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Graphene quantum dot-[beta]-cyclodextrin modified glassy carbon electrode was used as a new nanosensor for determination of L-tyrosine (L-Tyr). It was found that graphene quantum dot- [beta]-cyclodextrin has been stably electrodeposited on glassy carbon electrode modified by simple technique. The cyclic voltammograms of the modified electrode in an aqueous solution displayed a pair of well- defined, stable and irreversible reductive/oxidation redox systems. The apparent electron transfer rate constant ([k.sub.s]) and transfer coefficient ([alpha]) determined by cyclic voltammetry were approximately equal to 8.0 [s.sup.-1] and 0.7, respectively. The modified electrode showed excellent catalytic activity towards the oxidation of L-Tyr at positive potential in buffer solution. The nanosensor also displayed fast response time, high sensitivity, low detection limit and a remarkably positive potential oxidation of L-Tyr that decreased the effect of interferences in analysis. Keywords: L-tyrosine, graphene quantum dots, physiological pH, [beta]- cyclodextrin, nanosensor, amino acid DOI: 10.1134/S1061934818060096, Tyrosine is one of the 22 amino acids used by cells to synthesize proteins. It is a non-essential amino acid with a polar side group. It occurs in proteins that [...]

Electrochemical oxidation of peptides and proteins has been shown to lead to specific cleavage next to tyrosine (Tyr) and tryptophan (Trp) residues which makes the coupling of electrochemistry to mass spectrometry (EC-MS) a potential instrumental alternative to chemical and enzymatic cleavage. A set of Tyr and Trp-containing tripeptides has been studied to investigate the mechanistic aspects of electrochemical oxidation and the subsequent chemical reactions including peptide bond cleavage, making this the first detailed study of the electrochemistry of Trp-containing peptides. The effect of adjacent amino acids was studied leading to the conclusion that the ratios of oxidation and cleavage products are peptide-dependent and that the adjacent amino acid can influence the secondary chemical reactions occurring after the initial oxidation step. The effect of parameters such as potential and solvent conditions showed that control of the oxidation potential is crucial to avoid dimer formation for and an increasing number of oxygen insertions (hydroxylations) for Trp, which occur above 1000 mV (vs Pd/[H.sub.2]). While the formation of reactive intermediates after the first oxidation step is not strongly dependent on experimental conditions, an acidic pH is required for good cleavage yields. Working under strongly acidic conditions (pH 1.9-3.1) led to optimal cleavage yields (40-80%), whereas no or little cleavage occurred under basic conditions. Online EC-MS allowed determining the optimal potential for maximum cleavage yields, whereas EC--LC--MS/MS revealed the nature and distribution of the reaction products. 10.1021/ac101086w

The Wnt signaling pathway must be properly modulated to ensure an appropriate output: pathological conditions result from either insufficient or excessive levels of Wnt signal. For example, hyperactivation of the Wnt pathway is associated with various cancers and subnormal Wnt signaling can lead to increased invasiveness of tumor cells. We found that the Caenorhabditis elegans ortholog of the Fer nonreceptor tyrosine kinase, FRK-1, limits Wnt signaling by preventing the adhesion complex-associated [beta]-catenin, HMP-2, from participating in Wnt-dependent specification of the endoderm during embryogenesis. Removal of FRK-1 function results in relocalization of HMP-2 to the nucleus of epidermal cells, and allows it to substitute for WRM-1, the nuclear [beta]-catenin that normally transduces the Wnt signal during endoderm development. APR-1, the Co elegans APC ortholog, is similarly required to prevent HMP-2 relocalization and keeps it from participating in Wnt signal transduction; this finding partially explains the paradoxical observation that APR-1 acts either negatively or positively in Wnt signaling, depending on context. The apparent hyperactivation of the Wnt response in the absence of FRK-1 leads to hyperproliferation in the endoderm, as is also seen when WRM-1 is overexpressed in wildtype embryos. The specification and proliferation activities of Wnt signaling are separable: although the Tcf/Lef factor POP-1 acts in Wnt-dependent endoderm specification, it is not apparently required for hyperproliferation resulting from excessive Wnt signaling. These findings highlight a role for a Fer-type kinase in setting the proper levels of Wnt signaling and demonstrate the importance of this modulation in ensuring appropriate cell division. Caenorhabditis elegans | signal transduction | cell proliferation | beta-catenin | cell adhesion www.pnas.org/cgi/doi/ 10.1073/pnas.1006600107

