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Bioorganic & Medicinal Chemistry Jan 2016Kinase-catalyzed protein phosphorylation is involved in a wide variety of cellular events. Development of methods to monitor phosphoproteins in normal and diseased...
Kinase-catalyzed protein phosphorylation is involved in a wide variety of cellular events. Development of methods to monitor phosphoproteins in normal and diseased states is critical to fully characterize cell signaling. Towards phosphoprotein analysis tools, our lab reported kinase-catalyzed labeling where γ-phosphate modified ATP analogs are utilized by kinases to label peptides or protein substrates with a functional tag. In particular, the ATP-biotin analog was developed for kinase-catalyzed biotinylation. However, kinase-catalyzed labeling has been tested rigorously with only a few kinases, preventing use of ATP-biotin as a general tool. Here, biotinylation experiments, gel or HPLC-based quantification, and kinetic measurements indicated that twenty-five kinases throughout the kinome tree accepted ATP-biotin as a cosubstrate. With this rigorous characterization of ATP-biotin compatibility, kinase-catalyzed labeling is now immediately useful for studying phosphoproteins and characterizing the role of phosphorylation in various biological events.
Topics: Biotin; Biotinylation; Humans; Kinetics; Molecular Docking Simulation; Phosphorylation; Protein Kinases
PubMed: 26672511
DOI: 10.1016/j.bmc.2015.11.029 -
American Journal of Physiology.... Mar 2002Previous studies have demonstrated that glucocorticoids alter biotin metabolism. To extend these studies, the effect of dexamethasone on biotin pools was analyzed in...
Previous studies have demonstrated that glucocorticoids alter biotin metabolism. To extend these studies, the effect of dexamethasone on biotin pools was analyzed in rats consuming a purified diet containing a more physiological level of dietary biotin intake (0.06 mg/kg). Acute (5 h) dexamethasone administration (0.5 mg/kg) elicited elevated urinary glucose output as well as elevated urinary biotin excretion and serum biotin. Renal and hepatic free biotin was also significantly elevated by acute dexamethasone administration. Chow-fed rats treated with an acute administration of dexamethasone demonstrated significantly elevated urinary glucose excretion, urinary biotin excretion, and serum biotin, but no change in tissue associated biotin was detected. Chronic administration of dexamethasone (0.5 mg/kg ip) over 4 days significantly elevated urinary glucose excretion 42% but had no effect on urinary biotin excretion, serum biotin, or hepatic- or renal-associated free biotin. These results demonstrate the existence of potentially novel regulatory pathways for total biotin pools and the possibility that experimental models with high initial biotin status may mask potentially important regulatory mechanisms.
Topics: Animals; Biotin; Biotinylation; Dexamethasone; Diet; Glucocorticoids; Glycosuria; Kidney; Liver; Male; Rats; Rats, Sprague-Dawley
PubMed: 11832368
DOI: 10.1152/ajpendo.00357.2001 -
Science Advances Mar 2020Macromolecules tend to respond to applied forces in many different ways. Chemistry at high shear forces can be intriguing, with relatively soft bonds becoming very stiff...
Macromolecules tend to respond to applied forces in many different ways. Chemistry at high shear forces can be intriguing, with relatively soft bonds becoming very stiff in specific force-loading geometries. Largely used in bionanotechnology, an important case is the streptavidin (SA)/biotin interaction. Although SA's four subunits have the same affinity, we find that the forces required to break the SA/biotin bond depend strongly on the attachment geometry. With AFM-based single-molecule force spectroscopy (SMFS), we measured unbinding forces of biotin from different SA subunits to range from 100 to more than 400 pN. Using a wide-sampling approach, we carried out hundreds of all-atom steered molecular dynamics (SMD) simulations for the entire system, including molecular linkers. Our strategy revealed the molecular mechanism that causes a fourfold difference in mechanical stability: Certain force-loading geometries induce conformational changes in SA's binding pocket lowering the energy barrier, which biotin has to overcome to escape the pocket.
Topics: Biotin; Chemical Phenomena; Macromolecular Substances; Microscopy, Atomic Force; Models, Molecular; Molecular Conformation; Protein Binding; Streptavidin; Structure-Activity Relationship
PubMed: 32232150
DOI: 10.1126/sciadv.aay5999 -
Journal of Nutritional Science and... 2020Biotin is a water-soluble B-complex vitamin that functions as a cofactor of five carboxylases. Because biotin-dependent carboxylases catalyze indispensable cellular...
