SYPRO™ Orange Protein Gel Stain (5000X Concentrate in DMSO), 500 μL - Citations
SYPRO™ Orange Protein Gel Stain (5000X Concentrate in DMSO), 500 μL - Citations
View additional product information for SYPRO™ Protein Gel Stains - Citations (S12000, S12001, S6650, S6651, S12010, S12000X3, S6654, S6653, S21900)
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Abstract
LXXLL-related motifs in Dax-1 have target specificity for the orphan nuclear receptors Ad4BP/SF-1 and LRH-1.
AuthorsSuzuki T, Kasahara M, Yoshioka H, Morohashi K, Umesono K
JournalMol Cell Biol
PubMed ID12482977
The orphan receptor Ad4BP/SF-1 (NR5A1) is a constitutive activator, and its activity is repressed by another orphan receptor, Dax-1 (NR0B1). In the present study, we investigated the molecular mechanisms underlying this repression by Dax-1. Yeast two-hybrid and transient-transfection assays confirmed the necessity of three LXXLL-related motifs in Dax-1 for interaction ... More
Translational regulation of prostaglandin endoperoxide H synthase-1 mRNA in megakaryocytic MEG-01 cells. Specific protein binding to a conserved 20-nucleotide CIS element in the 3'-untranslated region.
AuthorsDuquette M, Laneuville O
JournalJ Biol Chem
PubMed ID12237309
Prostaglandin endoperoxide H synthase-1 (PGHS-1) is an abundant enzyme in platelets, where it plays a key role in the cascade of prostanoid formation. In platelets, the primary site of PGHS-1 synthesis is in precursor megakaryocytic cells. We have previously shown that in megakaryocytic MEG-01 cells, TPA induces an increase of ... More
Type I collagen is thermally unstable at body temperature.
AuthorsLeikina E, Mertts MV, Kuznetsova N, Leikin S
JournalProc Natl Acad Sci U S A
PubMed ID11805290
Molecular characterization of Saccharomyces cerevisiae TFIID.
AuthorsSanders SL, Garbett KA, Weil PA
JournalMol Cell Biol
PubMed ID12138208
'We previously defined Saccharomyces cerevisiae TFIID as a 15-subunit complex comprised of the TATA binding protein (TBP) and 14 distinct TBP-associated factors (TAFs). In this report we give a detailed biochemical characterization of this general transcription factor. We have shown that yeast TFIID efficiently mediates both basal and activator-dependent transcription ... More
Mechanism of calcium-independent synaptotagmin binding to target SNAREs.
AuthorsRickman C, Davletov B
JournalJ Biol Chem
PubMed ID12496268
'Synaptic vesicle exocytosis requires three SNARE (soluble N-ethylmaleimide-sensitive-factor attachment protein receptor) proteins: syntaxin and SNAP-25 on the plasma membrane (t-SNAREs) and synaptobrevin/VAMP on the synaptic vesicles (v-SNARE). Vesicular synaptotagmin 1 is essential for fast synchronous SNARE-mediated exocytosis and interacts with the SNAREs in brain material. To uncover the step at ... More
Energy-driven subunit rotation at the interface between subunit a and the c oligomer in the F(O) sector of Escherichia coli ATP synthase.
AuthorsHutcheon ML, Duncan TM, Ngai H, Cross RL
JournalProc Natl Acad Sci U S A
PubMed ID11438702
'Subunit rotation within the F(1) catalytic sector of the ATP synthase has been well documented, identifying the synthase as the smallest known rotary motor. In the membrane-embedded F(O) sector, it is thought that proton transport occurs at a rotor/stator interface between the oligomeric ring of c subunits (rotor) and the ... More
Analysis of tear protein patterns of dry-eye patients using fluorescent staining dyes and two-dimensional quantification algorithms.
AuthorsGrus FH, Sabuncuo P, Augustin AJ
JournalElectrophoresis
PubMed ID11425241
'Tear proteins of nonstimulated tears of 29 patients (healthy subjects, n = 8; dry-eye syndrome patients, n = 12; diabetic dry-eye patients, n = 9) were electrophoretically separated and stained by SYPRO Orange, followed by Coomassie blue staining. Both, the fluorescent and the Coomassie stains were subsequently analyzed by an ... More
Calreticulin recognizes misfolded HLA-A2 heavy chains.
AuthorsMancino L, Rizvi SM, Lapinski PE, Raghavan M
JournalProc Natl Acad Sci U S A
PubMed ID11983893
'Our studies investigated functional interactions between calreticulin, an endoplasmic reticulum chaperone, and major histocompatibility complex (MHC) class I molecules. Using in vitro thermal aggregation assays, we established that calreticulin can inhibit heat-induced aggregation of soluble, peptide-deficient HLA-A2 purified from supernatants of insect cells. The presence of HLA-A2-specific peptides also inhibits ... More
Regulation of microfilament organization by Kaposi sarcoma-associated herpes virus-cyclin.CDK6 phosphorylation of caldesmon.
AuthorsCuomo ME, Knebel A, Platt G, Morrice N, Cohen P, Mittnacht S
JournalJ Biol Chem
PubMed ID16115893
'Kaposi sarcoma-associated herpes virus (KSHV) encodes a D-like cyclin (K-cyclin) that is thought to contribute to the viral oncogenicity. K-cyclin activates cellular cyclin-dependent kinases (CDK) 4 and 6, generating enzymes with a substrate selectivity deviant from CDK4 and CDK6 activated by D-type cyclins, suggesting different biochemical and biological functions. Here ... More
Naturally occurring mutations in human mitochondrial pre-tRNASer(UCN) can affect the transfer ribonuclease Z cleavage site, processing kinetics, and substrate secondary structure.
AuthorsYan H, Zareen N, Levinger L
JournalJ Biol Chem
PubMed ID16361254
'tRNAs are transcribed as precursors with a 5'' end leader and a 3'' end trailer. The 5'' end leader is processed by RNase P, and in most organisms in all three kingdoms, transfer ribonuclease (tRNase) Z can endonucleolytically remove the 3'' end trailer. Long ((L)) and short ((S)) forms of ... More
Formation of a carboxy-terminal domain phosphatase (Fcp1)/TFIIF/RNA polymerase II (pol II) complex in Schizosaccharomyces pombe involves direct interaction between Fcp1 and the Rpb4 subunit of pol II.
AuthorsKimura M, Suzuki H, Ishihama A
JournalMol Cell Biol
PubMed ID11839823
'In transcriptional regulation, RNA polymerase II (pol II) interacts and forms complexes with a number of protein factors. To isolate and identify the pol II-associated proteins, we constructed a Schizosaccharomyces pombe strain carrying a FLAG tag sequence fused to the rpb3 gene encoding the pol II subunit Rpb3. By immunoaffinity ... More
Characterization and binding specificity of the monomeric STAT3-SH2 domain.
