recent interest as a proposed extracellular electron transfer (EET) pathway that links the bacterial electron transport chain to solid-phase electron acceptors away from the cell. Recent studies showed that Shewanella oneidensis MR-1 produces outer membrane (OM) and periplasmic extensions that contain EET components and hinted at their possible role as bacterial nanowires. However, their fine structure and distribution of cytochrome electron carriers under native conditions remained unclear, making it difficult to evaluate the potential electron transport (ET) mechanism along OM extensions. Here, we report high-resolution images of S. oneidensis OM extensions, using electron cryotomography (ECT). We...

  8. Stereoselective Enzymatic Synthesis of Heteroatom-Substituted Cyclopropanes

    Brandenberg, Oliver F.; Prier, Christopher K.; Chen, Kai; Knight, Anders M.; Wu, Zachary; Arnold, Frances H.
    The repurposing of hemoproteins for non-natural carbene transfer activities has generated enzymes for functions previously accessible only to chemical catalysts. With activities constrained to specific substrate classes, however, the synthetic utility of these new biocatalysts has been limited. To expand the capabilities of non-natural carbene transfer biocatalysis, we engineered variants of Cytochrome P450_(BM3) that catalyze the cyclopropanation of heteroatom-bearing alkenes, providing valuable nitrogen-, oxygen-, and sulfur-substituted cyclopropanes. Four or five active-site mutations converted a single parent enzyme into selective catalysts for the synthesis of both cis and trans heteroatom-substituted cyclopropanes, with high diastereoselectivities and enantioselectivities and up to 40 000 total...

  9. Evolution of Phototrophy in the Chloroflexi Phylum Driven by Horizontal Gene Transfer

    Ward, Lewis M.; Hemp, James; Shih, Patrick M.; McGlynn, Shawn E.; Fischer, Woodward W.
    The evolutionary mechanisms behind the extant distribution of photosynthesis is a point of substantial contention. Hypotheses range from the presence of phototrophy in the last universal common ancestor and massive gene loss in most lineages, to a later origin in Cyanobacteria followed by extensive horizontal gene transfer into the extant phototrophic clades, with intermediate scenarios that incorporate aspects of both end-members. Here, we report draft genomes of 11 Chloroflexi: the phototrophic Chloroflexia isolate Kouleothrix aurantiaca as well as 10 genome bins recovered from metagenomic sequencing of microbial mats found in Japanese hot springs. Two of these metagenome bins encode photrophic...

  10. Diverse engineered heme proteins enable stereodivergent cyclopropanation of unactivated alkenes

    Knight, Anders M.; Kan, S. B. Jennifer; Lewis, Russell D.; Brandenberg, Oliver F.; Chen, Kai; Arnold, Frances H.
    Developing catalysts that produce each stereoisomer of a desired product selectively is a longstanding synthetic challenge. Biochemists have addressed this challenge by screening nature’s diversity to discover enzymes that catalyze the formation of complementary stereoisomers. We show here that the same approach can be applied to a new-to-nature enzymatic reaction, alkene cyclopropanation via carbene transfer. By screening diverse native and engineered heme proteins, we identified globins and serine-ligated “P411” variants of cytochromes P450 with promiscuous activity for cyclopropanation of unactivated alkene substrates. We then enhanced their activities and stereoselectivities by directed evolution: just 1–3 rounds of site-saturation mutagenesis and screening...

  11. Improving membrane protein expression by optimizing integration efficiency

    Niesen, Michiel J. M.; Marshall, Stephen S.; Miller, Thomas F., III; Clemons, William M., Jr.
    The heterologous overexpression of integral membrane proteins in Escherichia coli often yields insufficient quantities of purifiable protein for applications of interest. The current study leverages a recently demonstrated link between co-translational membrane integration efficiency and protein expression levels to predict protein sequence modifications that improve expression. Membrane integration efficiencies, obtained using a coarse-grained simulation approach, robustly predicted effects on expression of the integral membrane protein TatC for a set of 140 sequence modifications, including loop-swap chimeras and single-residue mutations distributed throughout the protein sequence. Mutations that improve simulated integration efficiency were four-fold enriched with respect to improved experimentally observed expression...