Biotin is a water-soluble B-complex vitamin that functions as a cofactor of five carboxylases. Because biotin-dependent carboxylases catalyze indispensable cellular metabolic functions, biotin deficiency is considered to be involved in various pathological conditions. Moreover, biotin supplementation shows pharmacological effects in vivo. However, the precise mechanisms by which biotin deficiency induces pathological conditions remain unclear. Although abnormal metabolites are used as indicators for biotin deficiency, few comprehensive analyses of total metabolites have been reported. In this study, we analyzed the metabolomic profiles of liver extracts prepared from biotin-sufficient (BS) and -deficient (BD) mice. Thirteen of 126 metabolites showed significantly different concentrations between liver extracts from BD and BS mice. The concentrations of 5 essential amino acids, Met, Val, Thr, Ile, and Leu, and 2 conditionally essential amino acids, Cys and Tyr were significantly lower in BD mice than in BS mice. Among these, the concentrations of sulfur-containing amino acids, Cys and Met, were more than 1.5-fold lower in BD mice. The concentrations of Met metabolites, such as S-adenosylmethionine and S-adenosylhomocysteine were not significantly different between the two groups. The concentrations of glutathione and its reaction intermediates γ-Glu-Cys tendency to be lower in BD mice. The present study revealed that biotin deficiency induces an abnormal amino acids composition, especially among sulfur-containing amino acids and provide important information on the effect of biotin as a pharmacological agent.
Topics: Amino Acids, Essential; Amino Acids, Sulfur; Animals; Biotin; Biotinidase Deficiency; Diet; Liver; Metabolome; Mice
PubMed: 32115458
DOI: 10.3177/jnsv.66.82 -
Molecules (Basel, Switzerland) Dec 2022Single molecule interactions between biotin and streptavidin were characterized with functionalized DeepTip probes and used as a model system to develop a comprehensive...
Single molecule interactions between biotin and streptavidin were characterized with functionalized DeepTip probes and used as a model system to develop a comprehensive methodology for the high-yield identification and analysis of single molecular events. The procedure comprises the covalent binding of the target molecule to a surface and of the sensing molecule to the DeepTip probe, so that the interaction between both chemical species can be characterized by obtaining force-displacement curves in an atomic force microscope. It is shown that molecular resolution is consistently attained with a percentage of successful events higher than 90% of the total number of recorded curves, and a very low level of unspecific interactions. The combination of both features is a clear indication of the robustness and versatility of the proposed methodology.
Topics: Microscopy, Atomic Force; Streptavidin; Biotin; Models, Biological
PubMed: 36615422
DOI: 10.3390/molecules28010226 -
Current Opinion in Chemical Biology Feb 2011Nitric oxide (NO) is a cell-signaling molecule involved in a number of physiological and pathophysiological processes. Modification of cysteine residues by NO (or NO... (Review)
Review
Nitric oxide (NO) is a cell-signaling molecule involved in a number of physiological and pathophysiological processes. Modification of cysteine residues by NO (or NO metabolites), that is S-nitrosation, changes the function of a broad spectrum of proteins. This reaction represents an important post-translational modification that transduces NO-dependent signals. However, the detection and quantification of S-nitrosation in biological samples remain a challenge mainly because of the lability of S-nitrosation products: S-nitrosothiols (SNO). In this review we summarize recent developments of the methods to detect S-nitrosation. Our focus is on the methods which can be used to directly conjugate the site(s) of S-nitrosation.
Topics: Biotin; Nitric Oxide; Nitrosation; Phosphines; S-Nitrosothiols
PubMed: 21036657
DOI: 10.1016/j.cbpa.2010.10.006 -
Chemical Research in Toxicology Nov 2022Arsenic is a widespread environmental contaminant, and long-term exposure to arsenic in drinking water is known to be associated with the development of many human...
