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David M. Jameson - One of the best experts on this subject based on the ideXlab platform.
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Tetramethylrhodamine dimer formation as a spectroscopic probe of the conformation of Escherichia coli ribosomal protein L7/L12 dimers.
The Journal of biological chemistry, 1996Co-Authors: Brian D. Hamman, Andrew V. Oleinikov, George G. Jokhadze, Dmitry E. Bochkariov, Robert R. Traut, David M. JamesonAbstract:Abstract The fluorescent probe Tetramethylrhodamine iodoacetamide was attached to cysteine residues substituted at various specific locations in full-length and deletion variants of the homodimeric Escherichia coli ribosomal protein L7/L12. Ground-state Tetramethylrhodamine dimers form between the two subunits of L7/L12 depending upon the location of the probe. The formation of Tetramethylrhodamine dimers caused the appearance of a new absorption band at 518 nm that was used to estimate the extent of interaction of the probes in the different protein variants. Intersubunit Tetramethylrhodamine dimers form when Tetramethylrhodamine acetamide is attached to two different sites in the N-terminal domain of the L7/L12 dimer (residues 12 or 33), but not when attached to sites in the C-terminal domain (residues 63, 89, or 99). The Tetramethylrhodamine dimers do form at sites in the C-terminal domain in L7/L12 variants that contain deletions of 11 or 18 residues within the putative flexible hinge that separates the N- and C-terminal domains. The Tetramethylrhodamine dimers disappear rapidly (within 5 s) upon addition of excess unlabeled wild-type L7/L12. It appears that singly labeled L7/L12 dimers are formed by exchange with wild-type dimers. Binding of L7/L12:Tetramethylrhodamine cysteine 33 or cysteine 12 dimers either to L7/L12-depleted ribosomal core particles, or to ribosomal protein L10 alone, results in disappearance of the 518-nm absorption band. This result implies a conformational change in the N-terminal domain of L7/L12 upon its binding to the ribosome, or to L10.
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Tetramethylrhodamine dimer formation as a spectroscopic probe of the conformation of escherichia coli ribosomal protein l7 l12 dimers
Journal of Biological Chemistry, 1996Co-Authors: Brian D. Hamman, Andrew V. Oleinikov, George G. Jokhadze, Dmitry E. Bochkariov, Robert R. Traut, David M. JamesonAbstract:Abstract The fluorescent probe Tetramethylrhodamine iodoacetamide was attached to cysteine residues substituted at various specific locations in full-length and deletion variants of the homodimeric Escherichia coli ribosomal protein L7/L12. Ground-state Tetramethylrhodamine dimers form between the two subunits of L7/L12 depending upon the location of the probe. The formation of Tetramethylrhodamine dimers caused the appearance of a new absorption band at 518 nm that was used to estimate the extent of interaction of the probes in the different protein variants. Intersubunit Tetramethylrhodamine dimers form when Tetramethylrhodamine acetamide is attached to two different sites in the N-terminal domain of the L7/L12 dimer (residues 12 or 33), but not when attached to sites in the C-terminal domain (residues 63, 89, or 99). The Tetramethylrhodamine dimers do form at sites in the C-terminal domain in L7/L12 variants that contain deletions of 11 or 18 residues within the putative flexible hinge that separates the N- and C-terminal domains. The Tetramethylrhodamine dimers disappear rapidly (within 5 s) upon addition of excess unlabeled wild-type L7/L12. It appears that singly labeled L7/L12 dimers are formed by exchange with wild-type dimers. Binding of L7/L12:Tetramethylrhodamine cysteine 33 or cysteine 12 dimers either to L7/L12-depleted ribosomal core particles, or to ribosomal protein L10 alone, results in disappearance of the 518-nm absorption band. This result implies a conformational change in the N-terminal domain of L7/L12 upon its binding to the ribosome, or to L10.
Brian D. Hamman - One of the best experts on this subject based on the ideXlab platform.
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Tetramethylrhodamine dimer formation as a spectroscopic probe of the conformation of Escherichia coli ribosomal protein L7/L12 dimers.