Protein tyrosine phosphatases (PTPs) are regulated through reversible oxidation of the active-site cysteine. Previous studies have implied soluble reactive oxygen species (ROS), like [H.sub.2][O.sub.2], as the mediators of PTP oxidation. The potential role(s) of peroxidized lipids in PTP oxidation have not been described. This study demonstrates that increases in cellular lipid peroxides, induced by disruption of glutathione peroxidase 4, induce cellular PTP oxidation and reduce the activity of PDGF receptor targeting PTPs. These effects were accompanied by site-selective increased PDGF [beta]-receptor phosphorylation, sensitive to 12/15-lipoxygenase (12/15-LOX) inhibitors, and increased PDGF-induced cytoskeletal rearrangements. Importantly, the 12/15-LOX--derived 15-OOH-eicosatetraenoic acid lipid peroxide was much more effective than [H.sub.2][O.sub.2] in induction of in vitro PTP oxidation. Our study thus establishes that lipid peroxides are previously unrecognized inducers of oxidation of PTPs. This identifies a pathway for control of receptor tyrosine kinase signaling, which might also be involved in the etiology of diseases associated with increased lipid peroxidation. phospholipid hydroperoxide glutathione peroxidase | PDGF | redox regulation | reversible oxidation doi/ 10.1073/pnas.1007909107

An approach combining small-angle X-ray solution scattering (SAXS) data with coarse-grained (CG) simulations is developed to characterize the assembly states of Hck, a member of the Src-family kinases, under various conditions in solution. First, a basis set comprising a small number of assembly states is generated from extensive CG simulations. Second, a theoretical SAXS profile for each state in the basis set is computed by using the Fast-SAXS method. Finally, the relative population of the different assembly states is determined via a Bayesian-based Monte Carlo procedure seeking to optimize the theoretical scattering profiles against experimental SAXS data. The study establishes the concept of basis-set supported SAXS (BSS-SAXS) reconstruction combining computational and experimental techniques. Here, BSS-SAXS reconstruction is used to reveal the structural organization of Hck in solution and the different shifts in the equilibrium population of assembly states upon the binding of different signaling peptides. coarse-grained model | Bayesian analysis | folding | SAXS | simulation doi/ 10.1073/pnas.1004569107

Abl is a nonreceptor tyrosine kinase that is required for smooth muscle contraction. However, the mechanism by which Abl regulates smooth muscle contraction is not completely understood. In the present study, Abl underwent phosphorylation at Tyr412 (an index of Abl activation) in smooth muscle in response to contractile activation. Treatment with a cell-permeable decoy peptide, but not the control peptide, attenuated Abl phosphorylation during contractile stimulation. Treatment with the decoy peptide did not affect the association of Abl with the cytoskeletal protein vinculin and the spatial location of vinculin in smooth muscle. Inhibition of Abl phosphorylation by the decoy peptide attenuated the agonist-induced phosphorylation of Crk-associated substrate (CAS), an adapter protein participating in the signaling processes that regulate force development in smooth muscle. Additionally, previous studies have shown that contractile stimulation triggers the dissociation of CAS from the vimentin network, which is important for cytoskeletal signaling and contraction in smooth muscle. In this report, the decrease in the amount of CAS in cytoskeletal vimentin in response to contractile activation was reversed by the Abl inhibition with the decoy peptide. Moreover, force development and the enhancement of F-actin-to-G-actin ratios (an indication of actin polymerization) upon contractile activation were also attenuated by the Abl inhibition. However, myosin phosphorylation induced by contractile activation was not affected by the inhibition of Abl. These results suggest that Abl regulates the dissociation of CAS from the vimentin network, actin polymerization, and contraction by modulating CAS phosphorylation in smooth muscle. tyrosine kinase; Crk-associated substrate; cytoskeleton; contraction doi: 10.1152/ajpcell.00095.2010.

