47 research outputs found
Model-Driven Designs of an Oscillating Gene Network
ABSTRACT The current rapid expansion of biological knowledge offers a great opportunity to rationally engineer biological systems that respond to signals such as light and chemical inducers by producing specific proteins. Turning on and off the production of proteins on demand holds great promise for creating significant biotechnological and biomedical applications. With successful stories already registered, the challenge still lies with rationally engineering gene regulatory networks which, like electronic circuits, sense inputs and generate desired outputs. From the literature, we have found kinetic and thermodynamic information describing the molecular components and interactions of the transcriptionally repressing lac, tet, and ara operons. Connecting these components in a model gene network, we determine how to change the kinetic parameters to make this normally nonperiodic system one which has well-defined oscillations. Simulating the designed lac-tet-ara gene network using a hybrid stochastic-discrete and stochastic-continuous algorithm, we seek to elucidate the relationship between the strength and type of specific connections in the gene network and the oscillatory nature of the protein product. Modeling the molecular components of the gene network allows the simulation to capture the dynamics of the real biological system. Analyzing the effect of modifications at this level provides the ability to predict how changes to experimental systems will alter the network behavior, while saving the time and expense of trial and error experimental modifications
Divergent evolution of protein conformational dynamics in dihydrofolate reductase.
Molecular evolution is driven by mutations, which may affect the fitness of an organism and are then subject to natural selection or genetic drift. Analysis of primary protein sequences and tertiary structures has yielded valuable insights into the evolution of protein function, but little is known about the evolution of functional mechanisms, protein dynamics and conformational plasticity essential for activity. We characterized the atomic-level motions across divergent members of the dihydrofolate reductase (DHFR) family. Despite structural similarity, Escherichia coli and human DHFRs use different dynamic mechanisms to perform the same function, and human DHFR cannot complement DHFR-deficient E. coli cells. Identification of the primary-sequence determinants of flexibility in DHFRs from several species allowed us to propose a likely scenario for the evolution of functionally important DHFR dynamics following a pattern of divergent evolution that is tuned by cellular environment
Gcn4-Mediator Specificity Is Mediated by a Large and Dynamic Fuzzy Protein-Protein Complex.
Transcription activation domains (ADs) are inherently disordered proteins that often target multiple coactivator complexes, but the specificity of these interactions is not understood. Efficient transcription activation by yeast Gcn4 requires its tandem ADs and four activator-binding domains (ABDs) on its target, the Mediator subunit Med15. Multiple ABDs are a common feature of coactivator complexes. We find that the large Gcn4-Med15 complex is heterogeneous and contains nearly all possible AD-ABD interactions. Gcn4-Med15 forms via a dynamic fuzzy protein-protein interface, where ADs bind the ABDs in multiple orientations via hydrophobic regions that gain helicity. This combinatorial mechanism allows individual low-affinity and specificity interactions to generate a biologically functional, specific, and higher affinity complex despite lacking a defined protein-protein interface. This binding strategy is likely representative of many activators that target multiple coactivators, as it allows great flexibility in combinations of activators that can cooperate to regulate genes with variable coactivator requirements
Large expert-curated database for benchmarking document similarity detection in biomedical literature search
Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe
Side Chain Conformational Averaging in Human Dihydrofolate Reductase
The three-dimensional structures
of the dihydrofolate reductase
enzymes from <i>Escherichia coli</i> (ecDHFR or ecE) and <i>Homo sapiens</i> (hDHFR or hE) are very similar, despite a rather
low level of sequence identity. Whereas the active site loops of ecDHFR
undergo major conformational rearrangements during progression through
the reaction cycle, hDHFR remains fixed in a closed loop conformation
in all of its catalytic intermediates. To elucidate the structural
and dynamic differences between the human and <i>E. coli</i> enzymes, we conducted a comprehensive analysis of side chain flexibility
and dynamics in complexes of hDHFR that represent intermediates in
the major catalytic cycle. Nuclear magnetic resonance relaxation dispersion
experiments show that, in marked contrast to the functionally important
motions that feature prominently in the catalytic intermediates of
ecDHFR, millisecond time scale fluctuations cannot be detected for
hDHFR side chains. Ligand flux in hDHFR is thought to be mediated
by conformational changes between a hinge-open state when the substrate/product-binding
pocket is vacant and a hinge-closed state when this pocket is occupied.
Comparison of X-ray structures of hinge-open and hinge-closed states
shows that helix αF changes position by sliding between the
two states. Analysis of χ<sub>1</sub> rotamer populations derived
from measurements of <sup>3</sup><i>J</i><sub>CγCO</sub> and <sup>3</sup><i>J</i><sub>CγN</sub> couplings
indicates that many of the side chains that contact helix αF
exhibit rotamer averaging that may facilitate the conformational change.