AuthorsHaan S, Hemmann U, Hassiepen U, Schaper F, Schneider-Mergener J, Wollmer A, Heinrich PC, Grötzinger J
JournalJ Biol Chem
PubMed ID9880505
'Signal transducers and activators of transcription (STATs) are important mediators of cytokine signal transduction. STAT factors are recruited to phosphotyrosine-containing motifs of activated receptor chains via their SH2 domains. The subsequent tyrosine phosphorylation of the STATs leads to their dissociation from the receptor, dimerization, and translocation to the nucleus. Here ... More
Social exploitation of vitellogenin.
AuthorsAmdam GV, Norberg K, Hagen A, Omholt SW
JournalProc Natl Acad Sci U S A
PubMed ID12566563
'Vitellogenin is a female-specific glucolipoprotein yolk precursor produced by all oviparous animals. Vitellogenin expression is under hormonal control, and the protein is generally synthesized directly before yolk deposition. In the honeybee (Apis mellifera), vitellogenin is not only synthesized by the reproductive queen, but also by the functionally sterile workers. In ... More
High processivity of the reverse transcriptase from a non-long terminal repeat retrotransposon.
AuthorsBibillo A, Eickbush TH
JournalJ Biol Chem
PubMed ID12101182
'R2 is a retrotransposable element that specifically inserts into the 28 S rRNA genes of arthropods. The element encodes a single protein with endonuclease activity that cleaves the 28 S gene target site and reverse transcriptase (RT) activity that uses the cleaved DNA to prime reverse transcription. Here we compare ... More
Membrane binding modulates the quaternary structure of CTP:phosphocholine cytidylyltransferase.
AuthorsXie M, Smith JL, Ding Z, Zhang D, Cornell RB
JournalJ Biol Chem
PubMed ID15069071
'CTP:phosphocholine cytidylyltransferase (CCT), a key enzyme that controls phosphatidylcholine synthesis, is regulated by reversible interactions with membranes containing anionic lipids. Previous work demonstrated that CCT is a homodimer. In this work we show that the structure of the dimer interface is altered upon encountering membranes that activate CCT. Chemical cross-linking ... More
Mapping the binding site of the neuroprotectant ifenprodil on NMDA receptors.
AuthorsPerin-Dureau F, Rachline J, Neyton J, Paoletti P
JournalJ Neurosci
PubMed ID12122058
'Ifenprodil is a noncompetitive antagonist of NMDA receptors highly selective for the NMDA receptor 2B (NR2B) subunit. It is widely used as a pharmacological tool to discriminate subpopulations of NMDA receptors, and derivatives are currently being developed as candidate neuroprotectants. Despite numerous studies on the mechanism of action of ifenprodil ... More
Distinct peptide signals in the UmuD and UmuD' subunits of UmuD/D' mediate tethering and substrate processing by the ClpXP protease.
AuthorsNeher SB, Sauer RT, Baker TA
JournalProc Natl Acad Sci U S A
PubMed ID14595014
'The Escherichia coli UmuD'' protein is a component of DNA polymerase V, an error-prone polymerase that carries out translesion synthesis on damaged DNA templates. The intracellular concentration of UmuD'' is strictly controlled by regulated transcription, by posttranslational processing of UmuD to UmuD'', and by ClpXP degradation. UmuD'' is a substrate ... More
Practical aspects of fluorescent staining for proteomic applications.
'SYPRO Orange and SYPRO Ruby staining methods, modified for use with large-format two dimensional (2-D) gels, are compared to the manufacturer''s recommended protocols to determine sensitivity and reproducibility of the new methods. This study examines the critical aspects of fixation, washing, and staining to develop an optimized fluorescent staining method. ... More
Two lipid-packing sensor motifs contribute to the sensitivity of ArfGAP1 to membrane curvature.
AuthorsMesmin B, Drin G, Levi S, Rawet M, Cassel D, Bigay J, Antonny B
JournalBiochemistry
PubMed ID17253781
'ArfGAP1 (Arf GTPase activating protein 1) controls the cycling of the COPI coat on Golgi membranes by catalyzing GTP hydrolysis in the small G protein Arf1. ArfGAP1 contains a central motif named ALPS (ArfGAP1 lipid-packing sensor) that adsorbs preferentially onto highly curved membranes. This motif allows coupling of the rate ... More
Partial structure of glutamic acid and alanine-rich protein, a major surface glycoprotein of the insect stages of Trypanosoma congolense.
AuthorsThomson LM, Lamont DJ, Mehlert A, Barry JD, Ferguson MA
JournalJ Biol Chem
PubMed ID12368279
'The tsetse fly transmitted salivarian trypanosome, Trypanosoma congolense of the subgenus Nanomonas, is the most significant of the trypanosomes with respect to the pathology of livestock in sub-Saharan Africa. Unlike the related trypanosome Trypanosoma brucei of the subgenus Trypanozoon, the major surface molecules of the insect stages of T. congolense ... More
A photochemical approach to the lipid accessibility of engineered cysteinyl residues.
AuthorsLi J, Shi L, Karlin A
JournalProc Natl Acad Sci U S A
PubMed ID12533666
'Ordinary electrophilic reagents react too slowly in a nonpolar environment to be useful for the determination of the accessibility to lipid of continuous stretches of residues mutated to cysteine. By contrast, photoactivated 5-iodonaphthyl-1-azide (INA) reacted readily with 2-mercaptoethanol and dodecanethiol in nonpolar solvents and in liposomes. Continuous stretches of residues ... More
Adenovirus E1A N-terminal amino acid sequence requirements for repression of transcription in vitro and in vivo correlate with those required for E1A interference with TBP-TATA complex formation.
AuthorsBoyd JM, Loewenstein PM, Tang Qq QQ, Yu L, Green M
JournalJ Virol
PubMed ID11773419
'The adenovirus (Ad) E1A 243R oncoprotein encodes an N-terminal transcription repression domain that is essential for early viral functions, cell immortalization, and cell transformation. The transcription repression function requires sequences within amino acids 1 to 30 and 48 to 60. To elucidate the roles of the TATA-binding protein (TBP), p300, ... More
Inhibition of SAPK2a/p38 prevents hnRNP A0 phosphorylation by MAPKAP-K2 and its interaction with cytokine mRNAs.
AuthorsRousseau S, Morrice N, Peggie M, Campbell DG, Gaestel M, Cohen P
JournalEMBO J
PubMed ID12456657
'Lipopolysaccharide (LPS) stimulates production of inflammatory mediators, partly by stabilizing [interleukin-6 (IL-6), cyclooxygenase 2 (COX-2)] and/or stimulating translation [tumour necrosis factor-alpha (TNF-alpha)] of their mRNAs. Such regulation depends on AU-rich elements (AREs) within the 3''-untranslated regions and is partially suppressed by SB 203580 (which inhibits SAPK2a/p38). The LPS-induced production of ... More
Protein sizing on a microchip.
