排序方式: 共有43条查询结果,搜索用时 15 毫秒
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Gorokhovatsky AY Rudenko NV Marchenkov VV Skosyrev VS Arzhanov MA Burkhardt N Zakharov MV Semisotnov GV Vinokurov LM Alakhov YB 《Analytical biochemistry》2003,313(1):68-75
Here we describe a homogeneous assay for biotin based on bioluminescence resonance energy transfer (BRET) between aequorin and enhanced green fluorescent protein (EGFP). The fusions of aequorin with streptavidin (SAV) and EGFP with biotin carboxyl carrier protein (BCCP) were purified after expression of the corresponding genes in Escherichia coli cells. Association of SAV-aequorin and BCCP-EGFP fusions was followed by BRET between aequorin (donor) and EGFP (acceptor), resulting in significantly increasing 510 nm and decreasing 470 nm bioluminescence intensity. It was shown that free biotin inhibited BRET due to its competition with BCCP-EGFP for binding to SAV-aequorin. These properties were exploited to demonstrate competitive homogeneous BRET assay for biotin. 相似文献
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Marchenkov VV Sokolovskiĭ IV Kotova NV Galzitskaya OV Bochkareva ES Girshovich AS Semisotnov GV 《Biofizika》2004,49(6):987-994
The main function of the chaperone GroEL is to prevent nonspecific association of nonnative protein chains and provide their correct folding. In the present work, the renaturation kinetics of three globular proteins (human alpha-lactalbumin, bovine carbonic anhydrase, and yeast phosphoglycerate kinase) in the presence of different molar excess of GroEL (up to 10-fold) was studied. It was shown that the formation of the native structure during the refolding of these proteins is retarded with an increase in GroEL molar excess due to the interaction of kinetic protein intermediates with the chaperone. Mg(2+)-ATP and Mg(2+)-ADP weaken this interaction and decrease the retarding effect of GroEL on the protein refolding kinetics. The theoretical modeling of protein folding in the presence of GroEL showed that the experimentally observed linear increase in the protein refolding half-time with increasing molar excess of GroEL must occur only when the protein adopts its native structure outside of GroEL (i.e. in the free state), while the refolding of the protein in the complex with GroEL is inhibited. The dissociation constants of GroEL complexed with the kinetic intermediates of the proteins studied were evaluated, and a simple mechanism of the functioning of GroEL as a molecular chaperone was proposed. 相似文献
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A Iu Goncharov E K Goncharov I M Alutin V I Marchenkov 《Molekuliarnaia genetika, mikrobiologiia i virusologiia》1992,(11-12):10-14
Fragmentation of the pYT plasmid of the plague pathogen by ten restriction endonucleases has been studied. The evidence has been obtained in support of the possible presence of the movable genetic element containing a HindIII site within the plasmid pYT. The gene encoding the I fraction of the plague pathogen has been cloned. The physical map of the pYT plasmid has been constructed with the use of restriction endonucleases BamHI, XhoI, BstEII, SmaI, EcoRI, and HindIII. The fragment of the plasmid DNA coding for the synthesis of the plague pathogen fraction I has been mapped. 相似文献
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E. I. Grigorashvili O. M. Selivanova N. V. Dovidchenko U. F. Dzhus A. O. Mikhailina M. Yu. Suvorina V. V. Marchenkov A. K. Surin O. V. Galzitskaya 《Biochemistry. Biokhimii?a》2016,81(5):538-547
We have developed a highly efficient method for purification of the recombinant product Aβ(1-40) peptide. The concentration dependence of amyloid formation by recombinant Aβ(1-40) peptide was studied using fluorescence spectroscopy and electron microscopy. We found that the process of amyloid formation is preceded by lag time, which indicates that the process is nucleation-dependent. Further exponential growth of amyloid fibrils is followed by branching scenarios. Based on the experimental data on the concentration dependence, the sizes of the folding nuclei of fibrils were calculated. It turned out that the size of the primary nucleus is one “monomer” and the size of the secondary nucleus is zero. This means that the nucleus for new aggregates can be a surface of the fibrils themselves. Using electron microscopy, we have demonstrated that fibrils of these peptides are formed by the association of rounded ring structures. 相似文献
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Aruna Kasoju M Lakshmi Narasu Charuvaka Muvva Bathula VV SubbaRao 《Bioinformation》2012,8(14):684-686
Aflatoxins are polyketide-derived secondary metabolites produced by Aspergillus spp. The toxic effects of aflatoxins have adverse
consequences for human health and agricultural economics. The aflR gene, a regulatory gene for aflatoxin biosynthesis, encodes a
protein containing a zinc-finger DNA-binding motif. AFLR-Protein three-dimensional model was generated using Robetta server.
The modeled AFLR-Protein was further optimization and validation using Rampage. In the simulations, we monitored the
backbone atoms and the C-α-helix of the modeled protein. The low RMSD and the simulation time indicate that, as expected, the
3D structural model of AFLR-protein represents a stable folding conformation. This study paves the way for generating computer
molecular models for proteins whose crystal structures are not available and which would aid in detailed molecular mechanism of
inhibition of aflatoxin. 相似文献
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Visualization of molecular structures aids in the understanding of structural and functional roles of biological macromolecules.
Macromolecular transport between the cell nucleus and cytoplasm is facilitated by the nuclear pore complex (NPC). The ring
structure of the NPC is large and contains several distinct proteins (nucleoporins) which function as a selective gate for the passage
of certain molecules into and out of the nucleus. In this note we demonstrate the utility of a python code that allows direct
mapping of the physiochemical properties of the constituent nucleoporins on the scaffold of the yeast NPC׳s cytoplasmic view. We
expect this tool to be useful for researchers to visualize the NPC based on their physiochemical properties and how it alters when
specific mutations are introduced in one or more of the nucleoporins. The code developed using Python is available freely from the
authors. 相似文献
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