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Modification Sites (modification + site)
Selected AbstractsStructural modeling and mutational analysis of yeast eukaryotic translation initiation factor 5A reveal new critical residues and reinforce its involvement in protein synthesisFEBS JOURNAL, Issue 8 2008Camila A. O. Dias Eukaryotic translation initiation factor 5A (eIF5A) is a protein that is highly conserved and essential for cell viability. This factor is the only protein known to contain the unique and essential amino acid residue hypusine. This work focused on the structural and functional characterization of Saccharomyces cerevisiae eIF5A. The tertiary structure of yeast eIF5A was modeled based on the structure of its Leishmania mexicana homologue and this model was used to predict the structural localization of new site-directed and randomly generated mutations. Most of the 40 new mutants exhibited phenotypes that resulted from eIF-5A protein-folding defects. Our data provided evidence that the C-terminal ,-helix present in yeast eIF5A is an essential structural element, whereas the eIF5A N-terminal 10 amino acid extension not present in archaeal eIF5A homologs, is not. Moreover, the mutants containing substitutions at or in the vicinity of the hypusine modification site displayed nonviable or temperature-sensitive phenotypes and were defective in hypusine modification. Interestingly, two of the temperature-sensitive strains produced stable mutant eIF5A proteins , eIF5AK56A and eIF5AQ22H,L93F, and showed defects in protein synthesis at the restrictive temperature. Our data revealed important structural features of eIF5A that are required for its vital role in cell viability and underscored an essential function of eIF5A in the translation step of gene expression. [source] The carboxyl-terminal linker is important for chemoreceptor functionMOLECULAR MICROBIOLOGY, Issue 2 2006Mingshan Li Summary Sensory adaptation in bacterial chemotaxis is mediated by chemoreceptor methylation and demethylation. In Escherichia coli, methyltransferase CheR and methylesterase CheB bind both substrate sites and a carboxyl-terminal pentapeptide sequence carried by certain receptors. Pentapeptide binding enhances enzyme action, an enhancement required for effective adaptation and chemotaxis. Pentapeptides are linked to the conserved body of chemoreceptors through a notably variable sequence of 30,35 residues. We created nested deletions from the distal end of this linker in chemoreceptor Tar. Chemotaxis was eliminated by deletion of 20,40 residues and reduced by shorter deletions. This did not reflect generalized disruption, because all but the most extremely truncated receptors activated kinase, were substrates for adaptational modification and performed transmembrane signalling. In contrast, linker truncations reduced rates of adaptational modification in parallel with chemotaxis. We concluded the linker is important for chemotaxis because of its role in adaptational modification. Effects of linker truncations on CheR binding to receptor-borne pentapeptide implied linker (i) makes pentapeptide available to modification enzymes by separation from the helical receptor body, and (ii) is a flexible arm allowing dual binding of enzyme to pentapeptide and modification site. The data suggest linker and the helix from which it emerges are structurally dynamic. [source] Axoneme-dependent tubulin modifications in singlet microtubules of the Drosophila sperm tailCYTOSKELETON, Issue 4 2008Henry D. Hoyle Abstract Drosophila melanogaster sperm tubulins are posttranslationally glutamylated and glycylated. We show here that axonemes are the substrate for these tubulin C-terminal modifications. Axoneme architecture is required, but full length, motile axonemes are not necessary. Tubulin glutamylation occurs during or shortly after assembly into the axoneme; only glutamylated tubulins are glycylated. Tubulins in other testis microtubules are not modified. Only a small subset of total Drosophila sperm axoneme tubulins have these modifications. Biochemical fractionation of Drosophila sperm showed that central pair and accessory microtubules have the majority of poly-modified tubulins, whereas doublet microtubules have only small amounts of mono- and oligo-modified tubulins. Glutamylation patterns for different ,-tubulins experimentally assembled into axonemes were consistent with utilization of modification sites corresponding to those identified in other organisms, but surrounding sequence context was also important. We compared tubulin modifications in the 9 + 9 + 2 insect sperm tail axonemes of Drosophila with the canonical 9 + 2 axonemes