S James Remington

University of Oregon, Eugene, Oregon, United States

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Publications (126)741.67 Total impact

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    ABSTRACT: The gastric pathogen Helicobacter pylori forms biofilms on abiotic and biotic surfaces. We have shown previously that H. pylori perceives the quorum signal autoinducer-2 (AI-2) as a chemorepellent. We report here that H. pylori chemorepulsion from endogenous AI-2 influences the proportions and spatial organization of cells within biofilms. Strains that fail to produce AI-2 (∆luxS strains) or are defective for chemotaxis (∆cheA strains) formed more spatially homogenous biofilms with a greater proportion of adherent versus planktonic cells than wild-type biofilms. Reciprocally, a strain that overproduced AI-2 (luxSOP) formed biofilms with proportionally fewer adherent cells. Along with the known AI-2 chemoreceptor, TlpB, we identified AibA and AibB, two novel periplasmic binding proteins that are required for the AI-2 chemorepulsion response. Disruptions in any of the proteins required for AI-2 chemotaxis recapitulated the biofilm adherence and spatial organization phenotype of the ∆luxS mutant. Furthermore, exogenous administration of AI-2 was sufficient to decrease the proportion of adherent cells in biofilms and promote dispersal of cells from biofilms in a chemotaxis-dependent manner. Finally, we found that disruption of AI-2 production or AI-2 chemotaxis resulted in increased clustering of cells in microcolonies on cultured epithelial cells. We conclude that chemotaxis from AI-2 is a determinant of H. pylori biofilm spatial organization and dispersal.
    Preview · Article · Sep 2015 · mBio
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    ABSTRACT: The gastric pathogen Helicobacter pylori interacts intimately with the gastric mucosa to avoid the microbicidal acid in the stomach lumen. The cues H. pylori senses to locate and colonize the gastric epithelium have not been well defined. We show that metabolites emanating from human gastric organoids rapidly attract H. pylori. This response is largely controlled by the bacterial chemoreceptor TlpB, and the main attractant emanating from epithelia is urea. Our previous structural analyses show that TlpB binds urea with high affinity. Here we demonstrate that this tight binding controls highly sensitive responses, allowing detection of urea concentrations as low as 50 nM. Attraction to urea requires that H. pylori urease simultaneously destroys the signal. We propose that H. pylori has evolved a sensitive urea chemodetection and destruction system that allows the bacterium to dynamically and locally modify the host environment to locate the epithelium. Copyright © 2015 Elsevier Inc. All rights reserved.
    No preview · Article · Aug 2015 · Cell host & microbe
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    Full-text · Article · Oct 2014 · Nature
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    ABSTRACT: Mutations near the fluorescing chromophore of the green fluorescent protein (GFP) have direct effects on the absorption and emission spectra. Some mutants have significant band shifts and most of the mutants exhibit a loss of fluorescence intensity. In this study we continue our investigation of the factors controlling the excited state proton transfer (PT) process of GFP, in particular to study the effects of modifications to the key side chain Ser205 in wt-GFP, proposed to participate in the proton wire. To this aim we combined mutagenesis, X-ray crystallography, steady-state spectroscopy, time-resolved emission spectroscopy and all-atom explicit molecular dynamics (MD) simulations to study the double mutant T203V/S205A. Our results show that while in the previously described GFP double mutant T203V/S205V the PT process does not occur, in the T203V/S205A mutant the PT process does occur, but with a 350 times slower rate than in wild-type GFP (wt-GFP). Furthermore, the kinetic isotope effect in the GFP double mutant T203V/S205A is twice smaller than in the wt-GFP and in the GFP single mutant S205V, which forms a novel PT pathway. On the other hand, the crystal structure of GFP T203V/S205A does not reveal a viable proton transfer pathway. To explain PT in GFP T203V/S205A, we argue on the basis of the MD simulations for an alternative, novel proton-wire pathway which involves the phenol group of the chromophore and water molecules infrequently entering from the bulk. This alternative pathway may explain the dramatically slow PT in the GFP double mutant T203V/S205A compared to wt-GFP.
