Use of a distant reporter group as evidence for a conformational change in a sensory receptor

A highly sensitive method for demonstrating ligand-induced conformational changes in protein molecules in solution is described. The method utilizes an environmentally sensitive reporter group that is known to be distant from the active site. In the present application a conformational change is demonstrated in the galactose receptor of Salmonella typhimurium, involved in bacterial sensing and transport, by means of an extrinsic fluorophore, 5-iodoacetamidofluorescein, attached at a single methionine residue, and the intrinsic tryptophan fluorophore. Binding of the ligand galactose perturbs the microenvironment of both the fluorescein and tryptophan, as shown by both spectral and potassium iodide quenching changes. The distance between the two dyes is established by fluorescence energy transfer methods to be 41 ± 10 angstrom. Since only one molecule of galactose binds per molecule of receptor and since the galactose molecule is only about 5 angstrom in length, changes at one of these sites reflect the result of an indirect effect. Hence, there must be a ligand-induced conformational change that is propagated a minimum of 30 angstrom through the receptor molecule

Chemotactic response and adaptation dynamics in Escherichia coli

Adaptation of the chemotaxis sensory pathway of the bacterium Escherichia coli is integral for detecting chemicals over a wide range of background concentrations, ultimately allowing cells to swim towards sources of attractant and away from repellents. Its biochemical mechanism based on methylation and demethylation of chemoreceptors has long been known. Despite the importance of adaptation for cell memory and behavior, the dynamics of adaptation are difficult to reconcile with current models of precise adaptation. Here, we follow time courses of signaling in response to concentration step changes of attractant using in vivo fluorescence resonance energy transfer measurements. Specifically, we use a condensed representation of adaptation time courses for efficient evaluation of different adaptation models. To quantitatively explain the data, we finally develop a dynamic model for signaling and adaptation based on the attractant flow in the experiment, signaling by cooperative receptor complexes, and multiple layers of feedback regulation for adaptation. We experimentally confirm the predicted effects of changing the enzyme-expression level and bypassing the negative feedback for demethylation. Our data analysis suggests significant imprecision in adaptation for large additions. Furthermore, our model predicts highly regulated, ultrafast adaptation in response to removal of attractant, which may be useful for fast reorientation of the cell and noise reduction in adaptation.Comment: accepted for publication in PLoS Computational Biology; manuscript (19 pages, 5 figures) and supplementary information; added additional clarification on alternative adaptation models in supplementary informatio

Varieties of living things: Life at the intersection of lineage and metabolism

publication-status: Publishedtypes: Articl

Principles of genetic circuit design

Cells navigate environments, communicate and build complex patterns by initiating gene expression in response to specific signals. Engineers seek to harness this capability to program cells to perform tasks or create chemicals and materials that match the complexity seen in nature. This Review describes new tools that aid the construction of genetic circuits. Circuit dynamics can be influenced by the choice of regulators and changed with expression 'tuning knobs'. We collate the failure modes encountered when assembling circuits, quantify their impact on performance and review mitigation efforts. Finally, we discuss the constraints that arise from circuits having to operate within a living cell. Collectively, better tools, well-characterized parts and a comprehensive understanding of how to compose circuits are leading to a breakthrough in the ability to program living cells for advanced applications, from living therapeutics to the atomic manufacturing of functional materials.National Institute of General Medical Sciences (U.S.) (Grant P50 GM098792)National Institute of General Medical Sciences (U.S.) (Grant R01 GM095765)National Science Foundation (U.S.). Synthetic Biology Engineering Research Center (EEC0540879)Life Technologies, Inc. (A114510)National Science Foundation (U.S.). Graduate Research FellowshipUnited States. Office of Naval Research. Multidisciplinary University Research Initiative (Grant 4500000552

The kinetics and feedback inhibition of cytidine 5′-triphosphate synthetase in wild-type and mutant Chinese hamster cells

The kinetics and cytidine 5′-triphosphate (CTP) feedback inhibition of CTP synthetase in wild-type and four mutants of Chinese hamster V79 cells have been studied. The enzymes of the wild type and three of the four mutants exhibited positive cooperativity with the substrate uridine 5′-triphosphate (UTP). Three of the mutants had K m app and S 50 valuves distinctly greater than those of the wild type, while the fourth mutant had values similar to those of the wild type. all four mutants exhibited resistance to CTP feedback inhibition, while the wild type was sensitive to such inhibition. It is postulated that a single mutational event in each mutant had caused a concomitant change of the enzyme in its binding both to the substrate UTP and to the end-product CTP.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44151/1/10528_2004_Article_BF00485855.pd