The Journal of biological chemistry, 1996Co-Authors: Brian D. Hamman, Andrew V. Oleinikov, George G. Jokhadze, Dmitry E. Bochkariov, Robert R. Traut, David M. JamesonAbstract:Abstract The fluorescent probe Tetramethylrhodamine iodoacetamide was attached to cysteine residues substituted at various specific locations in full-length and deletion variants of the homodimeric Escherichia coli ribosomal protein L7/L12. Ground-state Tetramethylrhodamine dimers form between the two subunits of L7/L12 depending upon the location of the probe. The formation of Tetramethylrhodamine dimers caused the appearance of a new absorption band at 518 nm that was used to estimate the extent of interaction of the probes in the different protein variants. Intersubunit Tetramethylrhodamine dimers form when Tetramethylrhodamine acetamide is attached to two different sites in the N-terminal domain of the L7/L12 dimer (residues 12 or 33), but not when attached to sites in the C-terminal domain (residues 63, 89, or 99). The Tetramethylrhodamine dimers do form at sites in the C-terminal domain in L7/L12 variants that contain deletions of 11 or 18 residues within the putative flexible hinge that separates the N- and C-terminal domains. The Tetramethylrhodamine dimers disappear rapidly (within 5 s) upon addition of excess unlabeled wild-type L7/L12. It appears that singly labeled L7/L12 dimers are formed by exchange with wild-type dimers. Binding of L7/L12:Tetramethylrhodamine cysteine 33 or cysteine 12 dimers either to L7/L12-depleted ribosomal core particles, or to ribosomal protein L10 alone, results in disappearance of the 518-nm absorption band. This result implies a conformational change in the N-terminal domain of L7/L12 upon its binding to the ribosome, or to L10.
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Tetramethylrhodamine dimer formation as a spectroscopic probe of the conformation of escherichia coli ribosomal protein l7 l12 dimers
Journal of Biological Chemistry, 1996Co-Authors: Brian D. Hamman, Andrew V. Oleinikov, George G. Jokhadze, Dmitry E. Bochkariov, Robert R. Traut, David M. JamesonAbstract:Abstract The fluorescent probe Tetramethylrhodamine iodoacetamide was attached to cysteine residues substituted at various specific locations in full-length and deletion variants of the homodimeric Escherichia coli ribosomal protein L7/L12. Ground-state Tetramethylrhodamine dimers form between the two subunits of L7/L12 depending upon the location of the probe. The formation of Tetramethylrhodamine dimers caused the appearance of a new absorption band at 518 nm that was used to estimate the extent of interaction of the probes in the different protein variants. Intersubunit Tetramethylrhodamine dimers form when Tetramethylrhodamine acetamide is attached to two different sites in the N-terminal domain of the L7/L12 dimer (residues 12 or 33), but not when attached to sites in the C-terminal domain (residues 63, 89, or 99). The Tetramethylrhodamine dimers do form at sites in the C-terminal domain in L7/L12 variants that contain deletions of 11 or 18 residues within the putative flexible hinge that separates the N- and C-terminal domains. The Tetramethylrhodamine dimers disappear rapidly (within 5 s) upon addition of excess unlabeled wild-type L7/L12. It appears that singly labeled L7/L12 dimers are formed by exchange with wild-type dimers. Binding of L7/L12:Tetramethylrhodamine cysteine 33 or cysteine 12 dimers either to L7/L12-depleted ribosomal core particles, or to ribosomal protein L10 alone, results in disappearance of the 518-nm absorption band. This result implies a conformational change in the N-terminal domain of L7/L12 upon its binding to the ribosome, or to L10.
Emil Reisler - One of the best experts on this subject based on the ideXlab platform.
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Formation and Destabilization of Actin Filaments with Tetramethylrhodamine-Modified Actin
Biophysical journal, 2004Co-Authors: Dmitry S. Kudryashov, Martin L. Phillips, Emil ReislerAbstract:Actin labeling at Cys374 with tethramethylrhodamine derivatives (TMR-actin) has been widely used for direct observation of the in vitro filaments growth, branching, and treadmilling, as well as for the in vivo visualization of actin cytoskeleton. The advantage of TMR-actin is that it does not lock actin in filaments (as rhodamine-phalloidin does), possibly allowing for its use in investigating the dynamic assembly behavior of actin polymers. Although it is established that TMR-actin alone is polymerization incompetent, the impact of its copolymerization with unlabeled actin on filament structure and dynamics has not been tested yet. In this study, we show that TMR-actin perturbs the filaments structure when copolymerized with unlabeled actin; the resulting filaments are more fragile and shorter than the control filaments. Due to the increased severing of copolymer filaments, TMR-actin accelerates the polymerization of unlabeled actin in solution also at mole ratios lower than those used in most fluorescence microscopy experiments. The destabilizing and severing effect of TMR-actin is countered by filament stabilizing factors, phalloidin, S1, and tropomyosin. These results point to an analogy between the effects of TMR-actin and severing proteins on F-actin, and imply that TMR-actin may be inappropriate for investigations of actin filaments dynamics.