    No preview · Article · Apr 2014 · Physical Chemistry Chemical Physics
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    Ron Simkovitch · Amit Huppert · Dan Huppert · S James Remington · Yifat Miller
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    ABSTRACT: To study the dynamics and mechanisms of the proton wire of wild type green fluorescence protein (wt-GFP) and its S205V mutant, we applied molecular dynamics (MD) simulations and compared the results with the X-ray structures of both proteins and with the proton transfer kinetics of these proteins studied by the time-resolved emission technique. The MD simulations for the wt-GFP show that, in the proton wire, the Ser205 exists in two conformations with similar probabilities. One conformation supports the proton transfer, and the other does not. The fluctuation between the two conformers is relatively slow. This result may explain the time-resolved emission spectrum's long-time fluorescence tail of the wt-GFP chromophore's protonated form. The MD simulations of the S205V mutant show that the water molecule in the proton wire is replaced by other bulk water molecules along the simulations of 60 ns. Furthermore, as in the wt-GFP, the Thr203 also exists in two conformations in which only one conformation supports the proton transfer. These two findings give an insight into the relatively slow proton transfer rate in the S205V mutant in comparison to the wt-GFP.
    Full-text · Article · Oct 2013 · The Journal of Physical Chemistry B
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    ABSTRACT: pH sensing is crucial for survival of most organisms, yet the molecular basis of such sensing is poorly understood. Here, we present an atomic resolution structure of the periplasmic portion of the acid-sensing chemoreceptor, TlpB, from the gastric pathogen Helicobacter pylori. The structure reveals a universal signaling fold, a PAS domain, with a molecule of urea bound with high affinity. Through biophysical, biochemical, and in vivo mutagenesis studies, we show that urea and the urea-binding site residues play critical roles in the ability of H. pylori to sense acid. Our signaling model predicts that protonation events at Asp114, affected by changes in pH, dictate the stability of TlpB through urea binding.
    Preview · Article · Jun 2012 · Structure
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    ABSTRACT: To further explore excited state proton transfer (ESPT) pathways within green fluorescent protein (GFP), mutagenesis, X-ray crystallography, and time-resolved and steady-state optical spectroscopy were employed to create and study the GFP mutant S205A. In wild type GFP (wt-GFP), the proton transfer pathway includes the hydroxyl group of the chromophore, a water molecule, Ser205, and Glu222. We found that the ESPT rate constant of S205A is smaller by a factor of 20 than that of wt-GFP and larger by a factor of 2 in comparison to the ESPT rate of S205V mutant which we previously characterized. (1) High resolution crystal structures reveal that in both S205A and S205V mutants, an alternative proton transfer pathway is formed that involves the chromophore hydroxyl, a bridging water molecule, Thr203 and Glu222. The slow PT rate is explained by the long (∼3.2 Å and presumably weak) hydrogen bond between Thr203 and the water molecule, compared to the 2.7 Å normal hydrogen bond between the water molecule and Ser205 in wt-GFP. For data analysis of the experimental data from both GFP mutants, we used a two-rotamer kinetic model, assuming only one rotamer is capable of ESPT. Data analysis supports an agreement with the underlying assumption of this model.
    No preview · Article · Sep 2011 · The Journal of Physical Chemistry B
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    S James Remington
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    ABSTRACT: A brief personal perspective is provided for green fluorescent protein (GFP), covering the period 1994-2011. The topics discussed are primarily those in which my research group has made a contribution and include structure and function of the GFP polypeptide, the mechanism of fluorescence emission, excited state protein transfer, the design of ratiometric fluorescent protein biosensors and an overview of the fluorescent proteins derived from coral reef animals. Structure-function relationships in photoswitchable fluorescent proteins and nonfluorescent chromoproteins are also briefly covered.