of sperm of the sea urchin Lytichinus pictus and the 9 + 0 motile sperm axonemes of the eel Anguilla japonica. In contrast to Drosophila sperm, L. pictus sperm have equivalent levels of modified tubulins in both doublet and central pair microtubule fractions, whereas the doublets of A. japonica sperm exhibit little glutamylation but extensive glycylation. Tubulin C-terminal modifications are a prevalent feature of motile axonemes, but there is no conserved pattern for placement or amount of these modifications. We conclude their functions are likely species-specific. Cell Motil. Cytoskeleton 2008. © 2008 Wiley-Liss, Inc. [source] Probing protein structure by amino acid-specific covalent labeling and mass spectrometryMASS SPECTROMETRY REVIEWS, Issue 5 2009Vanessa Leah Mendoza Abstract For many years, amino acid-specific covalent labeling has been a valuable tool to study protein structure and protein interactions, especially for systems that are difficult to study by other means. These covalent labeling methods typically map protein structure and interactions by measuring the differential reactivity of amino acid side chains. The reactivity of amino acids in proteins generally depends on the accessibility of the side chain to the reagent, the inherent reactivity of the label and the reactivity of the amino acid side chain. Peptide mass mapping with ESI- or MALDI-MS and peptide sequencing with tandem MS are typically employed to identify modification sites to provide site-specific structural information. In this review, we describe the reagents that are most commonly used in these residue-specific modification reactions, details about the proper use of these covalent labeling reagents, and information about the specific biochemical problems that have been addressed with covalent labeling strategies. © 2008 Wiley Periodicals, Inc., Mass Spec Rev 28:785,815, 2009 [source] Functional site profiling and electrostatic analysis of cysteines modifiable to cysteine sulfenic acidPROTEIN SCIENCE, Issue 2 2008Freddie R. Salsbury Jr Abstract Cysteine sulfenic acid (Cys-SOH), a reversible modification, is a catalytic intermediate at enzyme active sites, a sensor for oxidative stress, a regulator of some transcription factors, and a redox-signaling intermediate. This post-translational modification is not random: specific features near the cysteine control its reactivity. To identify features responsible for the propensity of cysteines to be modified to sulfenic acid, a list of 47 proteins (containing 49 known Cys-SOH sites) was compiled. Modifiable cysteines are found in proteins from most structural classes and many functional classes, but have no propensity for any one type of protein secondary structure. To identify features affecting cysteine reactivity, these sites were analyzed using both functional site profiling and electrostatic analysis. Overall, the solvent exposure of modifiable cysteines is not different from the average cysteine. The combined sequence, structure, and electrostatic approaches reveal mechanistic determinants not obvious from overall sequence comparison, including: (1) pKas of some modifiable cysteines are affected by backbone features only; (2) charged residues are underrepresented in the structure near modifiable sites; (3) threonine and other polar residues can exert a large influence on the cysteine pKa; and (4) hydrogen bonding patterns are suggested to be important. This compilation of Cys-SOH modification sites and their features provides a quantitative assessment of previous observations and a basis for further analysis and prediction of these sites. Agreement with known experimental data indicates the utility of this combined approach for identifying mechanistic determinants at protein functional sites. [source] Reactivity of the NS2/3(907,1206)ASK4 protein with ,-mercaptoethanol studied by electrospray ion trap mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 20 2002Laura Orsatti The present work reports a mass spectrometric investigation of the NS2/3 protein, a protease from hepatitis C virus (HCV). During routine protein manipulation, in the presence of 100,mM ,-mercaptoethanol and under denatured conditions, the protein was unexpectedly modified at its cysteine residues, and the increased molecular weight corresponded to one molecule of ,-mercaptoethanol bound. The modified protein, once refolded, was found to be less active than the unmodified one. The aim of this work was to investigate whether the reactivity of cysteines with ,-mercaptoethanol involves one specific, highly reactive residue of the sequence, or if the modification is a random process. Liquid chromatography (LC) coupled on-line with an electrospray ion trap mass spectrometer was used to identify the modification sites. It was found that five cysteines out of nine had reacted with ,-mercaptoethanol, none of them showing a significantly higher reactivity than the others. 