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Solution Properties of Tetramethylrhodamine-Modified G-Actin
Biophysical journal, 2003Co-Authors: Dmitry S. Kudryashov, Emil ReislerAbstract:In the recently solved structure of TMR-modified ADP-G-actin, the nucleotide cleft is in a closed state conformation, and the D-loop contains an α-helix (L. R. Otterbein, P. Graceffa, and R. Dominguez, 2001, Science, 293:708–711). Subsequently, questions were raised regarding the possible role of the TMR label on Cys374 in determining these aspects of G-actin structure. We show here that the susceptibility of D-loop on G-actin to subtilisin cleavage, and ATP/ADP-dependent changes in this cleavage, are not affected by TMR-labeling of actin. The TMR modification inhibits nucleotide exchange, but has no effect on DNase I binding and the fast phase of tryptic digestion of actin. These results show an absence of allosteric effects of TMR on subdomain 2, while confirming ATP/ADP-dependent changes in D-loop structure. In conjunction with similar results obtained on actin-gelsolin segment 1 complex, this works reveals the limitations of solution methods in probing the putative open and closed nucleotide cleft states of G-actin.
Enrico Grazi - One of the best experts on this subject based on the ideXlab platform.
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On the mechanics of the actin filament: the linear relationship between stiffness and yield strength allows estimation of the yield strength of thin filament in vivo.
Journal of muscle research and cell motility, 2004Co-Authors: Enrico Grazi, Orietta Cintio, Giorgio TrombettaAbstract:Comparison of the behaviour of actin filaments either modified with Tetramethylrhodamine iodoacetamide or decorated with Tetramethylrhodamine-phalloidin or with tropomyosin or with myosin subfragment 1 shows that, in all the cases, yield strength is linearly related to stiffness.
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Effects of chemical modification, tropomyosin, and myosin subfragment 1 on the yield strength and critical concentration of F-actin
Biochemistry, 2002Co-Authors: Raffaella Adami, Orietta Cintio, Daniel Choquet, Giorgio Trombetta, Enrico GraziAbstract:The effects of coupling with Tetramethylrhodamine-5-iodoacetamide and of the decoration with tropomyosin and with myosin subfragment 1 on the elastic properties of F-actin filament are investigated. At 22 degrees C, in 15 mM orthophosphate and 3 mM MgCl2, Tetramethylrhodamine F-actin displays a yield strength of 3.69 +/- 0.213 pN and an elastic modulus by stretching of 0.91 MPa. Decoration with tropomyosin increases the yield strength of Tetramethylrhodamine F-actin to 10.51 +/- 0.24 pN and the elastic modulus by stretching to 23-75 MPa. Mixtures of myosin subfragment 1 and Tetramethylrhodamine F-actin at the 0.2:1, 0.4:1, 0.6:1, 0.8:1, and 1:1 molar ratios are also studied. Both yield strength and the elastic modulus by stretching are found to increase progressively with the ratio. At the 1:1 molar ratio, the yield strength is 15.81 +/- 0.26 pN and the elastic modulus by stretching is 13.45 to 40 MPa. Decoration of Tetramethylrhodamine F-actin with both tropomyosin and myosin subfragment 1, at the 1:1 molar ratio with the actin monomer, produces filaments with an yield strength of 22.3 +/- 0.48 pN.
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On the elastic properties of Tetramethylrhodamine F-actin.