    Preview · Article · Jun 2011 · Protein Science
  • S. Haga · T. Takezawa · T. Ozawa · S.J. Remington · N. Morita · M. Ozaki
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    ABSTRACT: Background/Aims. Ischemia/reperfusion (I/R)- induced injury is an acute injury mainly caused by surgical procedures. And HGF (hepatocyte growth factor) is known to protect against I/R-injury effectively. The sufficient dose of HGF must be topically delivered to the target organ to minimize the organ damage on surgery (reperfusion) and the dose of drug. For this purpose, we developed a novel technology for converting a fragile disk of the conventional type-I collagen gel into a strong and transparent 'vitrigel membrane', based on a concept for the vitrification of heat-denatured proteins. In the present study, we examined the efficacy of the newly designed 'HGF-containing collagen vitrigel sheet' as an effective drug delivery system in mouse liver I/R model. Materials and methods. Male C57Black6 mice were subjected to 60 min-liver ischemia (middle and left liver lobes) and reperfusion. Immediately after liver ischemia, the ischemic liver lobes were covered by collagen vitrigel sheet containing hHGF (3..g/ml). Reperfused liver injury was evaluated by biochemical/ histological methods and bio-imaging of hepatic oxidative stress (redox-sensitive GFP probe) and apoptosis (bioluminescent caspase-3 activity probe). Results. Collagen vitrigel sheet (containing hHGF) released hHGF until 96 hr in vitro and successfully delivered hHGF to the liver tissue suffering I/R. This effectively protected liver from I/R-induced injury. Blood biochemistry showed reduced levels of serum GOT/GPT/LDH, and liver histology revealed reduced necrotic area in the hHGF-treated liver. Oxidative stress and caspase-3 activity of the liver were examined by bioimaging, which revealed that hHGF delivered by vitrigel sheet significantly reduced oxidative stress and apoptotic cell death in the post-ischemic liver. Conclusions. hHGF-containing collagen vitrigel sheet protected liver from I/R-induced injury. Collagen vitrigel sheet may have good therapeutic potential in topical drug delivery in various clinical settings.
    No preview · Article · Jan 2011 · IFMBE proceedings
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    ABSTRACT: The lifetime exposure of organisms to oxidative stress influences many aging processes which involve the turnover of the extracellular matrix. In this study, we identify the redox-responsive molecular signals that drive senescence-associated (SA) matrix metalloproteinase-1 (MMP-1) expression. Precise biochemical monitoring revealed that senescent fibroblasts increase steady-state (H(2)O(2)) 3.5-fold (13.7-48.6 pM) relative to young cells. Restricting H(2)O(2) production through low O(2) exposure or by antioxidant treatments prevented SA increases in MMP-1 expression. The H(2)O(2)-dependent control of SA MMP-1 is attributed to sustained JNK activation and c-jun recruitment to the MMP-1 promoter. SA JNK activation corresponds to increases and decreases in the levels of its activating kinase (MKK-4) and inhibitory phosphatase (MKP-1), respectively. Enforced MKP-1 expression negates SA increases in JNK phosphorylation and MMP-1 production. Overall, these studies define redox-sensitive signaling networks regulating SA MMP-1 expression and link the free radical theory of aging to initiation of aberrant matrix turnover.
    Full-text · Article · Oct 2010 · Journal of Cellular Physiology
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    ABSTRACT: mKeima is an unusual monomeric red fluorescent protein (lambda(em)(max) approximately 620 nm) that is maximally excited in the blue (lambda(ex)(max) approximately 440 nm). The large Stokes shift suggests that the chromophore is normally protonated. A 1.63 A resolution structure of mKeima reveals the chromophore to be imbedded in a novel hydrogen bond network, different than in GFP, which could support proton transfer from the chromophore hydroxyl, via Ser142, to Asp157. At low temperatures the emission contains a green component (lambda(em)(max) approximately 535 nm), enhanced by deuterium substitution, presumably resulting from reduced proton transfer efficiency. Ultrafast pump/probe studies reveal a rising component in the 610 nm emission with a lifetime of approximately 4 ps, characterizing the rate of proton transfer. Mutation of Asp157 to neutral Asn changes the chromophore resting charge state to anionic (lambda(ex)(max) approximately 565 nm, lambda(em)(max) approximately 620 nm). Thus, excited state proton transfer (ESPT) explains the large Stokes shift. This work unambiguously characterizes green emission from the protonated acylimine chromophore of red fluorescent proteins.