AuthorsBousse L, Mouradian S, Minalla A, Yee H, Williams K, Dubrow R
JournalAnal Chem
PubMed ID11305653
'We have developed a microfabricated analytical device on a glass chip that performs a protein sizing assay, by integrating the required separation, staining, virtual destaining, and detection steps. To obtain a universal noncovalent fluorescent labeling method, we have combined on-chip dye staining with a novel electrophoretic dilution step. Denatured protein-sodium ... More
Detection technologies in proteome analysis.
AuthorsPatton WF
JournalJ Chromatogr B Analyt Technol Biomed Life Sci
PubMed ID12015990
'Common strategies employed for general protein detection include organic dye, silver stain, radiolabeling, reverse stain, fluorescent stain, chemiluminescent stain and mass spectrometry-based approaches. Fluorescence-based protein detection methods have recently surpassed conventional technologies such as colloidal Coomassie blue and silver staining in terms of quantitative accuracy, detection sensitivity, and compatibility with ... More
PRiMA: the membrane anchor of acetylcholinesterase in the brain.
'As a tetramer, acetylcholinesterase (AChE) is anchored to the basal lamina of the neuromuscular junction and to the membrane of neuronal synapses. We have previously shown that collagen Q (ColQ) anchors AChE at the neuromuscular junction. We have now cloned the gene PRiMA (proline-rich membrane anchor) encoding the AChE anchor ... More
New aldehyde tag sequences identified by screening formylglycine generating enzymes in vitro and in vivo.
AuthorsRush JS, Bertozzi CR,
JournalJ Am Chem Soc
PubMed ID18722427
'Formylglycine generating enzyme (FGE) performs a critical posttranslational modification of type I sulfatases, converting cysteine within the motif CxPxR to the aldehyde-bearing residue formylglycine (FGly). This concise motif can be installed within heterologous proteins as a genetically encoded ' ... More
Protein stains for proteomic applications: which, when, why?
AuthorsMiller I, Crawford J, Gianazza E,
JournalProteomics
PubMed ID16991193
'This review recollects literature data on sensitivity and dynamic range for the most commonly used colorimetric and fluorescent dyes for general protein staining, and summarizes procedures for the most common PTM-specific detection methods. It also compiles some important points to be considered in imaging and evaluation. In addition to theoretical ... More
VprBP binds full-length RAG1 and is required for B-cell development and V(D)J recombination fidelity.
AuthorsKassmeier MD, Mondal K, Palmer VL, Raval P, Kumar S, Perry GA, Anderson DK, Ciborowski P, Jackson S, Xiong Y, Swanson PC,
JournalEMBO J
PubMed ID22157821
'The N-terminus of full-length RAG1, though dispensable for RAG1/2 cleavage activity, is required for efficient V(D)J recombination. This region supports RING E3 ubiquitin ligase activity in vitro, but whether full-length RAG1 functions as a single subunit or a multi-subunit E3 ligase in vivo is unclear. We show the multi-subunit cullin ... More
Thermofluor-based high-throughput stability optimization of proteins for structural studies.
AuthorsEricsson UB, Hallberg BM, Detitta GT, Dekker N, Nordlund P
JournalAnal Biochem
PubMed ID16962548
'Production of proteins well suited for structural studies is inherently difficult and time-consuming. Protein sample homogeneity, stability, and solubility are strongly correlated with the proteins'' probability of yielding crystals, and optimization of these properties will improve success rates of crystallization. In the current study, we applied the thermofluor method as ... More
The DNA binding properties of the Escherichia coli RecQ helicase.
AuthorsDou SX, Wang PY, Xu HQ, Xi XG
JournalJ Biol Chem
PubMed ID14665634
'The RecQ helicase family is highly conserved from bacteria to men and plays a conserved role in the preservation of genome integrity. Its deficiency in human cells leads to a marked genomic instability that is associated with premature aging and cancer. To determine the thermodynamic parameters for the interaction of ... More
The Cdc42 binding and scaffolding activities of the fission yeast adaptor protein Scd2.
AuthorsEndo M, Shirouzu M, Yokoyama S
JournalJ Biol Chem
PubMed ID12409291
'The small GTP-binding protein Cdc42, the guanine nucleotide exchange factor Scd1, the p21-activated kinase Shk1, and the adaptor protein Scd2 are involved in the Cdc42-dependent signaling cascade in fission yeast. In the present study, we analyzed the Cdc42 binding and scaffolding activities of Scd2 by co-precipitation assays. We found that ... More
Fluorescent imaging and analysis with Typhoon 8600.
AuthorsMcNamara P, Lew W, Han L
JournalElectrophoresis
PubMed ID11332750
'Current technologies in laser-based imaging systems for life science applications offer highly sensitive and quantitative detection of multiple labels. Typhoon 8600 variable mode imager is capable of fluorescence, storage phosphor, and chemiluminescence detection of gels and blots. The system delivers sensitive fluorescent detection of nucleic acids and proteins in both ... More
Evaluation of fluorescence-based thermal shift assays for hit identification in drug discovery.
AuthorsLo MC, Aulabaugh A, Jin G, Cowling R, Bard J, Malamas M, Ellestad G
JournalAnal Biochem
PubMed ID15301960
'The fluorescence-based thermal shift assay is a general method for identification of inhibitors of target proteins from compound libraries. Using an environmentally sensitive fluorescent dye to monitor protein thermal unfolding, the ligand-binding affinity can be assessed from the shift of the unfolding temperature (Delta Tm) obtained in the presence of ... More
Analysis of the interaction between the transcription factor sigmaG and the anti-sigma factor SpoIIAB of Bacillus subtilis.
AuthorsEvans L, Clarkson J, Yudkin MD, Errington J, Feucht A
JournalJ Bacteriol
PubMed ID12867473
'The activation of sigma(G), a transcription factor, in Bacillus subtilis is coupled to the completion of engulfment during sporulation. SpoIIAB, an anti-sigma factor involved in regulation of sigma(F), is also shown to form a complex with sigma(G) in vitro. SpoIIAA, the corresponding anti-anti-sigma factor, can disrupt the SpoIIAB:sigma(G) complex, releasing ... More
Biochemical and molecular analyses of the Streptococcus pneumoniae acyl carrier protein synthase, an enzyme essential for fatty acid biosynthesis.
AuthorsMcAllister KA, Peery RB, Meier TI, Fischl AS, Zhao G
JournalJ Biol Chem
PubMed ID10903317
'Acyl carrier protein synthase (AcpS) is an essential enzyme in the biosynthesis of fatty acids in all bacteria. AcpS catalyzes the transfer of 4''-phosphopantetheine from coenzyme A (CoA) to apo-ACP, thus converting apo-ACP to holo-ACP that serves as an acyl carrier for the biosynthesis of fatty acids and lipids. To ... More
The recombinant Azotobacter vinelandii mannuronan C-5-epimerase AlgE4 epimerizes alginate by a nonrandom attack mechanism.