Biophysical chemistry, 2001Co-Authors: Orietta Cintio, Daniel Choquet, Raffaella Adami, Enrico GraziAbstract:(Iodoacetamido)Tetramethylrhodamine disrupts F-actin. At the 1:1 fluorophore to actin (as monomer) ratio approximately 80% of the protein becomes non-sedimentable. The fluorescent, non-sedimentable actin copolymerizes with G-actin to yield fluorescent filaments. The tensile strength of these filaments changes with the ratio of the fluorescent non-sedimentable actin to the G-actin, being 1.6 pN, 2.9 pN and 3.6 pN at the 1/4, 2/3 and 1/1 ratios, respectively. These tensile strengths are approximately two orders of magnitude lower than those obtained by decoration of F-actin with phalloidin.
Qiang Zhao - One of the best experts on this subject based on the ideXlab platform.
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An immunoassay for ochratoxin A using Tetramethylrhodamine-labeled ochratoxin A as a probe based on a binding-induced change in fluorescence intensity.
The Analyst, 2020Co-Authors: Ning Zhang, Hailin Wang, Qiang ZhaoAbstract:Ochratoxin A (OTA) is a mycotoxin that can cause health risks to human/animal health. Contamination by OTA can occur in various foods and agricultural products, so sensitive and rapid detection of OTA is crucial. We describe a simple and sensitive fluorescence immunoassay for OTA using Tetramethylrhodamine (TMR)-labeled OTA as a fluorescent probe. We conjugated Tetramethylrhodamine to OTA through a covalent reaction, and obtained three TMR-OTA isomer probes after purification by high-performance liquid chromatography. All of the fluorescent probes showed high binding affinity to the anti-OTA antibody. Binding of the TMR-OTA probe to the antibody induced strong fluorescence of TMR-OTA due to the possible change in the local environment of TMR caused by affinity binding. In the presence of OTA, the OTA target competitively displaced the bound TMR-OTA probe from the antibody, causing a decrease in fluorescence. Measuring the change in fluorescence enabled rapid detection of OTA. This method was selective and allowed the detection of 1 nM OTA, showing potential for rapid OTA analysis in applications.
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Direct fluorescence anisotropy assay for cocaine using Tetramethylrhodamine-labeled aptamer.
Analytical and bioanalytical chemistry, 2017Co-Authors: Yingxiong Liu, Qiang ZhaoAbstract:Development of simple, sensitive, and rapid method for cocaine detection is important in medicine and drug abuse monitoring. Taking advantage of fluorescence anisotropy and aptamer, this study reports a direct fluorescence anisotropy (FA) assay for cocaine by employing an aptamer probe with Tetramethylrhodamine (TMR) labeled on a specific position. The binding of cocaine and the aptamer causes a structure change of the TMR-labeled aptamer, leading to changes of the interaction between labeled TMR and adjacent G bases in aptamer sequence, so FA of TMR varies with increasing of cocaine. After screening different labeling positions of the aptamer, including thymine (T) bases and terminals of the aptamer, we obtained a favorable aptamer probe with TMR labeled on the 25th base T in the sequence, which exhibited sensitive and significant FA-decreasing responses upon cocaine. Under optimized assay conditions, this TMR-labeled aptamer allowed for direct FA detection of cocaine as low as 5 μM. The maximum FA change reached about 0.086. This FA method also enabled the detection of cocaine spiked in diluted serum and urine samples, showing potential for applications.
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Fluorescence anisotropy assay for D-vasopressin with a Tetramethylrhodamine-labeled aptamer
Analytical Methods, 2016Co-Authors: Yingxiong Liu, Qiang ZhaoAbstract:Here we report a fluorescence anisotropy (FA) assay for D-vasopressin (D-Vas), an important active peptide, by using a Tetramethylrhodamine (TMR) labeled 55-nt aptamer that contains 19 guanine (G) bases. This assay is based on a target-binding induced change of the intramolecular interaction between TMR and G base and the subsequent FA alteration of labeled TMR. We conjugated single TMR on different labeling positions of thymine (T) bases or terminals of the aptamer, obtaining 14 TMR-labeled aptamers. We screened the FA response of each TMR-labeled aptamer upon D-Vas addition. An aptamer probe with TMR labeled on the 34th base T in sequence exhibited sensitive and significant FA-decreasing responses upon the binding of D-Vas. Under optimized assay conditions this favorable TMR-labeled aptamer allowed direct FA detection of D-Vas ranging from 0.2 μM to 200 μM with good selectivity. This FA method also enabled the detection of D-Vas spiked in diluted serum and urine samples.