  12. Improving membrane protein expression by optimizing integration efficiency

    Niesen, Michiel J. M.; Marshall, Stephen S.; Miller, Thomas F., III; Clemons, William M., Jr.
    The heterologous overexpression of integral membrane proteins in Escherichia coli often yields insufficient quantities of purifiable protein for applications of interest. The current study leverages a recently demonstrated link between co-translational membrane integration efficiency and protein expression levels to predict protein sequence modifications that improve expression. Membrane integration efficiencies, obtained using a coarse-grained simulation approach, robustly predicted effects on expression of the integral membrane protein TatC for a set of 140 sequence modifications, including loop-swap chimeras and single-residue mutations distributed throughout the protein sequence. Mutations that improve simulated integration efficiency were four-fold enriched with respect to improved experimentally observed expression...

  13. Improving membrane protein expression by optimizing integration efficiency

    Niesen, Michiel J. M.; Marshall, Stephen S.; Miller, Thomas F., III; Clemons, William M., Jr.
    The heterologous overexpression of integral membrane proteins in Escherichia coli often yields insufficient quantities of purifiable protein for applications of interest. The current study leverages a recently demonstrated link between co-translational membrane integration efficiency and protein expression levels to predict protein sequence modifications that improve expression. Membrane integration efficiencies, obtained using a coarse-grained simulation approach, robustly predicted effects on expression of the integral membrane protein TatC for a set of 140 sequence modifications, including loop-swap chimeras and single-residue mutations distributed throughout the protein sequence. Mutations that improve simulated integration efficiency were 4-fold enriched with respect to improved experimentally observed expression...

  14. Going Deeper: Biomolecular Tools for Acoustic and Magnetic Imaging and Control of Cellular Function

    Piraner, Dan I.; Farhadi, Arash; Davis, Hunter C.; Wu, Di; Maresca, David; Szablowski, Jerzy O.; Shapiro, Mikhail G.
    Most cellular phenomena of interest to mammalian biology occur within the context of living tissues and organisms. However, today’s most advanced tools for observing and manipulating cellular function, based on fluorescent or light-controlled proteins, work best in cultured cells, transparent model species, or small, surgically accessed anatomical regions. Their reach into deep tissues and larger animals is limited by photon scattering. To overcome this limitation, we must design biochemical tools that interface with more penetrant forms of energy. For example, sound waves and magnetic fields easily permeate most biological tissues, allowing the formation of images and delivery of energy for...

  15. Going Deeper: Biomolecular Tools for Acoustic and Magnetic Imaging and Control of Cellular Function

    Piraner, Dan I.; Farhadi, Arash; Davis, Hunter C.; Wu, Di; Maresca, David; Szablowski, Jerzy O.; Shapiro, Mikhail G.
    Most cellular phenomena of interest to mammalian biology occur within the context of living tissues and organisms. However, today’s most advanced tools for observing and manipulating cellular function, based on fluorescent or light-controlled proteins, work best in cultured cells, transparent model species, or small, surgically accessed anatomical regions. Their reach into deep tissues and larger animals is limited by photon scattering. To overcome this limitation, we must design biochemical tools that interface with more penetrant forms of energy. For example, sound waves and magnetic fields easily permeate most biological tissues, allowing the formation of images and delivery of energy for...

  16. Going Deeper: Biomolecular Tools for Acoustic and Magnetic Imaging and Control of Cellular Function

    Piraner, Dan I.; Farhadi, Arash; Davis, Hunter C.; Wu, Di; Maresca, David; Szablowski, Jerzy O.; Shapiro, Mikhail G.
    Most cellular phenomena of interest to mammalian biology occur within the context of living tissues and organisms. However, today’s most advanced tools for observing and manipulating cellular function, based on fluorescent or light-controlled proteins, work best in cultured cells, transparent model species, or small, surgically accessed anatomical regions. Their reach into deep tissues and larger animals is limited by photon scattering. To overcome this limitation, we must design biochemical tools that interface with more penetrant forms of energy. For example, sound waves and magnetic fields easily permeate most biological tissues, allowing the formation of images and delivery of energy for...