AuthorsHøidal HK, Ertesvåg H, Skjåk-Braek G, Stokke BT, Valla S
JournalJ Biol Chem
PubMed ID10212201
'The Ca2+-dependent mannuronan C-5-epimerase AlgE4 is a representative of a family of Azotobacter vinelandii enzymes catalyzing the polymer level epimerization of beta-D-mannuronic acid (M) to alpha-L-guluronic acid (G) in the commercially important polysaccharide alginate. The reaction product of recombinantly produced AlgE4 is predominantly characterized by an alternating sequence distribution of ... More
Vitamin analysis using capillary zone electrophoresis.
AuthorsNeubert R, Schiewe J
JournalAm Biotechnol Lab
PubMed ID8867838
Perspective: proteomics--see "spots" run.
AuthorsKopchick JJ, List EO, Kohn DT, Keidan GM, Qiu L, Okada S
JournalEndocrinology
PubMed ID12021159
Structural evidence that human acetylcholinesterase inhibited by tabun ages through O-dealkylation.
AuthorsCarletti E, Colletier JP, Dupeux F, Trovaslet M, Masson P, Nachon F,
JournalJ Med Chem
PubMed ID20408548
Tabun is a warfare agent that inhibits human acetylcholinesterase (hAChE) by rapid phosphylation of the catalytic serine. A time-dependent reaction occurs on the tabun adduct, leading to an ... More
Introducing genetically encoded aldehydes into proteins.
AuthorsCarrico IS, Carlson BL, Bertozzi CR
JournalNat Chem Biol
PubMed ID17450134
Methods for introducing bioorthogonal functionalities into proteins have become central to protein engineering efforts. Here we describe a method for the site-specific introduction of aldehyde groups into recombinant proteins using the 6-amino-acid consensus sequence recognized by the formylglycine-generating enzyme. This genetically encoded 'aldehyde tag' is no larger than a His(6) ... More
High resolution crystal structure of the catalytic domain of ADAMTS-5 (aggrecanase-2).
Aggrecanase-2 (a disintegrin and metalloproteinase with thrombospondin motifs-5 (ADAMTS-5)), a member of the ADAMTS protein family, is critically involved in arthritic diseases because of its direct role in cleaving the cartilage component aggrecan. The catalytic domain of aggrecanase-2 has been refolded, purified, and crystallized, and its three-dimensional structure determined to ... More
Detection of fluorescence dye-labeled proteins in 2-D gels using an Arthur 1442 Multiwavelength Fluoroimager.
AuthorsHerick K, Jackson P, Wersch G, Burkovski A
JournalBiotechniques
PubMed ID11464508
Labeling of proteins with SYPRO Orange, SYPRO Red, and SYPRO Ruby after 2-D polyacrylamide gel electrophoresis (PAGE) using plastic-backed immobilized pH gradient (IPG) strips and precast SDS polyacrylamide gels was tested. Protein spots were detected using an Arthur 1442 Multiwavelength Fluoroimager. The labeling methods described allow detection of proteins both ... More
High throughput methods of assessing protein stability and aggregation.
AuthorsSenisterra GA, Finerty PJ,
JournalMol Biosyst
PubMed ID19225610
The significant increase in the demand for purified protein for crystallization and structural studies has made necessary the development of multi-sample methods for identifying solution conditions that affect protein stability and aggregation. Conditions that stabilize proteins can improve protein purification and crystallization. These methods can be used to identify small ... More
Altering dimerization specificity by changes in surface electrostatics.
AuthorsNohaile MJ, Hendsch ZS, Tidor B, Sauer RT
JournalProc Natl Acad Sci U S A
PubMed ID11248040
Arc repressor forms a homodimer in which the subunits intertwine to create a single globular domain. To obtain Arc sequences that fold preferentially as heterodimers, variants with surface patches of excess positive or negative charge were designed. Several but not all oppositely charged sequence pairs showed preferential heterodimer formation. In ... More
Latent ClpX-recognition signals ensure LexA destruction after DNA damage.
AuthorsNeher SB, Flynn JM, Sauer RT, Baker TA
JournalGenes Dev
PubMed ID12730132
The DNA-damage response genes in bacteria are up-regulated when LexA repressor undergoes autocatalytic cleavage stimulated by activated RecA protein. Intact LexA is stable to intracellular degradation but its auto-cleavage fragments are degraded rapidly. Here, both fragments of LexA are shown to be substrates for the ClpXP protease. ClpXP recognizes these ... More
Drosophila RNase Z processes mitochondrial and nuclear pre-tRNA 3' ends in vivo.
AuthorsDubrovsky EB, Dubrovskaya VA, Levinger L, Schiffer S, Marchfelder A
JournalNucleic Acids Res
PubMed ID14715923
Although correct tRNA 3' ends are crucial for protein biosynthesis, generation of mature tRNA 3' ends in eukaryotes is poorly understood and has so far only been investigated in vitro. We report here for the first time that eukaryotic tRNA 3' end maturation is catalysed by the endonuclease RNase Z ... More
Enhanced binding of altered H-NS protein to flagellar rotor protein FliG causes increased flagellar rotational speed and hypermotility in Escherichia coli.
AuthorsDonato GM, Kawula TH
JournalJ Biol Chem
PubMed ID9727020
H-NS is an Escherichia coli nucleoid protein known only to function as a modulator of gene expression. In this study, we found that specific single amino acid substitutions in H-NS caused an approximately 50% increase in flagellum rotational speed. In fluorescence anisotropy and chemical cross-linking assays, H-NS interacted with the ... More
Assessment of fluorochromes for two-photon laser scanning microscopy of biofilms.
AuthorsNeu TR, Kuhlicke U, Lawrence JR
JournalAppl Environ Microbiol
PubMed ID11823234
A major limitation for the use of two-proton laser scanning microscopy (2P-LSM) in biofilm and other studies is the lack of a thorough understanding of the excitation-emission responses of potential fluorochromes. In order to use 2P-LSM, the utility of various fluorochromes and probes specific for a range of biofilm constituents ... More
Cell-free N-terminal protein labeling using initiator suppressor tRNA.
A highly efficient method for the introduction of fluorophores and other markers at the N terminus of proteins produced in a cell-free extract has been developed. The method utilizes an amber (CUA) initiator suppressor tRNA chemically aminoacylated with a fluorophore-amino acid conjugate which is introduced into an Escherichia coli S30 ... More
Adhesion of single bacterial cells in the micronewton range.