    Full-text · Article · Sep 2009 · Journal of the American Chemical Society
  • Sanae Haga · S James Remington · Naoki Morita · Keita Terui · Michitaka Ozaki
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    ABSTRACT: Noninvasive evaluation of organ redox states provides invaluable information in many clinical settings. We evaluated a newly developed reduction/oxidation-sensitive green fluorescent protein (roGFP) probe that reports cellular redox potentials and their dynamic changes in live cells. On hypoxia/reoxygenation (H/R) of AML12 liver cells, roGFP indicated mild reduction during hypoxia, but immediate transient oxidation after reoxygenation. The roGFP probe confirmed the antioxidative effects of N-acetylcysteine, catalase, redox factor-1, and Mn-SOD/CuZn-SOD against H/R-induced cellular oxidative stress (OS). In a mouse liver ischemia/reperfusion (I/R) model, roGFP transduced by using an adenoviral vector revealed immediate reduction of the liver under ischemia, and two distinct peaks of OS: (a) early, observed within 60 min after reperfusion, similar to the in vitro study; and (b) later, at 24 h. The early peak levels paralleled the ischemic time up to 75 min and the postischemic liver injury (sGOT/GPT/LDH) in the later phase (6 and 24 h after I/R). The roGFP probe successfully indicated postischemic OS of the liver in living mice, accurately predicting postischemic liver injury. This probe may represent an effective OS marker indicating organ redox states and also predicting the damage/function.
    No preview · Article · Jul 2009 · Antioxidants & Redox Signaling
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    ABSTRACT: Crystal structures of the photoactivatable green fluorescent protein T203H variant (PA-GFP) have been solved in the native and photoactivated states, which under 488 nm illumination are dark and brightly fluorescent, respectively. We demonstrate that photoactivation of PA-GFP is the result of a UV-induced decarboxylation of the Glu222 side chain that shifts the chromophore equilibrium to the anionic form. Coupled with the T203H mutation, which stabilizes the native PA-GFP neutral chromophore, Glu222 decarboxylation yields a 100-fold contrast enhancement relative to wild-type GFP (WT). Additionally, the structures provide insights into the spectroscopic differences between WT and PA-GFP steady-state fluorescence maxima and excited-state proton transfer dynamics.
    No preview · Article · May 2009 · Journal of the American Chemical Society
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    Mark B Cannon · S James Remington
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    ABSTRACT: The quantification of transient redox events within subcellular compartments, such as those involved in certain signal transduction pathways, requires specific probes with high spatial and temporal resolution. Redox-sensitive variants of the green fluorescent protein (roGFP) have recently been developed that allow the noninvasive monitoring of intracellular thiol-disulfide equilibria. In this chapter, the biophysical properties of these probes are discussed, including recent efforts to enhance their response times. Several recent applications of roGFPs are highlighted, including roGFP expression within Arabidopsis to monitor redox status during root elongation, expression in neurons to measure oxidative stress during ischemia, and targeting of roGFPs to endosomal compartments demonstrating unexpectedly oxidizing potentials within these compartments. Possible future directions for the optimization of roGFPs or new classes of redox-sensitive fluorescent probes are also discussed.
    Full-text · Article · Feb 2009 · Methods in molecular biology (Clifton, N.J.)
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    Xiaokun Shu · Lei Wang · Leslie Colip · Karen Kallio · S James Remington
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    ABSTRACT: mPlum is a far-red fluorescent protein with emission maximum at approximately 650 nm and was derived by directed evolution from DsRed. Two residues near the chromophore, Glu16 and Ile65, were previously revealed to be indispensable for the far-red emission. Ultrafast time-resolved fluorescence emission studies revealed a time dependent shift in the emission maximum, initially about 625 nm, to about 650 nm over a period of 500 ps. This observation was attributed to rapid reorganization of the residues solvating the chromophore within mPlum. Here, the crystal structure of mPlum is described and compared with those of two blue shifted mutants mPlum-E16Q and -I65L. The results suggest that both the identity and precise orientation of residue 16, which forms a unique hydrogen bond with the chromophore, are required for far-red emission. Both the far-red emission and the time dependent shift in emission maximum are proposed to result from the interaction between the chromophore and Glu16. Our findings suggest that significant red shifts might be achieved in other fluorescent proteins using the strategy that led to the discovery of mPlum.
