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One Protein (one + protein)
Selected AbstractsA Facile System for Genetic Incorporation of Two Different Noncanonical Amino Acids into One Protein in Escherichia coli,ANGEWANDTE CHEMIE, Issue 18 2010Wei Wan Dr. Ein ideales Paar: Indem die Ochre-Mutation mit einem Wildtyp- oder einem modifizierten PylRS-pylTUUA -Paar und die Amber-Mutation mit einem modifizierten MjTyrRS- Mj -Paar unterdrückt wurde, konnten zwei verschiedene nichtkanonische Aminosäuren (NAAs) gemeinsam hoch effizient in ein Protein von E.,coli eingebaut werden (siehe Bild, mit den NAAs 1,4; GFP=grün fluoreszierendes Protein). [source] Analysis of FimX, a phosphodiesterase that governs twitching motility in Pseudomonas aeruginosaMOLECULAR MICROBIOLOGY, Issue 4 2006Barbara I. Kazmierczak Summary Type IV pili (Tfp) are polar surface structures of Pseudomonas aeruginosa required for twitching motility, biofilm formation and adherence. One protein required for the assembly of tfp is FimX, which possesses both GGDEF and EAL domains characteristic of diguanylate cyclases and phosphodiesterases respectively. In this work we demonstrate that FimX has phosphodiesterase activity towards bis-(3,-5,)-cyclic dimeric guanosine monophosphate (c-di-GMP), but does not show diguanylate cyclase activity. Instead, the imperfect GGDEF domain of FimX likely serves to activate phosphodiesterase activity when bound to GTP, as has recently been described for the Caulobacter crescentus composite GGDEF-EAL protein, CC3396. Bacteria expressing FimX in which either the GGDEF or EAL domain is deleted or mutated have phenotypes indistinguishable from a ,fimX strain, demonstrating the importance of both domains to function. Previous work has shown that FimX localizes to the bacterial pole. In this work we show that restriction of FimX to a single pole requires intact GGDEF and EAL domains. Deletion of the amino-terminal REC domain of FimX, which contains a putative polar localization signal, results in a protein that still supports intermediate levels of pilus assembly and function. RFP,FimX,REC, unlike RFP,FimX, is no longer localized to the bacterial pole, while transmission electron microscopy shows that surface pili can originate from non-polar sites in this mutant. Although ,fimX mutants show limited in vitro cytotoxicity, they are as virulent as the wild-type strain in a murine model of acute pneumonia. [source] Proteomic changes in the crucian carp brain during exposure to anoxiaPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 8 2009Richard W. Smith Dr. Abstract During exposure to anoxia, the crucian carp brain is able to maintain normal overall protein synthesis rates. However, it is not known if there are alterations in the synthesis or expression of specific proteins. This investigation addresses this issue by comparing the normoxic and anoxic brain proteome. Nine proteins were found to be reduced by anoxia. Reductions in the glycolytic pathway proteins creatine kinase, fructose biphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase and lactate dehydrogenase reflect the reduced production and requirement for adenosine tri-phosphate during anoxia. In terms of neural protection, voltage-dependent anion channel, a protein associated with neuronal apoptosis, was reduced, along with gefiltin, a protein associated with the subsequent need for neuronal repair. Additionally the expression of proteins associated with neural degeneration and impaired cognitive function also declined; dihydropyrimidinase-like protein-3 and vesicle amine transport protein-1. One protein was found to be increased by anoxia; pre-proependymin, the precursor to ependymin. Ependymin fulfils multiple roles in neural plasticity, memory formation and learning, neuron growth and regeneration, and is able to reverse the possibility of apoptosis, thus further protecting the anoxic brain. [source] Phenotology of disease-linked proteins,HUMAN MUTATION, Issue 1 2005Jeffrey K. Myers Abstract Are there analogous sequence positions in families of related proteins where disease-linked mutations occur with unusually high frequency? We attempt to answer this question by examining sequence alignments for G-protein coupled receptors (GPCRs) and voltage-gated potassium channels that have a significant number of missense mutations linked to some form of human disease. When the disease-linked mutations are mapped onto the sequences for each family, there are a large number of aligned sites at which disease-linked mutations occur in more than one protein. The statistical significance of the aligned sites is judged by analysis of artificially-generated random datasets. There are a modest number of aligned sites that are statistically significant,we refer to these as "phenotologous" sequence positions. Phenotologous sites represent aligned positions at which mutations linked to disease phenotypes occur with high frequency within a family of proteins. The most interesting of these sites are those which are not conserved,such sites are apparently critical in defining structural or functional differences between related proteins. Phenotology may be used to make experimentally testable predictions