Tyrosine nitration in proteins is an important posttranslational modification (PTM) linked to various pathological conditions. When multiple potential sites of nitration exist, tandem mass spectrometry (MS/MS) methods provide unique tools to locate the nitro-tyrosine(a) precisely. Electron capture dissociation (ECD) is a powerful MS/MS method, different in its mechanisms to the 'slow-heating' threshold fragmentation methods, such as collision-induced dissociation (CID) and infrared multiphoton dissociation (IRMPD). Generally, ECD provides more homogeneous cleavage of the protein backbone and preserves labile PTMs. However recent studies in our laboratory demonstrated that ECD of doubly charged nitrated peptides is inhibited by the large electron affinity of the nitro group, while CID efficiency remains unaffected by nitration. Here, we have investigated the efficiency of ECD versus CID and IRMPD for top-down MS/MS analysis of multiply charged intact nitrated protein ions of myoglobin, lysozyme, and cytochrome c in a commercial Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. CID and IRMPD produced more cleavages in the vicinity of the sites of nitration than ECD. However the total number of ECD fragments was greater than those from CID or IRMPD, and many ECD fragments contained the site(s) of nitration. We conclude that ECD can be used in the top-down analysis of nitrated proteins, but precise localization of the sites of nitration may require either of the 'slow-heating' methods. 10.1021/ac101177r

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2010.07.015 Byline: Diego Alvarado (1), Daryl E. Klein (1), Mark A. Lemmon (1) Keywords: SIGNALING; PROTEINS Abstract: Transmembrane signaling by the epidermal growth factor receptor (EGFR) involves ligand-induced dimerization and allosteric regulation of the intracellular tyrosine kinase domain. Crystallographic studies have shown how ligand binding induces dimerization of the EGFR extracellular region but cannot explain the 'high-affinity' and 'low-affinity' classes of cell-surface EGF-binding sites inferred from curved Scatchard plots. From a series of crystal structures of the Drosophila EGFR extracellular region, we show here how Scatchard plot curvature arises from negatively cooperative ligand binding. The first ligand-binding event induces formation of an asymmetric dimer with only one bound ligand. The unoccupied site in this dimer is structurally restrained, leading to reduced affinity for binding of the second ligand, and thus negative cooperativity. Our results explain the cell-surface binding characteristics of EGF receptors and suggest how individual EGFR ligands might stabilize distinct dimeric species with different signaling properties. Author Affiliation: (1) Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6059, USA Article History: Received 5 January 2010; Revised 19 April 2010; Accepted 16 June 2010 Article Note: (miscellaneous) Published: August 19, 2010

Elongation of the digit rays resulting in the formation of a defined number of phalanges is a process poorly understood in mammals, whereas in the chicken distal mesenchymal bone morphogenetic protein (BMP) signaling in the so-called phalanx-forming region (PFR) or digit crescent (DC) seems to be involved. The human brachydactylies (BDs) are inheritable conditions characterized by variable degrees of digit shortening, thus providing an ideal model to analyze the development and elongation of phalanges. We used a mouse model for BDB1 ([Ror2.sup.W749X/W749X]) lacking middle phalanges and show that a signaling center corresponding to the chick PFR exists in the mouse, which is diminished in BDB1 mice. This resulted in a strongly impaired elongation of the digit condensations due to reduced chondrogenic commitment of undifferentiated distal mesenchymal cells. We further show that a similar BMP-based mechanism accounts for digit shortening in a mouse model for the closely related condition BDA1 ([Ihh.sup.E95K/E95K]), altogether indicating the functional significance of the PFR in mammals. Genetic interaction experiments as well as pathway analysis in BDB1 mice suggest that Indian hedgehog and WNT/[beta]-catenin signaling, which we show is inhibited by receptor tyrosine kinase-like orphan receptor 2 (ROR2) in distal limb mesenchyme, are acting upstream of BMP signaling in the PFR. bone morphogenetic protein signaling | brachydactyly | cartilage | limb development | Wnt signaling doi/ 10.1073/pnas.1009314107

The mutational diversity and phenotypic consequences of the RET (MIM 164761) tyrosine kinase in Hirschsprung disease (HSCR [MIM 142623]) was examined to explore the actual allelic diversity of a gene which impacts the phenotypic diversity and non-Mendelian features of a complex disease. The results demonstrated that both rare and common mutations, coding and noncoding, within the same gene contribute to HSCR in unique and specific ways that lead to recognizable genotype-phenotype associations.