    Preview · Article · Feb 2009 · Protein Science
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    Jeremy R Lohman · Andrew C Olson · S James Remington
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    ABSTRACT: Enzymes of the glyoxylate shunt are important for the virulence of pathogenic organisms such as Mycobacterium tuberculosis and Candida albicans. Two isoforms have been identified for malate synthase, the second enzyme in the pathway. Isoform A, found in fungi and plants, comprises approximately 530 residues, whereas isoform G, found only in bacteria, is larger by approximately 200 residues. Crystal structures of malate synthase isoform G from Escherichia coli and Mycobacterium tuberculosis were previously determined at moderate resolution. Here we describe crystal structures of E. coli malate synthase A (MSA) in the apo form (1.04 A resolution) and in complex with acetyl-coenzyme A and a competitive inhibitor, possibly pyruvate or oxalate (1.40 A resolution). In addition, a crystal structure for Bacillus anthracis MSA at 1.70 A resolution is reported. The increase in size between isoforms A and G can be attributed primarily to an inserted alpha/beta domain that may have regulatory function. Upon binding of inhibitor or substrate, several active site loops in MSA undergo large conformational changes. However, in the substrate bound form, the active sites of isoforms A and G from E. coli are nearly identical. Considering that inhibitors bind with very similar affinities to both isoforms, MSA is as an excellent platform for high-resolution structural studies and drug discovery efforts.
    Full-text · Article · Sep 2008 · Protein Science
  • Jeremy R Lohman · S James Remington
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    ABSTRACT: Green fluorescent protein (GFP) indicators were previously developed that rapidly and quantitatively respond to changes in the thiol/disulfide equilibrium within subcellular compartments. In these indicators, surface-exposed cysteines residues were introduced so as to form a labile redox-active disulfide that in turn controls the emission properties of the internal chromophore. The biosensors have been shown to be effective reporters of the thiol/disulfide status within reducing compartments such as the mitochondria and cytosol for several cell types. However, due to the high thermodynamic stability of the introduced disulfide bond, the indicators are not useful for quantitative analysis within more oxidizing compartments such as the endoplasmic reticulum. Here we report the development of a new family of GFP-based redox indicators (roGFP1-iX) in which the thermodynamic stability of the disulfide is substantially lowered by insertion of a single amino acid into the main chain, adjacent to cysteine 147. The insertions result in indicators with midpoint potentials of -229 to -246 mV and are thus better suited for study of relatively oxidizing subcellular compartments. Atomic resolution crystallographic analyses suggest that two important factors act to destabilize the disulfide linkage in roGFP1-iX. In the oxidized state, an unusual non-proline cis-peptide bond adjacent to one of the cysteines introduces geometric strain into the system, while in the reduced state, a dramatic loop opening lowers the effective concentration of the reacting species.
    No preview · Article · Aug 2008 · Biochemistry
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    ABSTRACT: Ultrahigh-pressure dependence of the excited-state proton transfer (ESPT) in the wild-type green fluorescence protein (wt-GFP) in D2O was measured using steady-state and picosecond time-resolved fluorescence spectroscopies. The proton dissociation rate of the chromophore is almost insensitive to a pressure increase up to about 1.1 GPa. In contrast, the diffusion-limited geminate recombination kinetics is strongly affected by pressure, decreasing the effective dimensionality of proton diffusion as pressure increased. The GFP β-barrel structure sustains the high pressure and unfolds only at P > 1.5 GPa.