regarding medical genetics, the molecular basis of disease, and protein structure,function relationships. Hum Mutat 25:90,97, 2005. © 2004 Wiley-Liss, Inc. [source] Investigation into the protein composition of human tear fluid using centrifugal filters and drop coating deposition Raman spectroscopyJOURNAL OF RAMAN SPECTROSCOPY, Issue 2 2009Jacob Filik Abstract Drop coating deposition Raman spectroscopy (DCDRS) is a simple method of analysing weak protein solutions. This study is another step in evaluating the analysis of tear fluid by DCDRS as a future medical diagnostic technique. The main aims of this study are to determine whether the DCDR spectra from tear samples contain signals from more than one protein (so relative levels can be measured) and, if so, are the proteins homogeneously distributed in the dried ring of the deposited material. Tear samples were collected from four healthy volunteers and pooled prior to analysis. Proteins were separated by mass into three groups using centrifugal filters. These groups contained proteins with (1) masses greater than 100 kDa, (2) masses between 100 and 50 kDa and (3) masses between 50 and 3 kDa. DCDR spectra from each of these protein group solutions displayed significant differences, confirming that the mass separation had been successful. When used as basis vectors for least-squares fitting, these spectra (and that of urea) produced excellent fits to the normal tear spectra. Least-squares fitting of spectra from the same point on a single sample and from several drops of the same sample showed that the tear DCDR spectra were highly reproducible. Raman point mapping of the tear ring showed significant radial ring variation, especially towards the outer edge of the ring. The specific peak changes in the protein signal across the ring suggested that the difference in the outer edge was due to protein desiccation as opposed to inhomogeneous protein deposition. Copyright © 2008 John Wiley & Sons, Ltd. [source] Tomographic reconstruction of treponemal cytoplasmic filaments reveals novel bridging and anchoring componentsMOLECULAR MICROBIOLOGY, Issue 3 2004Jacques Izard Summary An understanding of the involvement of bacterial cytoplasmic filaments in cell division requires the elucidation of the structural organization of those filamentous structures. Treponemal cytoplasmic filaments are composed of one protein, CfpA, and have been demonstrated to be involved in cell division. In this study, we used electron tomography to show that the filaments are part of a complex with a novel molecular organization that includes at least two distinct features decorating the filaments. One set of components appears to anchor the filaments to the cytoplasmic membrane. The other set of components appears to bridge the cytoplasmic filaments on the cytoplasmic side, and to be involved in the interfilament spacing within the cell. The filaments occupy between 3 and 18% of the inner surface of the cytoplasmic membrane. These results reveal a novel filamentous molecular organization of independent filaments linked by bridges and continuously anchored to the membrane. [source] Exploring functional roles of multibinding protein interfacesPROTEIN SCIENCE, Issue 8 2009Manoj Tyagi Abstract Cellular processes are highly interconnected and many proteins are shared in different pathways. Some of these shared proteins or protein families may interact with diverse partners using the same interface regions; such multibinding proteins are the subject of our study. The main goal of our study is to attempt to decipher the mechanisms of specific molecular recognition of multiple diverse partners by promiscuous protein regions. To address this, we attempt to analyze the physicochemical properties of multibinding interfaces and highlight the major mechanisms of functional switches realized through multibinding. We find that only 5% of protein families in the structure database have multibinding interfaces, and multibinding interfaces do not show any higher sequence conservation compared with the background interface sites. We highlight several important functional mechanisms utilized by multibinding families. (a) Overlap between different functional pathways can be prevented by the switches involving nearby residues of the same interfacial region. (b) Interfaces can be reused in pathways where the substrate should be passed from one protein to another sequentially. (c) The same protein family can develop different specificities toward different binding partners reusing the same interface; and finally, (d) inhibitors can attach to substrate binding sites as substrate mimicry and thereby prevent substrate binding. [source] Three monophyletic superfamilies account for the majority of the known glycosyltransferasesPROTEIN SCIENCE, Issue 7 2003Jing Liu Abstract Sixty-five families of glycosyltransferases (EC 2.4.x.y) have been recognized on the basis of high-sequence similarity to a founding member with experimentally demonstrated enzymatic activity. Although distant sequence relationships between some of these families have been reported, the natural history of glycosyltransferases is poorly understood. We used iterative searches of sequence