Iodination of tyrosine residues in proteins has many uses in chemistry, biology, and medicine. Site specific identification of the sites of iodination is important for many of these uses. Reported herein is a facile method employing photodissociation and mass spectrometry to localize sites of iodination in whole proteins. Absorption of ultraviolet photons by iodotyrosine results in loss of iodine via homolytic bond dissociation. The resulting protein radical fragments in the vicinity of the iodotyrosine upon collisional activation. Analysis of the fragments within the vicinity of each tyrosine residue in the protein enables quantitative evaluation of the likelihood for iodination at each site. The results are compared with both traditional bottom up and top down mass spectrometric methods. Radical directed dissociation yields results in agreement with traditional approaches but requires significantly less effort and is inherently more sensitive. One limitation occurs when multiple tyrosine residues are in close proximity, in which case the extent of iodination at each residue may be difficult to determine. This limitation is frequently problematic for traditional approaches as well. 10.1021/ac100256v

Protein tyrosine phosphatases are often exploited and subverted by pathogenic bacteria to cause human diseases. The tyrosine phosphatase mPTPB from Mycobacterium tuberculosis is an essential virulence factor that is secreted by the bacterium into the cytoplasm of macrophages, where it mediates mycobacterial survival in the host. Consequently, there is considerable interest in understanding the mechanism by which mPTPB evades the host immune responses, and in developing potent and selective mPTPB inhibitors as unique antituberculosis (antiTB) agents. We uncovered that mPTPB subverts the innate immune responses by blocking the ERK1/2 and p38 mediated IL-6 production and promoting host cell survival by activating the Akt pathway. We identified a potent and selective mPTPB inhibitor I-A09 with highly efficacious cellular activity, from a combinatorial library of bidentate benzofuran salicylic acid derivatives assembled by click chemistry. We demonstrated that inhibition of mPTPB with I-A09 in macrophages reverses the altered host immune responses induced by the bacterial phosphatase and prevents TB growth in host cells. The results provide the necessary proof-of-principle data to support the notion that specific inhibitors of the mPTPB may serve as effective antiTB therapeutics. combinatorial chemistry | pathogen-host interaction | phosphatase inhibitor | signaling mechanism www.pnas.org/cgi/doi/10.1073/pnas.0909133107

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2009.05.028 Byline: Jae Hyun Bae (1), Erin Denise Lew (1), Satoru Yuzawa (1), Francisco Tome (1), Irit Lax (1), Joseph Schlessinger (1) Keywords: SIGNALING; PROTEINS Abstract: SH2 domain-mediated interactions represent a crucial step in transmembrane signaling by receptor tyrosine kinases. SH2 domains recognize phosphotyrosine (pY) in the context of particular sequence motifs in receptor phosphorylation sites. However, the modest binding affinity of SH2 domains to pY containing peptides may not account for and likely represents an oversimplified mechanism for regulation of selectivity of signaling pathways in living cells. Here we describe the crystal structure of the activated tyrosine kinase domain of FGFR1 in complex with a phospholipase C[gamma] fragment. The structural and biochemical data and experiments with cultured cells show that the selectivity of phospholipase C[gamma] binding and signaling via activated FGFR1 are determined by interactions between a secondary binding site on an SH2 domain and a region in FGFR1 kinase domain in a phosphorylation independent manner. These experiments reveal a mechanism for how SH2 domain selectivity is regulated in vivo to mediate a specific cellular process. Author Affiliation: (1) Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA Article History: Received 11 November 2008; Revised 26 March 2009; Accepted 7 May 2009 Article Note: (miscellaneous) Published: August 6, 2009

The accumulation of proteins damaged by reactive oxygen species (ROS), conventionally regarded as having pathological potentials, is associated with age-related diseases such as Alzheimer's, atherosclerosis, and cataractogenesis. Exposure of the aromatic amino acid phenylalanine to ROS-generating systems produces multiple isomers of tyrosine: m-tyrosine (m-Tyr), o-tyrosine (o-Tyr), and the standard p-tyrosine (Tyr). Previously it was demonstrated that exogenously supplied, oxidized amino acids could be incorporated into bacterial and eukaryotic proteins. It is, therefore, likely that in many cases, in vivo-damaged amino acids are available for de novo synthesis of proteins. Although the involvement of aminoacyl-tRNA synthetases in this process has been hypothesized, the specific pathway by which ROS-damaged amino acids are incorporated into proteins remains unclear. We provide herein evidence that mitochondrial and cytoplasmic phenylalanyl-tRNA synthetases (HsmtPheRS and HsctPheRS, respectively) catalyze direct attachment of m-Tyr to [tRNA.sup.Phe], thereby opening the way for delivery of the misacylated tRNA to the ribosome and incorporation of ROS-damaged amino acid into eukaryotic proteins. Crystal complexes of mitochondrial and bacterial PheRSs with m-Tyr reveal the net of highly specific interactions within the synthetic and editing sites.

