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1.  Comparison of the Complete Protein Sets of Worm and Yeast: Orthology and Divergence 
Science (New York, N.Y.)  1998;282(5396):2022-2028.
Comparative analysis of predicted protein sequences encoded by the genomes of Caenorhabditis elegans and Saccharomyces cerevisiae suggests that most of the core biological functions are carried out by orthologous proteins (proteins of different species that can be traced back to a common ancestor) that occur in comparable numbers. The specialized processes of signal transduction and regulatory control that are unique to the multicellular worm appear to use novel proteins, many of which re-use conserved domains. Major expansion of the number of some of these domains seen in the worm may have contributed to the advent of multicellularity. The proteins conserved in yeast and worm are likely to have orthologs throughout eukaryotes; in contrast, the proteins unique to the worm may well define metazoans.
PMCID: PMC3057080  PMID: 9851918
2.  Expanding Yeast Knowledge Online 
Yeast (Chichester, England)  1998;14(16):1453-1469.
The completion of the Saccharomyces cerevisiae genome sequencing project11 and the continued development of improved technology for large-scale genome analysis have led to tremendous growth in the amount of new yeast genetics and molecular biology data. Efficient organization, presentation, and dissemination of this information are essential if researchers are to exploit this knowledge. In addition, the development of tools that provide efficient analysis of this information and link it with pertinent information from other systems is becoming increasingly important at a time when the complete genome sequences of other organisms are becoming available. The aim of this review is to familiarize biologists with the type of data resources currently available on the World Wide Web (WWW).
doi:10.1002/(SICI)1097-0061(199812)14:16<1453::AID-YEA348>3.0.CO;2-G
PMCID: PMC3037831  PMID: 9885151
World Wide Web; Saccharomyces Genome Database; Munich Information Center for Protein Sequences; Yeast Protein Database
3.  Transmembrane Signaling by the Aspartate Receptor: Engineered Disulfides Reveal Static Regions of the Subunit Interface† 
Biochemistry  1995;34(30):9722-9733.
Ligand binding to the periplasmic domain of the transmembrane aspartate receptor generates an intramolecular conformational change which spans the bilayer and ultimately signals the cytoplasmic CheA histidine kinase, thereby triggering chemotaxis. The receptor is a homodimer stabilized by the interface between its two identical subunits: the present study investigates the role of the periplasmic and transmembrane regions of this interface in the mechanism of transmembrane signaling. Free cysteines and disulfide bonds are engineered into selected interfacial positions, and the resulting effects on the transmembrane signal are assayed by monitoring in vitro regulation of kinase activity. Three of the 14 engineered cysteine pairs examined, as well as six of the 14 engineered disulfides, cause perturbations of the interface structure which essentially destroy transmembrane regulation of the kinase. The remaining 11 cysteine pairs, and eight engineered disulfides covalently linking the two subunits at locations spanning positions 18–75, are observed to retain significant transmembrane kinase regulation. The eight functional disulfides positively identify adjacent faces of the two N-terminal helices in the native receptor dimer and indicate that large regions of the periplasmic and transmembrane subunit interface remain effectively static during the transmembrane signal. The results are consistent with a model in which the subunit interface plays a structural role, while the second membrane-spanning helix transmits the ligand-induced signal across the bilayer to the kinase binding domain. The effects of engineered cysteines and disulfides on receptor methylation in vitro are also measured, enabling direct comparison of the in vitro methylation and phosphorylation assays.
PMCID: PMC2904562  PMID: 7626643
4.  Lock On/Off Disulfides Identify the Transmembrane Signaling Helix of the Aspartate Receptor* 
The Journal of biological chemistry  1995;270(41):24043-24053.