databases, motif extraction, structural comparison, and analysis of completely sequenced genomes to track the origins of modern-type glycosyltransferases. We show that >75% of recognized glycosyltransferase families belong to one of only three monophyletic superfamilies of proteins, namely, (1) a recently described GPGTF/GT-B superfamily; (2) a nucleoside-diphosphosugar transferase (GT-A) superfamily, which is characterized by a DxD sequence signature and also includes nucleotidyltransferases; and (3) a GT-C superfamily of integral membrane glycosyltransferases with a modified DxD signature in the first extracellular loop. Several developmental regulators in Metazoans, including Fringe and Egghead homologs, belong to the second superfamily. Interestingly, Tout-velu/Exostosin family of developmental proteins found in all multicellular eukaryotes, contains separate domains belonging to the first and the second superfamilies, explaining multiple glycosyltransferase activities in one protein. [source] Stably folded de novo proteins from a designed combinatorial libraryPROTEIN SCIENCE, Issue 1 2003Yinan Wei Abstract Binary patterning of polar and nonpolar amino acids has been used as the key design feature for constructing large combinatorial libraries of de novo proteins. Each position in a binary patterned sequence is designed explicitly to be either polar or nonpolar; however, the precise identities of these amino acids are varied extensively. The combinatorial underpinnings of the "binary code" strategy preclude explicit design of particular side chains at specified positions. Therefore, packing interactions cannot be specified a priori. To assess whether the binary code strategy can nonetheless produce well-folded de novo proteins, we constructed a second-generation library based upon a new structural scaffold designed to fold into 102-residue four-helix bundles. Characterization of five proteins chosen arbitrarily from this new library revealed that (1) all are ,-helical and quite stable; (2) four of the five contain an abundance of tertiary interactions indicative of well-ordered structures; and (3) one protein forms a well-folded structure with native-like features. The proteins from this new 102-residue library are substantially more stable and dramatically more native-like than those from an earlier binary patterned library of 74-residue sequences. These findings demonstrate that chain length is a crucial determinant of structural order in libraries of de novo four-helix bundles. Moreover, these results show that the binary code strategy,if applied to an appropriately designed structural scaffold,can generate large collections of stably folded and/or native-like proteins. [source] Crystallization and preliminary X-ray analysis of Escherichia coli MutT in binary and ternary complex formsACTA CRYSTALLOGRAPHICA SECTION D, Issue 9 2004Teruya Nakamura During replication, Escherichia coli MutT prevents the misincorporation of mutagenic 8-oxoguanine into nascent DNA strands opposite adenine by hydrolyzing 8-oxo-dGTP in nucleotide pools to 8-oxo-dGMP. E. coli MutT is the most widely investigated member of the Nudix hydrolase family, which is large and found in all organisms. By co-crystallization of MutT with 8-oxo-dGMP, a reaction product, crystals of the binary complex were obtained using ammonium sulfate as a precipitant. The crystals belong to space group P212121, with unit-cell parameters a = 37.9, b = 56.0, c = 59.4,Å. Assuming the presence of one protein,nucleotide complex in the asymmetric unit, the Matthews coefficient VM is 2.1,Å3,Da,1. Crystals of the ternary complex were prepared by soaking crystals of the binary complex in 1,mM MnCl2 solution. They diffracted to 1.96 and 2.56,Å resolution, respectively. [source] PROTEOMIC ANALYSIS IN CARDIOVASCULAR DISEASESCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 4 2008C Cieniewski-Bernard SUMMARY 1Cardiovascular diseases are a major cause of morbidity and mortality in western countries. The molecular mechanisms responsible for heart dysfunction are still largely unknown, except in cases of genetic defects or alteration of genes and proteins. 2The publication of genome sequences from humans and other species has demonstrated the complexity of biology, including the finding that one gene does not encode for only one protein but for several, due to mRNA splicing and post-translational modifications. 3Proteomic analysis can provide an overall understanding of changes in the levels of protein expression. Differential proteomics is a powerful tool for improving our understanding of integrated biochemical responses. The main techniques used are two-dimensional electrophoresis (2D-gel) and Surface-Enhanced Laser Desorption/Ionization Time of Flight (SELDI-TOF) to separate proteins associated with mass spectrometry. Bioinformatic tools make it possible to compare protein profiles obtained from diverse biological samples. 4The combination of these approaches has proved to be particularly interesting for studying cardiovascular diseases and thereby improving our understanding of the mechanisms involved and identifying new biochemical factors and biomarkers involved in these diseases. [source] | |||||||||||||