Recursos de colección

Caltech Authors (160.010 recursos)

Repository of works by Caltech published authors.

Group = Caltech Center for Environmental Microbial Interactions (CEMI)

Mostrando recursos 1 - 20 de 58

  1. 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...

  2. 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...

  3. 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...

  4. 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...

  5. 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...

  6. The ultrastructure of Shewanella oneidensis MR-1 nanowires revealed by electron cryo-tomography

    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. In vivo fluorescence Light Microscopy (fLM) imaging recently showed that Shewanella oneidensis MR-1 nanowires are extensions of the outer membrane that contain EET components. However, their fine structure and distribution of cytochrome electron carriers remained unclear, making it difficult to evaluate the electron transport mechanism along the nanowires. Here, we report high-resolution images of nanowires using Electron Cryo-Tomography (ECT). We developed a robust method for fLM imaging of nanowire growth on...

  7. 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...

  8. 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...

  9. 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...

  10. N-H Bond Dissociation Enthalpies and Facile H-atom Transfers for Early Intermediates of Fe-N_2 and Fe-CN Reductions

    Rittle, Jonathan; Peters, Jonas C.
    Fe-mediated biological nitrogen fixation is thought to proceed either via a sequence of proton and electron transfer steps, concerted H-atom transfer steps, or some combination thereof. Regardless of the specifics, and whether the intimate mechanism for N_2-to-NH_3 conversion involves a distal pathway, an alternating pathway, or some hybrid of these limiting scenarios, Fe-N_xH_y intermediates are implicated that feature reactive N-H bonds. Thermodynamic knowledge of the N-H bond strengths of such species is scant, and is especially difficult to obtain for the most reactive early stage candidate intermediates (e.g., Fe-N=NH, Fe=N-NH_2, Fe-NH=NH). Such knowledge is essential to considering various mechanistic hypotheses...

  11. Draft Genome Sequence of Hydrogenibacillus schlegelii MA48, a Deep-Branching Member of the Bacilli Class of Firmicutes

    Maker, Allison; Hemp, James; Pace, Laura A.; Ward, Lewis M.; Fischer, Woodward W.
    We report here the draft genome sequence of Hydrogenibacillus schlegelii MA48, a thermophilic facultative anaerobe that can oxidize hydrogen aerobically. H. schlegelii MA48 belongs to a deep-branching clade of the Bacilli class and provides important insight into the acquisition of aerobic respiration within the Firmicutes phylum.

  12. BONCAT enables time-resolved analysis of protein synthesis in native plant tissue

    Glenn, Weslee S.; Stone, Shannon E.; Ho, Samuel H.; Sweredoski, Michael J.; Moradian, Annie; Hess, Sonja; Bailey-Serres, Julia; Tirrell, David A.
    Proteomic plasticity undergirds stress responses in plants, and understanding such responses requires accurate measurement of the extent to which proteins levels are adjusted to counter external stimuli. Here, we adapt bioorthogonal non-canonical amino acid tagging (BONCAT) to interrogate protein synthesis in vegetative Arabidopsis thaliana seedlings. BONCAT relies on the translational incorporation of a non-canonical amino acid (ncAA) probe into cellular proteins. In this study, the probe is the methionine surrogate azidohomoalanine (Aha), which carries a reactive azide moiety in its amino acid side chain. The azide handle in Aha can be selectively conjugated to dyes and functionalized beads to enable...

  13. Cell-selective proteomics for biological discovery

    Stone, Shannon E.; Glenn, Weslee S.; Hamblin, Graham D.; Tirrell, David A.
    Cells alter the proteome to respond to environmental and developmental cues. Global analysis of proteomic responses is of limited value in heterogeneous environments, where there is no ‘average’ cell. Advances in sequencing, protein labeling, mass spectrometry, and data analysis have fueled recent progress in the investigation of specific subpopulations of cells in complex systems. Here we highlight recently developed chemical tools that enable cell-selective proteomic analysis of complex biological systems, from bacterial pathogens to whole animals.

  14. Mapping the Microscale Origins of MRI Contrast with Subcellular NV Diamond Magnetometry

    Davis, Hunter C.; Ramesh, Pradeep; Bhatnagar, Aadyot; Lee-Gosselin, Audrey; Barry, John F.; Glenn, David R.; Walsworth, Ronald L.; Shapiro, Mikhail G.
    Magnetic resonance imaging (MRI) is a widely used biomedical imaging modality that derives much of its contrast from microscale magnetic field gradients in biological tissues. However, the connection between these sub-voxel field patterns and MRI contrast has not been studied experimentally. Here, we describe a new method to map subcellular magnetic fields in mammalian cells and tissues using nitrogen vacancy diamond magnetometry and connect these maps to voxel-scale MRI contrast, providing insights for in vivo imaging and contrast agent design.