Size Doesn't Matter: Towards a More Inclusive Philosophy of Biology

notes: As the primary author, O’Malley drafted the paper, and gathered and analysed data (scientific papers and talks). Conceptual analysis was conducted by both authors.publication-status: Publishedtypes: ArticlePhilosophers of biology, along with everyone else, generally perceive life to fall into two broad categories, the microbes and macrobes, and then pay most of their attention to the latter. ‘Macrobe’ is the word we propose for larger life forms, and we use it as part of an argument for microbial equality. We suggest that taking more notice of microbes – the dominant life form on the planet, both now and throughout evolutionary history – will transform some of the philosophy of biology’s standard ideas on ontology, evolution, taxonomy and biodiversity. We set out a number of recent developments in microbiology – including biofilm formation, chemotaxis, quorum sensing and gene transfer – that highlight microbial capacities for cooperation and communication and break down conventional thinking that microbes are solely or primarily single-celled organisms. These insights also bring new perspectives to the levels of selection debate, as well as to discussions of the evolution and nature of multicellularity, and to neo-Darwinian understandings of evolutionary mechanisms. We show how these revisions lead to further complications for microbial classification and the philosophies of systematics and biodiversity. Incorporating microbial insights into the philosophy of biology will challenge many of its assumptions, but also give greater scope and depth to its investigations

Robustness in Glyoxylate Bypass Regulation

The glyoxylate bypass allows Escherichia coli to grow on carbon sources with only two carbons by bypassing the loss of carbons as CO2 in the tricarboxylic acid cycle. The flux toward this bypass is regulated by the phosphorylation of the enzyme isocitrate dehydrogenase (IDH) by a bifunctional kinase–phosphatase called IDHKP. In this system, IDH activity has been found to be remarkably robust with respect to wide variations in the total IDH protein concentration. Here, we examine possible mechanisms to explain this robustness. Explanations in which IDHKP works simultaneously as a first-order kinase and as a zero-order phosphatase with a single IDH binding site are found to be inconsistent with robustness. Instead, we suggest a robust mechanism where both substrates bind the bifunctional enzyme to form a ternary complex

Control of Canalization and Evolvability by Hsp90

Partial reduction of Hsp90 increases expression of morphological novelty in qualitative traits of Drosophila and Arabidopsis, but the extent to which the Hsp90 chaperone also controls smaller and more likely adaptive changes in natural quantitative traits has been unclear. To determine the effect of Hsp90 on quantitative trait variability we deconstructed genetic, stochastic and environmental components of variation in Drosophila wing and bristle traits of genetically matched flies, differing only by Hsp90 loss-of-function or wild-type alleles. Unexpectedly, Hsp90 buffering was remarkably specific to certain normally invariant and highly discrete quantitative traits. Like the qualitative trait phenotypes controlled by Hsp90, highly discrete quantitative traits such as scutellor and thoracic bristle number are threshold traits. When tested across genotypes sampled from a wild population or in laboratory strains, the sensitivity of these traits to many types of variation was coordinately controlled, while continuously variable bristle types and wing size, and critically invariant left-right wing asymmetry, remained relatively unaffected. Although increased environmental variation and developmental noise would impede many types of selection response, in replicate populations in which Hsp90 was specifically impaired, heritability and ‘extrinsic evolvability’, the expected response to selection, were also markedly increased. However, despite the overall buffering effect of Hsp90 on variation in populations, for any particular individual or genotype in which Hsp90 was impaired, the size and direction of its effects were unpredictable. The trait and genetic-background dependence of Hsp90 effects and its remarkable bias toward invariant or canalized traits support the idea that traits evolve independent and trait-specific mechanisms of canalization and evolvability through their evolution of non-linearity and thresholds. Highly non-linear responses would buffer variation in Hsp90-dependent signaling over a wide range, while over a narrow range of signaling near trait thresholds become more variable with increasing probability of triggering all-or-none developmental responses

Structural Discrimination of Robustness in Transcriptional Feedforward Loops for Pattern Formation

Signaling pathways are interconnected to regulatory circuits for sensing the environment and expressing the appropriate genetic profile. In particular, gradients of diffusing molecules (morphogens) determine cell fate at a given position, dictating development and spatial organization. The feedforward loop (FFL) circuit is among the simplest genetic architectures able to generate one-stripe patterns by operating as an amplitude detection device, where high output levels are achieved at intermediate input ones. Here, using a heuristic optimization-based approach, we dissected the design space containing all possible topologies and parameter values of the FFL circuits. We explored the ability of being sensitive or adaptive to variations in the critical morphogen level where cell fate is switched. We found four different solutions for precision, corresponding to the four incoherent architectures, but remarkably only one mode for adaptiveness, the incoherent type 4 (I4-FFL). We further carried out a theoretical study to unveil the design principle for such structural discrimination, finding that the synergistic action and cooperative binding on the downstream promoter are instrumental to achieve absolute adaptive responses. Subsequently, we analyzed the robustness of these optimal circuits against perturbations in the kinetic parameters and molecular noise, which has allowed us to depict a scenario where adaptiveness, parameter sensitivity and noise tolerance are different, correlated facets of the robustness of the I4-FFL circuit. Strikingly, we showed a strong correlation between the input (environment-related) and the intrinsic (mutation-related) susceptibilities. Finally, we discussed the evolution of incoherent regulations in terms of multifunctionality and robustness