AuthorsTsang PH, Li G, Brun YV, Freund LB, Tang JX
JournalProc Natl Acad Sci U S A
PubMed ID16585522
The adhesion of bacteria to surfaces plays critical roles in the environment, disease, and industry. In aquatic environments, Caulobacter crescentus is one of the first colonizers of submerged surfaces. Using a micromanipulation technique, we measured the adhesion force of single C. crescentus cells attached to borosilicate substrates through their adhesive ... More
Functional assignment of solute-binding proteins of ABC transporters using a fluorescence-based thermal shift assay.
AuthorsGiuliani SE, Frank AM, Collart FR,
JournalBiochemistry
PubMed ID19063603
We have used a fluorescence-based thermal shift (FTS) assay to identify amino acids that bind to solute-binding proteins in the bacterial ABC transporter family. The assay was validated with a set of six proteins with known binding specificity and was consistently able to map proteins with their known binding ligands. ... More
High-throughput thermal scanning: a general, rapid dye-binding thermal shift screen for protein engineering.
AuthorsLavinder JJ, Hari SB, Sullivan BJ, Magliery TJ,
JournalJ Am Chem Soc
PubMed ID19292479
The low stability of natural proteins often limits their use in therapeutic, industrial, and research applications. The scale and throughput of methods such as circular dichroism, fluorescence spectroscopy, and calorimetry severely limit the number of variants that can be examined. Here we demonstrate a high-throughput thermal scanning (HTTS) method for ... More
Methods for protein characterization by mass spectrometry, thermal shift (ThermoFluor) assay, and multiangle or static light scattering.
AuthorsNettleship JE, Brown J, Groves MR, Geerlof A,
JournalMethods Mol Biol
PubMed ID18542872
Mass spectrometry (MS) is widely used within structural and functional proteomics for a variety of tasks including protein quality assessment, identification, and characterization. MS is used routinely for the determination of the total mass of proteins, including N-glycosylated proteins, analysis of selenomethionine incorporation, crystal content verification, and analysis of N-glycosylation ... More
Application of high-throughput isothermal denaturation to assess protein stability and screen for ligands.
AuthorsSenisterra GA, Soo Hong B, Park HW, Vedadi M,
JournalJ Biomol Screen
PubMed ID18448703
Many diseases in humans are caused by mutations that decrease the stability of specific proteins or increase their susceptibility to aggregation. Consequently, the availability of high-throughput methods for assessing protein stability and aggregation properties under physiological conditions (e.g., 37 degrees C) is necessary to analyze physicochemical properties under conditions that ... More
Chemical screening methods to identify ligands that promote protein stability, protein crystallization, and structure determination.
AuthorsVedadi M, Niesen FH, Allali-Hassani A, Fedorov OY, Finerty PJ, Wasney GA, Yeung R, Arrowsmith C, Ball LJ, Berglund H, Hui R, Marsden BD, Nordlund P, Sundstrom M, Weigelt J, Edwards AM,
JournalProc Natl Acad Sci U S A
PubMed ID17035505
The 3D structures of human therapeutic targets are enabling for drug discovery. However, their purification and crystallization remain rate determining. In individual cases, ligands have been used to increase the success rate of protein purification and crystallization, but the broad applicability of this approach is unknown. We implemented two screening ... More
New inhibitors of prion replication that target the amyloid precursor.
AuthorsCharvériat M, Reboul M, Wang Q, Picoli C, Lenuzza N, Montagnac A, Nhiri N, Jacquet E, Guéritte F, Lallemand JY, Deslys JP, Mouthon F,
JournalJ Gen Virol
PubMed ID19264641
At present, there is no effective therapy for any of the neurodegenerative amyloidoses, despite renewed efforts to identify compounds active against the various implicated pathogenetic molecules. We have screened a library of 2960 natural and synthetic compounds in two cell lines chronically infected with mouse prions, and have identified eight ... More
Cellulase production from spent lignocellulose hydrolysates by recombinant Aspergillus niger.
AuthorsAlriksson B, Rose SH, van Zyl WH, Sjöde A, Nilvebrant NO, Jönsson LJ,
JournalAppl Environ Microbiol
PubMed ID19251882
A recombinant Aspergillus niger strain expressing the Hypocrea jecorina endoglucanase Cel7B was grown on spent hydrolysates (stillage) from sugarcane bagasse and spruce wood. The spent hydrolysates served as excellent growth media for the Cel7B-producing strain, A. niger D15[egI], which displayed higher endoglucanase activities in the spent hydrolysates than in standard ... More
Histometrics: improvement of the dynamic range of fluorescently stained proteins resolved in electrophoretic gels using hyperspectral imaging.
AuthorsWoodward AM, Kaderbhai N, Kaderbhai M, Shaw A, Rowland J, Kell DB,
JournalProteomics
PubMed ID11922594
Most image-based analyses, using absorbance or fluorescence of the spatial distribution of identifiable structures in complex biological systems, use only a very small number of dimensions of possible spectral data for the generation and interpretation of the image. We here extend the concepts of hyperspectral imaging, being developed in remote ... More
The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability.
AuthorsNiesen FH, Berglund H, Vedadi M,
JournalNat Protoc
PubMed ID17853878
Differential scanning fluorimetry (DSF) is a rapid and inexpensive screening method to identify low-molecular-weight ligands that bind and stabilize purified proteins. The temperature at which a protein unfolds is measured by an increase in the fluorescence of a dye with affinity for hydrophobic parts of the protein, which are exposed ... More
Protein gel staining methods: an introduction and overview.
AuthorsSteinberg TH,
JournalMethods Enzymol
PubMed ID19892191
Laboratory scientists who encounter protein biochemistry in many of its myriad forms must often ask: is my protein pure? The most frequent response: run a denaturing SDS polyacrylamide gel. Running this gel raises another series of considerations regarding detection, quantitation, and characterization and so the next questions invariably center on ... More
Reversible, allosteric small-molecule inhibitors of regulator of g protein signaling proteins.
AuthorsBlazer LL, Roman DL, Chung A, Larsen MJ, Greedy BM, Husbands SM, Neubig RR,
JournalMol Pharmacol
PubMed ID20571077
Regulators of G protein signaling (RGS) proteins are potent negative modulators of G protein signaling and have been proposed as potential targets for small-molecule inhibitor development. We report a high-throughput time-resolved fluorescence resonance energy transfer screen to identify inhibitors of RGS4 and describe the first reversible small-molecule inhibitors of an ... More
Multiparameter assessments to determine the effects of sugars and antimicrobials on a polymicrobial oral biofilm.
AuthorsYang Y, Sreenivasan PK, Subramanyam R, Cummins D,
JournalAppl Environ Microbiol
PubMed ID17021225
Clinical studies indicate relationships between dental plaque, a naturally formed biofilm, and oral diseases. The crucial role of nonmicrobial biofilm constituents in maintaining biofilm structure and biofilm-specific attributes, such as resistance to shear and viscoelasticity, is increasingly recognized. Concurrent analyses of the diverse nonmicrobial biofilm components for multiparameter assessments formed ... More
The pepR gene of Lactobacillus sakei is positively regulated by anaerobiosis at the transcriptional level.