  17. Ultrastructure of Shewanella oneidensis MR-1 nanowires revealed by electron cryotomography

    Subramanian, Poorna; Pirbadian, Sahand; El-Naggar, Mohamed Y.; Jensen, Grant J.
    Bacterial nanowires have garnered recent interest as a proposed extracellular electron transfer (EET) pathway that links the bacterial electron transport chain to solid-phase electron acceptors away from the cell. Recent studies showed that Shewanella oneidensis MR-1 produces outer membrane (OM) and periplasmic extensions that contain EET components and hinted at their possible role as bacterial nanowires. However, their fine structure and distribution of cytochrome electron carriers under native conditions remained unclear, making it difficult to evaluate the potential electron transport (ET) mechanism along OM extensions. Here, we report high-resolution images of S. oneidensis OM extensions, using electron cryotomography (ECT). We...

  18. Cellular Electron Cryotomography: Toward Structural Biology In Situ

    Oikonomou, Catherine M.; Jensen, Grant J.
    Electron cryotomography (ECT) provides three-dimensional views of macromolecular complexes inside cells in a native frozen–hydrated state. Over the last two decades, ECT has revealed the ultrastructure of cells in unprecedented detail. It has also allowed us to visualize the structures of macromolecular machines in their native context inside intact cells. In many cases, such machines cannot be purified intact for in vitro study. In other cases, the function of a structure is lost outside the cell, so that the mechanism can be understood only by observation in situ. In this review, we describe the technique and its history and provide...

  19. Structure-guided SCHEMA recombination generates diverse chimeric channelrhodopsins

    Bedbrook, Claire N.; Rice, Austin J.; Yang, Kevin K.; Ding, Xiaozhe; Chen, Siyuan; LeProust, Emily M.; Gradinaru, Viviana; Arnold, Frances H.
    Integral membrane proteins (MPs) are key engineering targets due to their critical roles in regulating cell function. In engineering MPs, it can be extremely challenging to retain membrane localization capability while changing other desired properties. We have used structure-guided SCHEMA recombination to create a large set of functionally diverse chimeras from three sequence-diverse channelrhodopsins (ChRs). We chose 218 ChR chimeras from two SCHEMA libraries and assayed them for expression and plasma membrane localization in human embryonic kidney cells. The majority of the chimeras express, with 89% of the tested chimeras outperforming the lowest-expressing parent; 12% of the tested chimeras express...

  20. Directed Evolution of a Bright Near-Infrared Fluorescent Rhodopsin Using a Synthetic Chromophore

    Herwig, Lukas; Rice, Austin J.; Bedbrook, Claire N.; Zhang, Ruijie K.; Lignell, Antti; Cahn, Jackson K. B.; Renata, Hans; Dodani, Sheel C.; Cho, Inha; Cai, Long; Gradinaru, Viviana; Arnold, Frances H.
    By engineering a microbial rhodopsin, Archaerhodopsin-3 (Arch), to bind a synthetic chromophore, merocyanine retinal, in place of the natural chromophore all-trans-retinal (ATR), we generated a protein with exceptionally bright and unprecedentedly red-shifted near-infrared (NIR) fluorescence. We show that chromophore substitution generates a fluorescent Arch complex with a 200-nm bathochromic excitation shift relative to ATR-bound wild-type Arch and an emission maximum at 772 nm. Directed evolution of this complex produced variants with pH-sensitive NIR fluorescence and molecular brightness 8.5-fold greater than the brightest ATR-bound Arch variant. The resulting proteins are well suited to bacterial imaging; expression and stability have not been...

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