Arsenic is a widespread environmental contaminant, and long-term exposure to arsenic in drinking water is known to be associated with the development of many human diseases. Identification of arsenic-binding proteins is important for understanding the mechanisms underlying the toxic effects of arsenic species. Here, we developed a chemoproteomic strategy, relying on the use of a biotin-As(III) probe, stable isotope labeling by amino acids in cell culture, and liquid chromatography-tandem mass spectrometry analysis, to identify quantitatively As(III)-binding proteins. Over 400 proteins were enriched from the lysate of HEK293T cells with streptavidin beads immobilized with the biotin-As(III) probe. Competitive labeling experiments in the presence or absence of -aminophenylarsenoxide (PAPAO) revealed 51 candidate As(III)-binding proteins, including several molecular chaperones and cochaperones, that is, HSPA4, HSPA4L, HSPH1, HOP1, FKBP51, and FKBP52. We also validated, by employing western blot analysis, the ability of HSPA4, a member of heat shock protein 70 (HSP70) family, in binding with PAPAO and sodium arsenite in vitro. Together, our work led to the identification of a number of new As(III)-interaction proteins, and our results suggest that As(III) may perturb proteostasis partly through binding directly with molecular chaperones.
Topics: Humans; Carrier Proteins; Arsenic; Biotin; HEK293 Cells; Molecular Chaperones
PubMed: 36269594
DOI: 10.1021/acs.chemrestox.2c00244 -
Acta Crystallographica. Section D,... Sep 2011Atomic resolution crystallographic studies of streptavidin and its biotin complex have been carried out at 1.03 and 0.95 Å, respectively. The wild-type protein...
Atomic resolution crystallographic studies of streptavidin and its biotin complex have been carried out at 1.03 and 0.95 Å, respectively. The wild-type protein crystallized with a tetramer in the asymmetric unit, while the crystals of the biotin complex contained two subunits in the asymmetric unit. Comparison of the six subunits shows the various ways in which the protein accommodates ligand binding and different crystal-packing environments. Conformational variation is found in each of the polypeptide loops connecting the eight strands in the β-sandwich subunit, but the largest differences are found in the flexible binding loop (residues 45-52). In three of the unliganded subunits the loop is in an `open' conformation, while in the two subunits binding biotin, as well as in one of the unliganded subunits, this loop `closes' over the biotin-binding site. The `closed' loop contributes to the protein's high affinity for biotin. Analysis of the anisotropic displacement parameters included in the crystallographic models is consistent with the variation found in the loop structures and the view that the dynamic nature of the protein structure contributes to the ability of the protein to bind biotin so tightly.
Topics: Biotin; Crystallography, X-Ray; Protein Structure, Secondary; Streptavidin
PubMed: 21904034
DOI: 10.1107/S0907444911027806 -
Analytical Chemistry Apr 2011In recent years, there has been a growing interest in using porous microbeads such as agarose beads as solid supports to bind target molecules from complex fluid...
In recent years, there has been a growing interest in using porous microbeads such as agarose beads as solid supports to bind target molecules from complex fluid samples. Porous beads have large surface area to volume ratios and high receptor concentrations, and they facilitate relatively high sensitivity detection and multiplexing. Unfortunately, to take full advantage of the porous beads' attributes, long incubation times are needed due to the relatively slow mass transfer of target molecules from the exterior solution into the beads' interior. To accelerate the mass transfer process, we propose a novel assay in which functionalized porous beads are periodically compressed and expanded. Preliminary experiments were carried out to compare the performance of the pulsating beads with that of conventional, nonpulsating beads. These experiments indicate that the pulsating beads significantly accelerate binding rates with minimal increase in nonspecific binding. Thus, pulsing has the potential of significantly reducing assay time.
Topics: Biotin; Microfluidic Analytical Techniques; Particle Size; Porosity; Quantum Dots; Sepharose; Streptavidin; Surface Properties
PubMed: 21438559
DOI: 10.1021/ac200410v -
International Journal of Molecular... Feb 2023In this study, we fabricated three different ZnO tetrapodal nanostructures (ZnO-Ts) by a combustion process and studied their physicochemical properties by different...
In this study, we fabricated three different ZnO tetrapodal nanostructures (ZnO-Ts) by a combustion process and studied their physicochemical properties by different techniques to evaluate their potentiality for label-free biosensing purposes. Then, we explored the chemical reactivity of ZnO-Ts by quantifying the available functional hydroxyl groups (-OH) on the transducer surface necessary for biosensor development. The best ZnO-T sample was chemically modified and bioconjugated with biotin as a model bioprobe by a multi-step procedure based on silanization and carbodiimide chemistry. The results demonstrated that the ZnO-Ts could be easily and efficiently biomodified, and sensing experiments based on the streptavidin target detection confirmed these structures' suitability for biosensing applications.
Topics: Zinc Oxide; Nanostructures; Biotin; Biosensing Techniques
PubMed: 36901879
DOI: 10.3390/ijms24054449