Phosphotyrosine-containing peptide monoanions [M - H] exhibit extensive neutral loss of phosphoric acid (98 Da) upon quadrupole time-of-flight and ion-trap collision-induced dissociation (CID). In contrast, a neutral loss of metaphosphoric acid HP[O.sub.3] (80 Da) is negligible from the deprotonated phosphotyrosine peptides. The efficient [H.sub.3]P[O.sub.4] release is unexpected, given the structure of phosphotyrosine. Our study reveals that the abundant [M - H - 98]- product ions of pTyrpeptides are not a result of consecutive losses of HP[O.sub.3] and [H.sub.2]O but, rather, are induced by an intramolecular interaction of the phosphotyrosine phosphate with deprotonated peptide functions such as hydroxyl, carboxyl, and to a small extent, amide. As a result, an internal phosphotyrosine phosphate shift occurs, and the obtained phosphorylated functionalities undergo elimination of [H.sub.3]P[O.sub.4] to give rise to the [[M - H - 98].sup.-] fragments. The mechanism proposed for the phosphoric acid neutral loss is based on extensive CID studies of Ala-substituted model phosphorylated peptides and oxygen-18 labeling. The proposed mechanistic pathway explains the fact that the pTyr phosphate transfer and the subsequent [H.sub.3]P[O.sub.4] neutral loss are not observed for multiply charged anions of pTyr-peptides. Monoanions of pSer-containing peptides undergo the intramolecular phosphate shift as well, although its efficiency is much lower compared to the aromatic phosphorylation sites. These observations facilitate correct identification of pSer-, pThr-, and pTyr-peptides in CID studies. This work demonstrates that the established phosphate-specific neutral loss fragmentation rules of protonated pTyr-peptides cannot be applied to the CID spectra of their [M - H]- ions.

Enterohemorrhagic Escherichia coli O157:H7 translocates 2 effectors to trigger localized actin assembly in mammalian cells, resulting in filamentous actin 'pedestals.' One effector, the translocated intimin receptor (Tir), is localized in the plasma membrane and clustered upon binding the bacterial outer membrane protein intimin. The second, the proline-rich effector [EspF.sub.u] (aka TccP) activates the actin nucleation-promoting factor WASP/N-WASP, and is recruited to sites of bacterial attachment by a mechanism dependent on an Asn-Pro-Tyr ([NPY.sub.458]) sequence in the Tir C-terminal cytoplasmic domain. Tir, [EspF.sub.u], and N-WASP form a complex, but neither [EspF.sub.u] nor N-WASP bind Tir directly, suggesting involvement of another protein in complex formation. Screening of the mammalian SH3 proteome for the ability to bind [EspF.sub.u] identified the SH3 domain of insulin receptor tyrosine kinase substrate (IRTKS), a factor known to regulate the cytoskeleton. Derivatives of WASP, [EspF.sub.u], and the IRTKS SH3 domain were capable of forming a ternary complex in vitro, and replacement of the C terminus of Tir with the IRTKS SH3 domain resulted in a fusion protein competent for actin assembly in vivo. A second domain of IRTKS, the IRSp53/MIM homology domain (IMD), bound to Tir in a manner dependent on the C-terminal [NPY.sub.458] sequence, thereby recruiting IRTKS to sites of bacterial attachment. Ectopic expression of either the IRTKS SH3 domain or the IMD, or genetic depletion of IRTKS, blocked pedestal formation. Thus, enterohemorrhagic E. coli translocates 2 effectors that bind to distinct domains of a common host factor to promote the formation of a complex that triggers robust actin assembly at the plasma membrane. enterohemorrhagic Escherichia coli | IRSp53/MIM homology domain | IRTKS | N-WASP | SH3 domain