AuthorsChampomier-Vergès MC, Marceau A, Méra T, Zagorec M
JournalAppl Environ Microbiol
PubMed ID12147484
Lactobacillus sakei is a lactic acid bacterium belonging to the natural flora of meat products. It constitutes the main flora of vacuum-packed meat and is largely used in western Europe as a starter for the manufacturing of fermented sausages. This species is able to grow both under aerobiosis and anaerobiosis. ... More
Optimal filter combinations for photographing SYPRO orange or SYPRO red dye-stained gels.
AuthorsSteinberg TH, White HM, Singer VL
JournalAnal Biochem
PubMed ID9177736
Photography or electronic image acquisition is required to document results obtained from staining protein gels with the fluorescent SYPRO dyes. We found that, when using Polaroid type 667 or 57 instant films, the choice of optical filter combination and photographic exposure time strongly influences protein detection sensitivity limits. Ultraviolet light-blocking ... More
Problems associated with determining protein concentration: a comparison of techniques for protein estimations.
AuthorsKnight MI, Chambers PJ
JournalMol Biotechnol
PubMed ID12611266
Although a range of methods are available for determining protein concentration, many scientists encounter problems when quantifying proteins in the laboratory. The most commonly used methods for determining protein concentration in a modern biochemistry laboratory would probably be the Lowry and/or the Bradford protein assays. Other techniques, including direct spectrophotometric ... More
Mass spectrometry compatibility of two-dimensional gel protein stains.
As proteomic technology evolves, protein staining sensitivity is constantly being improved, enabling researchers to better visualize the proteome of their system. The current challenge is to balance the limits of detection of protein visualization with those of the mass spectrometric methods. In this report, mass spectra generated from human serum ... More
Regulation of mitochondrial D-loops by transcription factor A and single-stranded DNA-binding protein.
AuthorsTakamatsu C, Umeda S, Ohsato T, Ohno T, Abe Y, Fukuoh A, Shinagawa H, Hamasaki N, Kang D
JournalEMBO Rep
PubMed ID11964388
During replication, mitochondrial DNA (mtDNA) takes on a triple-stranded structure called a D-loop. Although their physiological roles are not understood, D-loops are implicated in replication and transcription of mtDNA. Little is known about the turnover of D-loops. We investigated the effects of mitochondrial transcription factor A (TFAM) and single-stranded DNA-binding ... More
Secretion of active-form Streptoverticillium mobaraense transglutaminase by Corynebacterium glutamicum: processing of the pro-transglutaminase by a cosecreted subtilisin-Like protease from Streptomyces albogriseolus.
AuthorsKikuchi Y, Date M, Yokoyama K, Umezawa Y, Matsui H
JournalAppl Environ Microbiol
PubMed ID12514016
The transglutaminase secreted by Streptoverticillium mobaraense is a useful enzyme in the food industry. A fragment of transglutaminase was secreted by Corynebacterium glutamicum when it was coupled on a plasmid to the promoter and signal peptide of a cell surface protein from C. glutamicum. We analyzed the signal peptide and ... More
Applications of SYPRO orange and SYPRO red protein gel stains.
AuthorsSteinberg TH, Haugland RP, Singer VL
JournalAnal Biochem
PubMed ID8811917
We have further characterized the sensitivity and specificity of SYPRO Orange protein gel stain and SYPRO Red protein gel stain with native and 2-dimensional polyacrylamide gels and for staining gels prior to Western blot analysis. We found that nucleic acids are not stained by the SYPRO protein gel stains, in ... More
High affinity interaction of syntaxin and SNAP-25 on the plasma membrane is abolished by botulinum toxin E.
AuthorsRickman C, Meunier FA, Binz T, Davletov B
JournalJ Biol Chem
PubMed ID14551199
The release of hormones and neurotransmitters requires the fusion of cargo-containing vesicles with the plasma membrane. This process of exocytosis relies on three SNARE proteins, namely syntaxin and SNAP-25 on the target plasma membrane and synaptobrevin on the vesicular membrane. In this study we examined the molecular assembly pathway that ... More
R2 retrotransposition on assembled nucleosomes depends on the translational position of the target site.
AuthorsYe J, Yang Z, Hayes JJ, Eickbush TH
JournalEMBO J
PubMed ID12486006
R2 retrotransposons insert into the 28S rRNA genes of insects. Integration occurs by specific cleavage of the target site and utilization of the released DNA end to prime reverse transcription of the RNA transcript. Specificity of the protein to the target site is dependent upon nucleotide sequence recognition extending from ... More
Single-molecule assay reveals strand switching and enhanced processivity of UvrD.
AuthorsDessinges MN, Lionnet T, Xi XG, Bensimon D, Croquette V
JournalProc Natl Acad Sci U S A
PubMed ID15079074
DNA helicases are enzymes capable of unwinding double-stranded DNA (dsDNA) to provide the single-stranded DNA template required in many biological processes. Among these, UvrD, an essential DNA repair enzyme, has been shown to unwind dsDNA while moving 3'-5' on one strand. Here, we use a single-molecule manipulation technique to monitor ... More
Postelectrophoretic staining of proteins separated by two-dimensional gel electrophoresis using SYPRO dyes.
AuthorsYan JX, Harry RA, Spibey C, Dunn MJ
JournalElectrophoresis
PubMed ID11271484
While the classical silver stain has been the method of choice for high sensitivity protein visualization on two-dimensional gel electrophoresis (2-D PAGE), post-electrophoretic fluorescent staining with the SYPRO group of dyes has emerged to challenge silver staining for proteome analysis. The latter offers improved sensitivity, higher dynamic range and easy ... More
Trax (translin-associated factor X), a primarily cytoplasmic protein, inhibits the binding of TB-RBP (translin) to RNA.
AuthorsChennathukuzhi VM, Kurihara Y, Bray JD, Hecht NB
JournalJ Biol Chem
PubMed ID11278549
Trax (Translin-associated factor X) has been shown to interact with TB-RBP/Translin by its coimmunoprecipitation and in yeast two-hybrid assays. Here we demonstrate that Trax is widely expressed, does not bind to DNA or RNA, but forms heterodimers with TB-RBP under reducing conditions. The heterodimer of TB-RBP and Trax inhibits TB-RBP ... More
The structure and transcriptional analysis of a global regulator from Neisseria meningitidis.