The aspartate receptor of the bacterial chemotaxis pathway regulates the autophosphorylation rate of a cytoplasmic histidine kinase in response to ligand binding. The transmembrane signal, which is transmitted from the periplasmic aspartate-binding domain to the cytoplasmic regulatory domain, is carried by an intramolecular conformational change within the homodimeric receptor structure. The present work uses engineered cysteines and disulfide bonds to probe the nature of this conformational change, focusing in particular on the role of the second transmembrane α-helix. Altogether 26 modifications, consisting of 13 cysteine pairs and the corresponding disulfide bonds, have been introduced into the contacts between the second transmembrane helix and adjacent helices. The effects of these modifications on the transmembrane signal have been quantified by in vitro assays which measure (i) ligand binding, (ii) receptor-mediated regulation of kinase activity, and (iii) receptor methylation. All three parameters are observed to be highly sensitive to perturbations of the second transmembrane helix. In particular, 13 of the 26 modifications (6 cysteine pairs and 7 disulfides) significantly increase or decrease aspartate affinity, while 15 of the 26 modifications (5 cysteine pairs and 10 disulfides) destroy transmembrane kinase regulation. Importantly, 3 of the perturbing disulfides are found to lock the receptor in the “on” or “off” signaling state by covalently constraining the second transmembrane helix, demonstrating that it is possible to use engineered disulfides to lock the signaling function of a receptor protein. A separate aspect of the study probes the thermal motions of the second transmembrane helix: 4 disulfides designed to trap large amplitude twisting motions are observed to disrupt function but form readily, suggesting that the helix is mobile. Together the results support a model in which the second transmembrane helix is a mobile signaling element responsible for communicating the transmembrane signal.
PMCID: PMC2899691  PMID: 7592603
5.  THE TWO-COMPONENT SIGNALING PATHWAY OF BACTERIAL CHEMOTAXIS: A Molecular View of Signal Transduction by Receptors, Kinases, and Adaptation Enzymes 
The chemosensory pathway of bacterial chemotaxis has become a paradigm for the two-component superfamily of receptor-regulated phosphorylation pathways. This simple pathway illustrates many of the fundamental principles and unanswered questions in the field of signaling biology. A molecular description of pathway function has progressed rapidly because it is accessible to diverse structural, biochemical, and genetic approaches. As a result, structures are emerging for most of the pathway elements, biochemical studies are elucidating the mechanisms of key signaling events, and genetic methods are revealing the intermolecular interactions that transmit information between components. Recent advances include (a) the first molecular picture of a conformational transmembrane signal in a cell surface receptor, (b) four new structures of kinase domains and adaptation enzymes, and (c) significant new insights into the mechanisms of receptor-mediated kinase regulation, receptor adaptation, and the phospho-activation of signaling proteins. Overall, the chemosensory pathway and the propulsion system it regulates provide an ideal system in which to probe molecular principles underlying complex cellular signaling and behavior.
doi:10.1146/annurev.cellbio.13.1.457
PMCID: PMC2899694  PMID: 9442881
transmembrane signaling; kinase regulation; histidine kinase; aspartate kinase; protein methylation
6.  Use of Site-Directed Cysteine and Disulfide Chemistry to Probe Protein Structure and Dynamics: Applications to Soluble and Transmembrane Receptors of Bacterial Chemotaxis 
Methods in enzymology  2007;423:25-51.
Site-directed cysteine and disulfide chemistry is broadly useful in the analysis of protein structure and dynamics, and applications of this chemistry to the bacterial chemotaxis pathway have illustrated the kinds of information that can be generated. Notably, in many cases, cysteine and disulfide chemistry can be carried out in the native environment of the protein whether it be aqueous solution, a lipid bilayer, or a multiprotein complex. Moreover, the approach can tackle three types of problems crucial to a molecular understanding of a given protein: (1) it can map out 2° structure, 3° structure, and 4° structure; (2) it can analyze conformational changes and the structural basis of regulation by covalently trapping specific conformational or signaling states; and (3) it can uncover the spatial and temporal aspects of thermal fluctuations by detecting backbone and domain dynamics. The approach can provide structural information for many proteins inaccessible to high-resolution methods. Even when a high-resolution structure is available, the approach provides complementary information about regulatory mechanisms and thermal dynamics in the native environment. Finally, the approach can be applied to an entire protein, or to a specific domain or subdomain within the full-length protein, thereby facilitating a divide-and-conquer strategy in large systems or multiprotein complexes.