Tyrosine recombinases mediate a wide range of important genetic rearrangement reactions. Models for tyrosine recombinases have been based largely on work done on the integrase of phage lambda and recombinases like Cre, FIp, and XerC/D. All of these recombinases share a common amino acid signature that is important for catalysis. Several conjugative transposons (CTns) encode recombinases that are also members of the tyrosine recombinase family, but the reaction that they catalyze differs in that recombination does not require homology in the attachment sites. In this study, we examine the role of the core-binding (CB) domain of the CTnDOT integrase (IntDOT) that is located adjacent to the catalytic domain of the protein. Since there is no crystal structure for any of the CTn integrases, we began with a predicted three-dimensional structure produced by homology-based modeling. Amino acid substitutions were made at positions predicted by the model to be close to the DNA. Mutant proteins were tested for the ability to mediate integration in vivo and for in vitro DNA-binding, cleavage, and ligation activities. We identified for the first time nonconserved amino acid residues in the CB domain that are important for catalytic activity. Mutant proteins with substitutions at three positions in the CB domain are defective for DNA cleavage but still proficient in ligation. The positions of the residues in the complex suggest that the mutant residues affect the positioning of the cleaved phosphodiester bond in the active site without disruption of the ligation step.

Accumulating evidence suggests that protein tyrosine phosphorylation-based signaling pathways are under the regulation of reactive oxygen species. Although protein tyrosine phosphatases are directly regulated by reversible oxidation, it is not clear whether protein tyrosine kinases (PTKs) are also directly regulated by reduction/oxidation (redox). In this study we report a mechanism of direct oxidative inactivation specific for the PTKs in the Src and fibroblast growth factor receptor (FGFR) families, key enzymes in mammalian signal transduction. Src is fully active when reduced and retains 8-25% of the full activity toward various substrates when oxidized. This inactivation is caused by oxidation of a specific cysteine residue (Cys-277), which results in homodimerization of Src linked by a disulfide bridge. Cys-277 is located in the Gly loop in the catalytic domain. This cysteine residue is conserved only in 8 of the >90 PTKs in the human kinome, including 3 of the 10 Src family kinases and all 4 kinases of the FGFR family. FGFR1 is also reversibly regulated by redox because of this cysteine residue, whereas Csk, a PTK that lacks a cysteine residue at the corresponding position, is not similarly regulated. These results demonstrate a mechanism of direct redox regulation conserved in certain specific PTKs.

Multimass ion imaging techniques were used to study the photodissociation of p-methylphenol, p-ethylphenol and p(2-aminoethyl)phenol which are chormophores of the amino acid tyrosine. The chromophores of amino acid tyrosine show side-chain size-dependent dissociation properties and one dissociation channel, H atom elimination is seen for p-methylphenol and p-ethylphenol.

Src kinase mediates growth factor signaling and causes oncogenic transformation, which includes dramatic changes in the actin cytoskeleton, cell shape, and motility. Cortactin was discovered as a substrate for Src. How phosphorylation of cortactin can enhance actin assembly is unknown. Here, using an actin assembly system reconstituted from purified components, we demonstrate for the first time a biochemical mechanism by which Src phosphorylation of cortactin affects actin assembly. The adaptor Nck is an important component of the system, linking phosphorylated cortactin with neuronal WASp (N-WASp) and WASp-interacting protein (WIP) to activate Arp2/3 complex. N-WASp | Nck | tyrosine phosphorylation

mechanism of [Y.sub.Z] oxidation at cryogenic temperatures. The results have shown the formation and breaking of hydrogen bonds between [Y.sub.Z] and Di-His190 and [Y.sub.D] and D2-His190, respectively, and the oxidation of respective tyrosine at 5 K has demanded that the hydrogen bond be well-defined.

Several techniques are employed to investigate the [super 13]C NMR chemical shifts in phenylalanine and tyrosine in dipeptides, as well as proteins. The analysis shows that the atomic charge correlation shown in such proteins is the same as that shown in aromatic carbocations.

The improved blue fluorescent protein (BFP) is constructed from the green fluorescent protein (GFP) by substituting histidine for tyrosine in the chromophore precursor sequence. These improved, new versions of BFP are useful tools in fluorescent imaging.

The analysis presents an ab intio study of the excites-state deactivation pathways, as well as the lifetimes of the protonated aromatic amino acids, namely tryptophan (Trp[H.sup.+]) and tyrosine ([TyrH.sup.+]). The results show that the lifetime of tyrosine is much longer than that of tryptophan, though various specific photofragments including the formation of a radical cation after hydrogen loss are observed in tryptophan and not tyrosine.

The remote SH2 domain in each of the substrates is required to achieve efficient tyrosine phosphorylation by Itk and extend this observation to two other Tec family kinases, Btk and Tec. At the same time a stable interaction between the substrate SH2 domains and the kinase domain of Itk is detected and found that addition of specific, exogenous SH2 domains to the in vitro kinase assay competes directly with substrate phosphorylation.