  15. Chemoenzymatic Labeling of Proteins for Imaging in Bacterial Cells

    Ho, Samuel H.; Tirrell, David A.
    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...

  16. Exposing the Three-Dimensional Biogeography and Metabolic States of Pathogens in Cystic Fibrosis Sputum via Hydrogel Embedding, Clearing, and rRNA Labeling

    DePas, William H.; Starwalt-Lee, Ruth; Van Sambeek, Lindsey; Kumar, Sripriya Ravindra; Gradinaru, Viviana; Newman, Dianne K.
    Physiological resistance to antibiotics confounds the treatment of many chronic bacterial infections, motivating researchers to identify novel therapeutic approaches. To do this effectively, an understanding of how microbes survive in vivo is needed. Though much can be inferred from bulk approaches to characterizing complex environments, essential information can be lost if spatial organization is not preserved. Here, we introduce a tissue-clearing technique, termed MiPACT, designed to retain and visualize bacteria with associated proteins and nucleic acids in situ on various spatial scales. By coupling MiPACT with hybridization chain reaction (HCR) to detect rRNA in sputum samples from cystic fibrosis (CF)...

  17. Exposing the Three-Dimensional Biogeography and Metabolic States of Pathogens in Cystic Fibrosis Sputum via Hydrogel Embedding, Clearing, and rRNA Labeling

    DePas, William H.; Starwalt-Lee, Ruth; Van Sambeek, Lindsey; Kumar, Sripriya Ravindra; Gradinaru, Viviana; Newman, Dianne K.
    Physiological resistance to antibiotics confounds the treatment of many chronic bacterial infections, motivating researchers to identify novel therapeutic approaches. To do this effectively, an understanding of how microbes survive in vivo is needed. Though much can be inferred from bulk approaches to characterizing complex environments, essential information can be lost if spatial organization is not preserved. Here, we introduce a tissue-clearing technique, termed MiPACT, designed to retain and visualize bacteria with associated proteins and nucleic acids in situ on various spatial scales. By coupling MiPACT with hybridization chain reaction (HCR) to detect rRNA in sputum samples from cystic fibrosis (CF)...

  18. A Link between Integral Membrane Protein Expression and Simulated Integration Efficiency

    Marshall, Stephen S.; Niesen, Michiel J. M.; Müller, Axel; Tiemann, Katrin; Saladi, Shyam M.; Galimidi, Rachel P.; Zhang, Bin; Clemons, William M., Jr.; Miller, Thomas F., III
    Integral membrane proteins (IMPs) control the flow of information and nutrients across cell membranes, yet IMP mechanistic studies are hindered by difficulties in expression. We investigate this issue by addressing the connection between IMP sequence and observed expression levels. For homologs of the IMP TatC, observed expression levels vary widely and are affected by small changes in protein sequence. The effect of sequence changes on experimentally observed expression levels strongly correlates with the simulated integration efficiency obtained from coarse-grained modeling, which is directly confirmed using an in vivo assay. Furthermore, mutations that improve the simulated integration efficiency likewise increase the...

  19. A Link between Integral Membrane Protein Expression and Simulated Integration Efficiency

    Marshall, Stephen S.; Niesen, Michiel J. M.; Müller, Axel; Tiemann, Katrin; Saladi, Shyam M.; Galimidi, Rachel P.; Zhang, Bin; Clemons, William M., Jr.; Miller, Thomas F., III
    Integral membrane proteins (IMPs) control the flow of information and nutrients across cell membranes, yet IMP mechanistic studies are hindered by difficulties in expression. We investigate this issue by addressing the connection between IMP sequence and observed expression levels. For homologs of the IMP TatC, observed expression levels vary widely and are affected by small changes in protein sequence. The effect of sequence changes on experimentally observed expression levels strongly correlates with the simulated integration efficiency obtained from coarse-grained modeling, which is directly confirmed using an in vivo assay. Furthermore, mutations that improve the simulated integration efficiency likewise increase the...

  20. A Link between Integral Membrane Protein Expression and Simulated Integration Efficiency

    Marshall, Stephen S.; Niesen, Michiel J. M.; Müller, Axel; Tiemann, Katrin; Saladi, Shyam M.; Galimidi, Rachel P.; Zhang, Bin; Clemons, William M., Jr.; Miller, Thomas F., III
    Integral membrane proteins (IMPs) control the flow of information and nutrients across cell membranes, yet IMP mechanistic studies are hindered by difficulties in expression. We investigate this issue by addressing the connection between IMP sequence and observed expression levels. For homologs of the IMP TatC, observed expression levels vary widely and are affected by small changes in protein sequence. The effect of sequence changes on experimentally observed expression levels strongly correlates with the simulated integration efficiency obtained from coarse-grained modeling, which is directly confirmed using an in vivo assay. Furthermore, mutations that improve the simulated integration efficiency likewise increase the...

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