Rigorous application of the approach to a given protein, domain, or subdomain requires careful experimental design that adequately resolves the structural and dynamical information provided by the method. A full structural and dynamical analysis begins by scanning engineered cysteines throughout the region of interest. To determine 2° structure, the solvent exposure of each cysteine is determined by measuring its chemical reactivity, and the periodicity of exposure is analyzed. To probe 3° structure, 4° structure, and conformational regulation, pairs of cysteines are identified that rapidly form disulfide bonds and that retain function when induced to form a disulfide bond in the folded protein or complex. Finally, to map out thermal fluctuations in a protein of known structure, disulfide formation rates are measured between distal pairs of nonperturbing surface cysteines. This chapter details these methods and illustrates applications to two proteins from the bacterial chemotaxis pathway: the periplasmic galactose binding protein and the transmembrane aspartate receptor.
doi:10.1016/S0076-6879(07)23002-2
PMCID: PMC2896970  PMID: 17609126
7.  Activation of the Phosphosignaling Protein CheY II. Analysis of activated mutants by 19F NMR and protein engineering* 
The Journal of biological chemistry  1993;268(18):13089-13096.
The Escherichia coli CheY protein is activated by phosphorylation, and in turn alters flagellar rotation. To investigate the molecular mechanism of activation, an extensive collection of mutant CheY proteins was analyzed by behavioral assays, in vitro phosphorylation, and 19F NMR chemical shift measurements. Substitution of a positively charged residue (Arg or Lys) in place of Asp13 in the CheY activation site results in activation, even for mutants which cannot be phos-phorylated. Thus phosphorylation plays an indirect role in the activation mechanism. Lys109, a residue proposed to act as a conformational “switch” in the activation site, is required for activation of CheY by either phosphorylation or mutation.
The 19F NMR chemical shift assay described in the preceding article (Drake, S. K., Bourret, R. B., Luck, L. A., Simon, M. I., and Falke, J. J. (1993) J. Biol Chem. 268, 13081–13088) was again used to monitor six phenylalanine positions in CheY, including one position which probed the vicinity of Lys109. Mutations which activate CheY were observed to perturb the Lys109 probe, providing further evidence that Lys109 is directly involved in the activating conformational change. Two striking contrasts were observed between activation by mutation and phosphorylation, (i) Each activating mutation generates a relatively localized perturbation in the activation site region, whereas phosphorylation triggers a global structural change. (ii) The perturbation of the Lys109 region observed for activating mutations is not detected in the phosphorylated protein. These results are consistent with a two-step model of activated CheY docking to the flagellar switch.
PMCID: PMC2892986  PMID: 8514750
8.  Comparative Genomics of the Eukaryotes 
Science (New York, N.Y.)  2000;287(5461):2204-2215.
A comparative analysis of the genomes of Drosophila melanogaster, Caenorhabditis elegans, and Saccharomyces cerevisiae—and the proteins they are predicted to encode—was undertaken in the context of cellular, developmental, and evolutionary processes. The nonredundant protein sets of flies and worms are similar in size and are only twice that of yeast, but different gene families are expanded in each genome, and the multidomain proteins and signaling pathways of the fly and worm are far more complex than those of yeast. The fly has orthologs to 177 of the 289 human disease genes examined and provides the foundation for rapid analysis of some of the basic processes involved in human disease.
PMCID: PMC2754258  PMID: 10731134
9.  Genoviz Software Development Kit: Java tool kit for building genomics visualization applications 
BMC Bioinformatics  2009;10:266.
Background
Visualization software can expose previously undiscovered patterns in genomic data and advance biological science.
Results
The Genoviz Software Development Kit (SDK) is an open source, Java-based framework designed for rapid assembly of visualization software applications for genomics. The Genoviz SDK framework provides a mechanism for incorporating adaptive, dynamic zooming into applications, a desirable feature of genome viewers. Visualization capabilities of the Genoviz SDK include automated layout of features along genetic or genomic axes; support for user interactions with graphical elements (Glyphs) in a map; a variety of Glyph sub-classes that promote experimentation with new ways of representing data in graphical formats; and support for adaptive, semantic zooming, whereby objects change their appearance depending on zoom level and zooming rate adapts to the current scale. Freely available demonstration and production quality applications, including the Integrated Genome Browser, illustrate Genoviz SDK capabilities.
Conclusion
Separation between graphics components and genomic data models makes it easy for developers to add visualization capability to pre-existing applications or build new applications using third-party data models. Source code, documentation, sample applications, and tutorials are available at .
doi:10.1186/1471-2105-10-266
PMCID: PMC2746221  PMID: 19706180

Results 1-9 (9)