AuthorsRen J, Sainsbury S, Combs SE, Capper RG, Jordan PW, Berrow NS, Stammers DK, Saunders NJ, Owens RJ
JournalJ Biol Chem
PubMed ID17374605
Neisseria meningitidis, a causative agent of bacterial meningitis, has a relatively small repertoire of transcription factors, including NMB0573 (annotated AsnC), a member of the Lrp-AsnC family of regulators that are widely expressed in both Bacteria and Archaea. In the present study we show that NMB0573 binds to l-leucine and l-methionine ... More
Protein-free phospholipid emulsion treatment improved cardiopulmonary function and survival in porcine sepsis.
AuthorsGoldfarb RD, Parker TS, Levine DM, Glock D, Akhter I, Alkhudari A, McCarthy RJ, David EM, Gordon BR, Saal SD, Rubin AL, Trenholme GM, Parrillo JE
JournalAm J Physiol Regul Integr Comp Physiol
PubMed ID12399248
Lipoprotein phospholipid (PL) plays a major role in neutralization of endotoxin. This study tested the hypothesis that prophylactic administration of a PL-enriched emulsion (PRE), which augments PL content of serum lipoproteins and neutralizes endotoxin in vitro, would preserve cardiovascular function and improve survival in porcine septic peritonitis. A control group ... More
Xenopus actin-interacting protein 1 (XAip1) enhances cofilin fragmentation of filaments by capping filament ends.
Xenopus actin-interacting protein 1 (XAip1) is thought to promote fragmentation of actin filaments by cofilin. To examine the mechanism of XAip1, we measured polymer lengths by fluorescence microscopy and the concentration of filament ends with an elongation assay. Cofilin creates ends by severing actin filaments. XAip1 alone does not sever ... More
Specificity of interaction of INI1/hSNF5 with retroviral integrases and its functional significance.
AuthorsYung E, Sorin M, Wang EJ, Perumal S, Ott D, Kalpana GV
JournalJ Virol
PubMed ID14963118
Integrase interactor 1 (INI1)/hSNF5 is a host factor that directly interacts with human immunodeficiency virus type 1 (HIV-1) integrase and is incorporated into HIV-1 virions. Here, we show that while INI1/hSNF5 is completely absent from purified microvesicular fractions, it is specifically incorporated into HIV-1 virions with an integrase-to-INI1/hSNF5 stoichiometry of ... More
Time-dependent changes in expression of troponin subunit isoforms in unloaded rat soleus muscle.
AuthorsStevens L, Bastide B, Kischel P, Pette D, Mounier Y
JournalAm J Physiol Cell Physiol
PubMed ID11940518
This study focuses on the effects of mechanical unloading of rat soleus muscle on the isoform patterns of the three troponin (Tn) subunits: troponin T (TnT), troponin I (TnI), and troponin C (TnC). Mechanical unloading was achieved by hindlimb unloading (HU) for time periods of 7, 15, and 28 days. ... More
End-to-end template jumping by the reverse transcriptase encoded by the R2 retrotransposon.
AuthorsBibillo A, Eickbush TH
JournalJ Biol Chem
PubMed ID14752111
The reverse transcriptase encoded by the non-long terminal repeat retrotransposon R2 has been shown to be able to jump from the 5'-end of one RNA template (the donor) to the 3'-end of a second RNA template (the acceptor) in the absence of preexisting sequence identity between the two templates. These ... More
Proinflammatory activities of S100: proteins S100A8, S100A9, and S100A8/A9 induce neutrophil chemotaxis and adhesion.
AuthorsRyckman C, Vandal K, Rouleau P, Talbot M, Tessier PA
JournalJ Immunol
PubMed ID12626582
S100A8 and S100A9 are small calcium-binding proteins that are highly expressed in neutrophil and monocyte cytosol and are found at high levels in the extracellular milieu during inflammatory conditions. Although reports have proposed a proinflammatory role for these proteins, their extracellular activity remains controversial. In this study, we report that ... More
Regulation of InsP3 receptor activity by neuronal Ca2+-binding proteins.
Inositol 1,4,5-trisphosphate receptors (InsP(3)Rs) were recently demonstrated to be activated independently of InsP(3) by a family of calmodulin (CaM)-like neuronal Ca(2+)-binding proteins (CaBPs). We investigated the interaction of both naturally occurring long and short CaBP1 isoforms with InsP(3)Rs, and their functional effects on InsP(3)R-evoked Ca(2+) signals. Using several experimental paradigms, ... More
Sem1p is a novel subunit of the 26 S proteasome from Saccharomyces cerevisiae.
AuthorsSone T, Saeki Y, Toh-e A, Yokosawa H
JournalJ Biol Chem
PubMed ID15117943
The 26 S proteasome, which catalyzes degradation of polyubiquitinated proteins, is composed of the 20 S proteasome and the 19 S regulatory particle (RP). The RP is composed of the lid and base subcomplexes and regulates the catalytic activity of the 20 S proteasome. In this study, we carried out ... More
De novo infection with rhesus monkey rhadinovirus leads to the accumulation of multiple intranuclear capsid species during lytic replication but favors the release of genome-containing virions.
AuthorsO'Connor CM, Damania B, Kedes DH
JournalJ Virol
PubMed ID14645602
Rhesus monkey rhadinovirus (RRV) is one of the closest phylogenetic relatives to the human pathogen Kaposi's sarcoma-associated herpesvirus (KSHV), yet it has the distinct experimental advantage of entering efficiently into lytic replication and growing to high titers in culture. RRV therefore holds promise as a potentially attractive model with which ... More
A thousand points of light: the application of fluorescence detection technologies to two-dimensional gel electrophoresis and proteomics.
AuthorsPatton WF
JournalElectrophoresis
PubMed ID10786886
As proteomics evolves into a high-throughput technology for the study of global protein regulation, new demands are continually being placed upon protein visualization and quantitation methods. Chief among these are increased detection sensitivity, broad linear dynamic range and compatibility with modern methods of microchemical analyses. The limitations of conventional protein ... More
SYPRO orange and SYPRO red protein gel stains: one-step fluorescent staining of denaturing gels for detection of nanogram levels of protein.
AuthorsSteinberg TH, Jones LJ, Haugland RP, Singer VL
JournalAnal Biochem
PubMed ID8811914
We have developed two new fluorescent dyes, SYPRO Orange protein gel stain and SYPRO Red protein gel stain, to detect proteins in electrophoretic gels. Stained protein bands can be excited by ultraviolet light at approximately 300 nm, or at visible wavelengths, with excitation maxima of 472 nm for the Orange ... More
A luminescent ruthenium complex for ultrasensitive detection of proteins immobilized on membrane supports.
SYPRO Ruby protein blot stain provides a sensitive, gentle, fluorescence-based method for detecting proteins on nitrocellulose or polyvinylidene difluoride (PVDF) membranes. SYPRO Ruby dye is a permanent stain composed of ruthenium as part of an organic complex that interacts noncovalently with proteins. Stained proteins can be excited by ultraviolet light ... More
She4p/Dim1p interacts with the motor domain of unconventional myosins in the budding yeast, Saccharomyces cerevisiae.