    No preview · Article · Apr 2008 · Chemical Physics Letters
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    ABSTRACT: Wild-type green fluorescent protein (wt-GFP) has a prominent absorbance band centered at approximately 395 nm, attributed to the neutral chromophore form. The green emission arising upon excitation of this band results from excited-state proton transfer (ESPT) from the chromophore hydroxyl, through a hydrogen-bond network proposed to consist of a water molecule and Ser205, to Glu222. Although evidence for Glu222 as a terminal proton acceptor has already been obtained, no evidence for the participation of Ser205 in the proton transfer process exists. To examine the role of Ser205 in the proton transfer, we mutated Ser205 to valine. However, the derived GFP variant S205V, upon excitation at 400 nm, still produces green fluorescence. Time-resolved emission spectroscopy suggests that ESPT contributes to the green fluorescence, and that the proton transfer takes place approximately 30 times more slowly than in wt-GFP. The crystal structure of S205V reveals rearrangement of Glu222 and Thr203, forming a new hydrogen-bonding network. We propose this network to be an alternative ESPT pathway with distinctive features that explain the significantly slowed rate of proton transfer. In support of this proposal, the double mutant S205V/T203V is shown to be a novel blue fluorescent protein containing a tyrosine-based chromophore, yet is incapable of ESPT. The results have implications for the detailed mechanism of ESPT and the photocycle of wt-GFP, in particular for the structures of spectroscopically identified intermediates in the cycle.
    Preview · Article · Jan 2008 · Protein Science
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    ABSTRACT: Steady-state emission, femtosecond pump-probe spectroscopy, and time-correlated single-photon counting (TCSPC) measurements were used to study the photophysics and the excited-state proton transfer (ESPT) reactions in the green fluorescent protein (GFP) variant S65T/H148D at three pH values: 6.0, 7.9, and 9.5. Selective mutation of GFP caused a dramatic change in the steady-state and excited-state behavior as compared to the wild-type GFP (wt-GFP) studied earlier. An excitation wavelength dependence of the quantum yield of the strong emission band at 510 nm (I* band) indicates the competition between adiabatic and non-adiabatic excited-state proton-transfer reactions. Pump-probe measurements show that the signal buildup probed at 510 nm is biphasic, where 0.8 of the signal amplitude is ultrashort, <150 fs, and 0.2 of the signal decreases with a approximately 10 ps time constant. This effect is a summary result of adiabatic ESPT to the carboxylate group of Asp148 and nonradiative processes. When compared with the luminescence of wt-GFP, the steady-state intensity at 450 nm is lower by a factor of about 10. This very weak emission at 450 nm has a nonexponential decay. It is relatively pH insensitive and, at about 25 ps, is almost twice as long as in wt-GFP. The former exhibits a surprisingly small kinetic deuterium isotope effect (KDIE), compared with the KDIE of about 5 for wt-GFP. Such weak proton dependence may indicate that this emission comes from the species not directly involved in the ESPT. In contrast, pH and H/D isotope dependence of the intense nonexponential luminescence decay of the S65T/H148D deprotonated form measured at 510 nm may result from an isomerization-induced deactivation that is accompanied by the proton recombination quenching. The data are complementary to the femtosecond time-resolved emission data obtained by ultrafast fluorescence up-conversion spectroscopy, found in the preceding paper (Shi et al.). The spectroscopic results are discussed on the basis of the detailed X-ray structure of the mutant published in the preceding paper (Shu et al.).
    No preview · Article · Oct 2007 · Biochemistry

Publication Stats

10k Citations
741.67 Total Impact Points


  • 1977-2015
    • University of Oregon
      • • Department of Physics
      • • Institute of Molecular Biology
      • • Department of Chemistry
      Eugene, Oregon, United States
  • 2005
    • Russian Academy of Sciences
      • Institute of Inorganic Chemistry
      Moskva, Moscow, Russia
    • University of Texas Southwestern Medical Center
      Dallas, Texas, United States
  • 2003-2005
    • Stanford University
      • Department of Chemistry
      Palo Alto, California, United States
  • 2004
    • University of California, San Diego
      San Diego, California, United States
  • 2002
    • Université Victor Segalen Bordeaux 2
      Burdeos, Aquitaine, France
  • 1985
    • Texas A&M University
      • Department of Biochemistry/Biophysics
      College Station, Texas, United States