Interleukin-2 tyrosine kinase (Itk) is used as a model system to gain insight into the regulatory apparatus of the Tec kinases. Use of a quantitative in vitro kinase assay uncovered an essential role for the short linker region flanked by the SH2 and kinase domains of Itk in positively regulating Itk catalytic activity.

Laser-induced fluorescence (LIF) spectroscopy and fluorescence-detected ultraviolet (UV)-UV and infrared (IR)-UV double-resonance spectroscopy are used to study the structure of jet-cooled L-tyrosine (L-Tyr). The comparison of the results of L-Tyr and L-phenylalanine have shown that conformers of L-Tyr existing under jet-cooled condition have side-chain conformers similar to conformers B, D, E, and X of L-phenylalanine.

A newly designed amino acid sequence containing one tyrosine residue is employed to study and describe the various proton-coupled electron-transfer reactions in a biomimetic peptide, which are often used as a model for the study of the enzyme regulatory mechanisms in proteins. The results of the analysis show that the thermodynamic coupling increases the yield of the tyrosyl radical at low pH by stabilizing it due to the interstrand [pi]-cation interaction.

The research mainly deals with azurin, a metalloprotein called cupredoxins that along with being involved in the electron transfer, also enters cancer cells and induces apoptosis in them and is very much similar to the ligand called cphrinB2. The binding of azurin with the cognate receptor tyrosine kinase EphB2 initiates Eph/ephrin signaling and highly interferes with the tyrosine phosphorylation, thus facilitating the cancer cell growth inhibition.

The crystal structure of Y98Q at 2.2 Angstrom resolutions was determined to reveal the role of residue Y98 in the photoactive yellow protein (PYP) photocycle. The recovery kinetics of Y98Q/M100A were found to be similar those of M100 than R52A/M100A, proving that the repositioning of R52 caused by the Y98Q mutation does not affect the dark state recovery.

A phosphoproteomic analysis of tyrosine-phosphorylated proteins is carried out in ErbB2-overexpressing breast and ovarian cancer cell lines in order to understand the cellular signaling networks activated by ErbB2. The treatment of cells with SiRNAs against a number of RNA binding proteins have shown that these proteins play a vital role in cell migration, a phenotype associated with tumor metastasis.

A remarkable enhancement of phosphorylation by Src family kinases with no apparent change in kinase activity was observed by mechanically extending bacterially expressed tyrosine phosphorylation of p130Cas (Cas). It is proposed based on the findings that Cas acts as a primary force sensor, transducing force into mechanical extension and thereby priming phosphorylation and activation of downstream signaling.

We compare electron capture dissociation (ECD) of doubly protonated and divalent metal-adducted tyrosine O-sulfated peptides without basic amino acid residues. ECD of doubly protonated Tyr2-sulfated cholecystokinin (CCKS) and doubly protonated Tyrl2-sulfated gastrin II (GST) resulted in complete loss of S[O.sub.3] from all product ions. Thus, contrary to typical ECD behavior, localization of the sulfate groups was not possible. By contrast, ECD of Ca-, Mn-, Zn-, and Fe-adducted CCKS and ECD of deprotonated GST with two calcium adducts, i.e., [[GST + 2Ca - H].sup.3+], resulted in sulfated c'- and [z.sup.*]-type product ions with high sequence coverage, thereby allowing both sequencing and sulfate localization. In addition, divalent metal adduction provided improved positive mode ionization efficiency for these peptides. The drastically different fragmentation behavior observed in ECD of protonated and metal-adducted CCKS and GST, respectively, is proposed to be a consequence of the absence of basic amino acid residues, promoting a mobile proton-like fragmentation mechanism, including abundant sulfate loss, for protonated species. Retention of sulfate groups was also observed in electron detachment dissociation (EDD) of CCKS and GST. However, the EDD fragmentation efficiency was much lower than that of ECD and very limited fragmentation was observed in EDD of GST, precluding localization of the sulfate group in that peptide.

Structural and chemical changes in the [P.sub.M) intermediate of Paracoccus denitrificans cytochrome c oxidase are investigated using infrared spectroscopy with labeled tyrosines and histidine. The results suggest a structural alteration in P(sub M) that is centered on the His276-Pro277-Glu278-Val279-Tyr280 pentapeptide formed by the His-Tyr covalent linkage to mediate the perturbation of the infrared band of the protonated Glu278 headgroup.