AuthorsToi H, Fujimura-Kamada K, Irie K, Takai Y, Todo S, Tanaka K
JournalMol Biol Cell
PubMed ID12808026
She4p/Dim1p, a member of the UNC-45/CRO1/She4p (UCS) domain-containing protein family, is required for endocytosis, polarization of actin cytoskeleton, and polarization of ASH1 mRNA in Saccharomyces cerevisiae. We show herein that She4p/Dim1p is involved in endocytosis and actin polarization through interactions with the type I myosins Myo3p and Myo5p. Two-hybrid and ... More
Reactions of cysteines substituted in the amphipathic N-terminal tail of a bacterial potassium channel with hydrophilic and hydrophobic maleimides.
AuthorsLi J, Xu Q, Cortes DM, Perozo E, Laskey A, Karlin A
JournalProc Natl Acad Sci U S A
PubMed ID12189213
Single cysteine-substitution mutants of KcsA, a K(+) channel from Streptomyces lividans, were expressed in Escherichia coli, and inner membranes were isolated. The rate constants for the reactions of these cysteines with three maleimides of increasing hydrophobicity, 4-(N-maleimido)phenyltrimethylammonium, N-phenylmaleimide, and N-(1-pyrenyl)maleimide, were determined by back titration of the remaining cysteines with ... More
High glucose and insulin promote O-GlcNAc modification of proteins, including alpha-tubulin.
AuthorsWalgren JL, Vincent TS, Schey KL, Buse MG
JournalAm J Physiol Endocrinol Metab
PubMed ID12397027
Increased flux through the hexosamine biosynthesis pathway has been implicated in the development of glucose-induced insulin resistance and may promote the modification of certain proteins with O-linked N-acetylglucosamine (O-GlcNAc). L6 myotubes (a model of skeletal muscle) were incubated for 18 h in 5 or 25 mM glucose with or without ... More
Structure-function analysis of the enteroaggregative Escherichia coli plasmid-encoded toxin autotransporter using scanning linker mutagenesis.
AuthorsDutta PR, Sui BQ, Nataro JP
JournalJ Biol Chem
PubMed ID12878602
The plasmid-encoded toxin (Pet) from enteroaggregative Escherichia coli is a cytopathic serine protease, which is prototypical of a large family of bacterial autotransporter toxins. To further elucidate the structure-function relationships of this toxin, we employed transposon-based scanning linker mutagenesis. A subset of insertions throughout the Pet mature toxin (passenger) domain ... More
Characterization of the protease of a fish retrovirus, walleye dermal sarcoma virus.
AuthorsFodor SK, Vogt VM
JournalJ Virol
PubMed ID11932400
Three fish retroviruses infecting walleyes constitute the recently recognized genus called epsilonretrovirus. The founding member of this group, walleye dermal sarcoma virus (WDSV), induces benign skin tumors in the infected fish and replicates near 4 degrees C. While the viral genomic sequence is known, biochemical characterization of the virus has ... More
Making blind robots see: the synergy between fluorescent dyes and imaging devices in automated proteomics.
AuthorsPatton WF
JournalBiotechniques
PubMed ID10818702
Proteomics investigations endeavor to provide a global understanding of gene product synthesis rate, degradation rate, functional competence, posttranslational modification, subcellular distribution and physical interactions with other cell components. Protein expression encompasses an enormous dynamic range. Since rare proteins cannot be amplified by any type of PCR method, sensitive detection is ... More
The ligand affinity of proteins measured by isothermal denaturation kinetics.
AuthorsEpps DE, Sarver RW, Rogers JM, Herberg JT, Tomich PK
JournalAnal Biochem
PubMed ID11319816
An isothermal denaturation kinetic method was developed for identifying potential ligands of proteins and measuring their affinity. The method is suitable for finding ligands specific toward proteins of unknown function and for large-scale drug screening. It consists of analyzing the kinetics of isothermal denaturation of the protein-with and without the ... More
Identification of the RNA binding domain of T4 RegA protein by structure-based mutagenesis.
AuthorsGordon J, Sengupta TK, Phillips CA, O'Malley SM, Williams KR, Spicer EK
JournalJ Biol Chem
PubMed ID10542265
The T4 translational repressor RegA protein folds into two structural domains, as revealed by the crystal structure (Kang, C.-H. , Chan, R., Berger, I., Lockshin, C., Green, L., Gold, L., and Rich, A. (1995) Science 268, 1170-1173). Domain I of the RegA protein contains a four-stranded beta-sheet and two alpha-helices. ... More
A key role for old yellow enzyme in the metabolism of drugs by Trypanosoma cruzi.
AuthorsKubata BK, Kabututu Z, Nozaki T, Munday CJ, Fukuzumi S, Ohkubo K, Lazarus M, Maruyama T, Martin SK, Duszenko M, Urade Y
JournalJ Exp Med
PubMed ID12417633
Trypanosoma cruzi is the etiological agent of Chagas' disease. So far, first choice anti-chagasic drugs in use have been shown to have undesirable side effects in addition to the emergence of parasite resistance and the lack of prospect for vaccine against T. cruzi infection. Thus, the isolation and characterization of ... More
Protein interaction domains of the ubiquitin-specific protease, USP7/HAUSP.
AuthorsHolowaty MN, Sheng Y, Nguyen T, Arrowsmith C, Frappier L
JournalJ Biol Chem
PubMed ID14506283
USP7 or HAUSP is a ubiquitin-specific protease in human cells that regulates the turnover of p53 and is bound by at least two viral proteins, the ICP0 protein of herpes simplex type 1 and the EBNA1 protein of Epstein-Barr virus. We have overexpressed and purified USP7 and shown that the ... More
Klotho is a novel beta-glucuronidase capable of hydrolyzing steroid beta-glucuronides.
AuthorsTohyama O, Imura A, Iwano A, Freund JN, Henrissat B, Fujimori T, Nabeshima Y
JournalJ Biol Chem
PubMed ID14701853
klotho mutant mice provide a unique model to analyze mechanisms of aging because their phenotypes resemble those of human aging-associated disorders. The klotho gene encodes Klotho, a type I membrane protein that shares sequence similarity with members of the glycosidase family 1. Because Klotho lacks the glutamic acid residues that ... More
DNMT3L stimulates the DNA methylation activity of Dnmt3a and Dnmt3b through a direct interaction.
AuthorsSuetake I, Shinozaki F, Miyagawa J, Takeshima H, Tajima S
JournalJ Biol Chem
PubMed ID15105426
In mammals, the resetting of DNA methylation patterns in early embryos and germ cells is crucial for development. Two DNA methyltransferases, Dnmt3a and Dnmt3b, are responsible for the creation of DNA methylation patterns. Dnmt3L, a member of the Dnmt3 family, has been reported to be necessary for maternal methylation imprinting, ... More