95% of sequence coverage was obtained. Copyright © 2002 John Wiley & Sons, Ltd. [source] Molecular Mechanisms of Microtubular Organelle Assembly in TetrahymenaTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 3 2000JACEK GAERTIG ABSTRACT. Thanks to recent technological advances, the ciliate Tetrahymena thermophila has emerged as an attractive model organism for studies on the assembly of microtubular organelles in a single cell. Tetrahymena assembles 17 types of distinct microtubules, which are localix.ed in cilia, cell cortex, nuclei, and the endoplasm. These diverse microtubules have distinct morphologies, stabilities, and associations with specific Microtubule-Assoeiated Proteins. For example, kinesin-111, a microtubular motor protein, is required for assembly of cilia and is preferentially targeted to microtubules of actively assembled, immature cilia. It is unlikely that the unique properties of individual microtubules are derived from the utilization of diverse tubulin genes, because Tetrahymena expresses only a single isotype of ,-and two isotypes of ,-tubulin. However, Tetrahymena tubulins are modified secondarily by a host of post-translational mechanisms. Each microtubule organelle type displays a unique set of secondary tubulin modifications. The results of systematic in vivo mutational analyses of modification sites indicate a divergence in significance among post-translational mechanisms affecting either ,-or ,-tubulin. Both acetylation and polyglycylation of ,-tubulin are not essential and their complete elimination does not change the cell's phenotype in an appreciable way. However, the multiple polyglycylation sites on ,-tubulin are essential for survival, and their partial elimination dramatically affects cell motility, growth and morphology. Thus, both high-precision targeting of molecular motors to individual organelles as well as organelle-specific tubulin modifications contribute to the creation of diverse microtubules in a single cytoplasm of Tetrahymena. [source] Selenium Derivatization of Nucleic Acids for X-Ray Crystal-Structure and Function StudiesCHEMISTRY & BIODIVERSITY, Issue 4 2010Jia Sheng Abstract It is estimated that over two thirds of all new crystal structures of proteins are determined via the protein selenium derivatization (selenomethionine (Se-Met) strategy). This selenium derivatization strategy via MAD (multi-wavelength anomalous dispersion) phasing has revolutionized protein X-ray crystallography. Through our pioneer research, similarly, Se has also been successfully incorporated into nucleic acids to facilitate the X-ray crystal-structure and function studies of nucleic acids. Currently, Se has been stably introduced into nucleic acids by replacing nucleotide O-atom at the positions 2,, 4,, 5,, and in nucleobases and non-bridging phosphates. The Se derivatization of nucleic acids can be achieved through solid-phase chemical synthesis and enzymatic methods, and the Se-derivatized nucleic acids (SeNA) can be easily purified by HPLC, FPLC, and gel electrophoresis to obtain high purity. It has also been demonstrated that the Se derivatization of nucleic acids facilitates the phase determination via MAD phasing without significant perturbation. A growing number of structures of DNAs, RNAs, and protein,nucleic acid complexes have been determined by the Se derivatization and MAD phasing. Furthermore, it was observed that the Se derivatization can facilitate crystallization, especially when it is introduced to the 2,-position. In addition, this novel derivatization strategy has many advantages over the conventional halogen derivatization, such as more choices of the modification sites via the atom-specific substitution of the nucleotide O-atom, better stability under X-ray radiation, and structure isomorphism. Therefore, our Se-derivatization strategy has great potentials to provide rational solutions for both phase determination and high-quality crystal growth in nucleic-acid crystallography. Moreover, the Se derivatization generates the nucleic acids with many new properties and creates a new paradigm of nucleic acids. This review summarizes the recent developments of the atomic site-specific Se derivatization of nucleic acids for structure determination and function study. Several applications of this Se-derivatization strategy in nucleic acid and protein research are also described in this review. [source] | |||||||||||||