NMR studies on the native and denatured states of green fluorescent protein are presented, which gives vital information on the first and last states of protein on the folding pathway. The data suggest that pH-denatured and GdnDCI-denatured states are similar in terms of the chemical environments of the tyrosine residues.

Various researches and studies are conducted to investigate the human topoisomerase 1-meditated DNA relaxation reaction, which changes with the modification of the enzyme at the active site tyrosine of the human DNA. The results demonstrate that only the tyrosine groups having the phenolic group in the normal position with respect to the protein backbone are active and hence lead to varying steric, electronic and stereochemical features in the substance.

The Fourier transform infrared (FT-IR) results obtained from the characterizations of tyrosinate, tyrosyl radical and their isotopologues at 77 K are illustrated. The obtained results are compared to vibrational spectra associated with the oxidation of a redox active tyrosine in photosystem II (PS II), the photosynthetic water splitting enzyme.

Receptor tyrosine kinases (RTK)-mediated signals play key roles in the regulation of various cellular processes, such as control of cell growth, differentiation, metabolism, and migration. Two models of RTK-mediated signaling are presented and the role of the transmembrane (TM) domains is discussed within the framework of these models.

A study in which the N7 hydrogen bond acceptor groups of each guanine and adenine within the consensus sequence are replaced with unnatural 7-deaza bases is described. The double modifications have shown equal destabilizing effects in the transition state and covalent complex leading to slower rates of cleavage and less damaging effects on ligation.

The role of a cluster of conserved tyrosines in rubisco structure and in vivo degradation is investigated by examining the site-directed mutants of these residues in Chlamydomonas reinhardtii. The results have shown that the tyrosine cluster around the betaA-betaB loop of rubisco small subunit plays a stabilizing role by affecting the catalytic activity and the degradation rate of the enzyme in stressed cells.

An experiment was carried out to show that dimeric radical cations of tryptophan and tyrosine derivatives could be generated by collision-induced dissociation of copper(II) complexes formed via electrospray. The results of experiments with a derivative of the methyl ether of tyrosine, a derivative that deters hydrogen bonding, are in accordance with an interpretation of intermolecular hydrogen bonding (between ligands) within the dimeric radical cations.

Alzheimer's disease (AD) is a progressive amnestic dementia typified by abnormal modifications of the microtubule (MT)-associated tau protein that promote its pathological self-assembly and displacement from the MT lattice. The results suggest that peroxynitrite-mediated modifications stabilize tau filaments through 3,3'-DT bonding and destabilize MTs by site-selective nitration of tau monomers.

A binuclear copper enzyme, a mu-eta(super 2):eta(super 2)-peroxodicopper (II) species is accepted generally to be the active oxidant, in tyrosinase. Reports reveal the characterization and reactivity of a mu-eta(super 2):eta(super 2)-peroxodicopper (II) complex synthesized by reacting the Cu(I) complex of the secondary diamine ligand N,N'-di-tert-butyl-ethylenediamine (DBED), ((DBED)Cu(MeCN))(X) with O2 at 193 K to give ({Cu(DBED)}2(O2))(X2).

A study investigates photochemical H/D exchange in L-tyrosine and related compounds in D2O solution. The regioselectivity of the reaction is determined and strong support obtained for a photoactivated Birch reduction/protonation/oxidation/deprotonation cycle as the mechanistic basis for H/D exchange.

The kinetics of reduction of ferric tyrosine hydroxylase (TyrH) by several reductants is determined by anaerobic stopped-flow spectrometry. The S40 TyrH, which mimics the phosphorylated state of the enzyme, has oxidation and reduction kinetics similar to those of wild-type enzyme, indicating that phosphorylation does not directly regulate the interconversion of the ferric and ferrous forms.

It is shown that laminin-1 binds to alphaDG (alpha-dystroglycan) via LG4-5, causing syntrophin to become phosphorylated on a tyrosine and allowing Grb2 to bind via its SH2 domain. A model for how this phosphorylation may initiate downstream events, such as binding of Sos1/2 or syntrophin phosphorylation on tryosine, in laminin signaling is presented.

The different methods for calculating the relative energetic of alternative bis(oxo) and peroxo isomers of Cu2O2 cores supported by 0, 2, 4 and 6 ammonia ligands are discussed. CCSD(T) model is less accurate and exhibits poor convergence in predicted relative energies.