The χ<sub>1</sub> rotamer adopted by the Phe31 side chain depends
upon whether the active site contains the substrate or product. In
the holoenzyme (the binary complex of hDHFR with reduced nicotinamide
adenine dinucleotide phosphate), a combination of hinge opening and
a change in the Phe31 χ<sub>1</sub> rotamer opens the active
site to facilitate entry of the substrate. Overall, the data suggest
that, unlike ecDHFR, hDHFR requires minimal backbone conformational
rearrangement as it proceeds through its enzymatic cycle, but that
ligand flux is brokered by more subtle conformational changes that
depend on the side chain motions of critical residues
Side-Chain Conformational Heterogeneity of Intermediates in the <i>Escherichia coli</i> Dihydrofolate Reductase Catalytic Cycle
<i>Escherichia coli</i> dihydrofolate reductase (DHFR)
provides a paradigm for the integrated study of the role of protein
dynamics in enzyme function. Previous studies of backbone and side
chain dynamics have yielded unprecedented insights into the mechanism
by which DHFR progresses through the structural changes that occur
during its catalytic cycle. Here we report a comprehensive study of
the χ<sub>1</sub> rotamer populations of the aromatic and γ-methyl
containing residues for complexes of the catalytic cycle, based on
NMR measurement of <sup>3</sup><i>J</i><sub>CγCO</sub> and <sup>3</sup><i>J</i><sub>CγN</sub> coupling
constants. We report conformational and dynamic information for eight
distinct complexes, where transitions between rotamer wells may occur
on a broad picosecond to millisecond time scale. This large volume
of <sup>3</sup><i>J</i> data has allowed us to fit new Karplus
parameterizations for aromatic side chains and to select the best
available of previously determined parameters for Ile, Thr, and Val.
The <sup>3</sup><i>J</i><sub>CγCO</sub> and <sup>3</sup><i>J</i><sub>CγN</sub> coupling constants are found
to be extremely sensitive measures of side chain χ<sub>1</sub> rotamers and to give important insights into the extent of conformational
averaging. For a subset of residues in DHFR, the extent of rotamer
averaging is invariant to the nature of the bound ligand, while for
other residues the rotamer averaging differs in one or more complexes
of the enzymatic cycle. These variable-averaging residues are generally
located near the active site, but the phenomenon extends into the
adenosine binding domain. For several residues, the rotamer populations
in different DHFR complexes appear to depend on whether the complex
is in the closed or occluded state, and some residues are exquisitely
sensitive to small changes in the nature of the bound ligand
Gcn4-Mediator Specificity Is Mediated by a Large and Dynamic Fuzzy Protein-Protein Complex
Summary: Transcription activation domains (ADs) are inherently disordered proteins that often target multiple coactivator complexes, but the specificity of these interactions is not understood. Efficient transcription activation by yeast Gcn4 requires its tandem ADs and four activator-binding domains (ABDs) on its target, the Mediator subunit Med15. Multiple ABDs are a common feature of coactivator complexes. We find that the large Gcn4-Med15 complex is heterogeneous and contains nearly all possible AD-ABD interactions. Gcn4-Med15 forms via a dynamic fuzzy protein-protein interface, where ADs bind the ABDs in multiple orientations via hydrophobic regions that gain helicity. This combinatorial mechanism allows individual low-affinity and specificity interactions to generate a biologically functional, specific, and higher affinity complex despite lacking a defined protein-protein interface. This binding strategy is likely representative of many activators that target multiple coactivators, as it allows great flexibility in combinations of activators that can cooperate to regulate genes with variable coactivator requirements. : Tuttle et al. report a “fuzzy free-for-all” interaction mechanism that explains how seemingly unrelated transcription activators converge on a limited number of coactivator targets. The mechanism provides a rationale for the observation that individually weak and low-specificity interactions can combine to produce biologically critical function without requiring highly ordered structure. Keywords: transcription activation, intrinsically disordered proteins, fuzzy bindin
Solution structure of sperm lysin yields novel insights into molecular dynamics of rapid protein evolution
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Ovomucoid specific immunoglobulin E as a predictor of tolerance to cooked egg
Background: Ovomucoid is the dominant allergen in hen's egg. Although several studies evaluated the utility of ovomucoid specific immunoglobulin E (sIgE) levels in predicting baked (e.g., muffin or cupcake) or raw egg food challenge outcomes, studies that evaluated ovomucoid sIgE as a predictor of cooked egg (e.g., scrambled or hard boiled) challenge outcomes are limited. Objective: To determine the relation of ovomucoid sIgE levels with cooked egg food challenge outcomes. Methods: A retrospective review of 44 children who underwent cooked egg food challenge and who had the ovomucoid sIgE level measured. Results: Thirty-six of 44 children (81.8%) passed cooked egg challenge. The ovomucoid sIgE level predicted cooked egg challenge outcome (passed median, <0.35 kU/L [range, <0.35–0.64 kU/L]; failed median, 0.40 kU/L [range, <0.35–3.13 kU/L]; p = 0.004). Ovomucoid sIgE levels correlated with egg white (EW) sIgE levels (Spearman correlation coefficient, 0.588; p < 0.001). Receiver operating characteristic curve analysis of ovomucoid and EW sIgE demonstrated areas under the curve of 0.711 and 0.766, respectively. No significant difference was observed among those immunologic parameters in their abilities to predict cooked egg challenge outcome (p = 0.559). Conclusion: The ovomucoid sIgE level may be helpful in predicting cooked egg challenge outcomes. However, our study did not support a role for ovomucoid sIgE replacing EW sIgE testing in evaluating egg allergy