Kinetic Capillary Electrophoresis (KCE):  A Conceptual Platform for Kinetic Homogeneous Affinity Methods

Petrov, Alexander P.; Okhonin, Victor; Berezovski, Maxim V.; Krylov, Sergey N.

doi:10.1021/ja056232l

Cited by 138 publications

(162 citation statements)

References 29 publications

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“…The rate constants of complex formation were also calculated as k on ϭ k off /K d . For both HS5ss and CS5ss, the k on were 1.3 ϫ 10 6 and 1.5 ϫ 10 6 M Ϫ1 s Ϫ1 , which are similar to k on values of other DNA binding proteins such as single-stranded binding protein (30). Thus, NECEEM confirmed the results by EMSA, namely, that there is no appreciable difference in the complex formation properties of AID on single-stranded substrates bearing tandem hot-or cold-spot motifs.…”

Section: Fig 4 Emsa Measuring Aid Binding To Bubble Substrates (A)supporting

confidence: 75%

“…NECEEM has been used for measuring equilibrium and kinetic parameters of protein-DNA interactions (3). This method is based on kinetic capillary electrophoresis, defined as capillary electrophoresis of species which interact under nonequilibrium conditions during electrophoresis (30). Binding reactions were carried out as in EMSA, except that the substrates were fluorescently labeled.…”

Section: Fig 4 Emsa Measuring Aid Binding To Bubble Substrates (A)mentioning

confidence: 99%

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AID Associates with Single-Stranded DNA with High Affinity and a Long Complex Half-Life in a Sequence-Independent Manner

Larijani

Petrov

Kolenchenko

et al. 2007

Molecular and Cellular Biology

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110

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Activation-induced cytidine deaminase (AID) initiates secondary antibody diversification processes by deaminating cytidines on single-stranded DNA. AID preferentially mutates cytidines preceded by W(A/ T)R(A/G) dinucleotides, a sequence specificity that is evolutionarily conserved from bony fish to humans. To uncover the biochemical mechanism of AID, we compared the catalytic and binding kinetics of AID on WRC (a hot-spot motif, where W equals A or T and R equals A or G) and non-WRC motifs. We show that although purified AID preferentially deaminates WRC over non-WRC motifs to the same degree observed in vivo, it exhibits similar binding affinities to either motif, indicating that its sequence specificity is not due to preferential binding of WRC motifs. AID preferentially deaminates bubble substrates of five to seven nucleotides rather than larger bubbles and preferentially binds to bubble-type rather than to single-stranded DNA substrates, suggesting that the natural targets of AID are either transcription bubbles or stem-loop structures. Importantly, AID displays remarkably high affinity for single-stranded DNA as indicated by the low dissociation constants and long half-life of complex dissociation that are typical of transcription factors and singlestranded DNA binding protein. These findings suggest that AID may persist on immunoglobulin and other target sequences after deamination, possibly acting as a scaffolding protein to recruit other factors.Activation-induced cytidine deaminase (AID) is the B-cellspecific enzyme responsible for the conversion of cytidine to uridine, the initiating event in somatic hypermutation (SHM) and class switch recombination (CSR) of antibody genes (20,22,26,29). This initial lesion can either be replicated, producing a transition mutation, or be engaged by the base excision (i.e., UNG) (34) or mismatch repair pathways (i.e., MSH2) (21, 33, 52), resulting in transversion mutations or A-T mutations, respectively. Cytidine deamination by AID also leads to the generation of double-stranded lesions in the switch regions of antibody genes, leading to CSR (9,22,42,43).While necessary for SHM and CSR, AID-induced lesions have been shown to cause chromosomal translocations contributing to lymphoma (13,36,37). Because of this inherently dangerous property of AID, it is likely that the activity of AID is controlled at different levels to focus it on immunoglobulin genes, such as through protein kinase A phosphorylation (2, 27) or the interaction with cofactors, such as replication protein A (RPA) (7). At the target DNA level, high levels of transcription have been shown to be necessary but not sufficient for AID activity (1,22,23,28,38). Since AID is able to deaminate cytidines only on single-stranded DNA (ssDNA) (6,8,10,17,18,25,47), it is likely that the requirement for transcription reflects the generation of single-stranded regions by transcription bubbles (8,22) or the generation of G4 DNA structures (11,12).Even prior to the discovery of AID, it was noted that SHM occurs more freq...

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Section: Fig 4 Emsa Measuring Aid Binding To Bubble Substrates (A)supporting

confidence: 75%

Section: Fig 4 Emsa Measuring Aid Binding To Bubble Substrates (A)mentioning

confidence: 99%

AID Associates with Single-Stranded DNA with High Affinity and a Long Complex Half-Life in a Sequence-Independent Manner

Larijani

Petrov

Kolenchenko

et al. 2007

Molecular and Cellular Biology

Self Cite

110

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“…40,42 Some methods based on CE have also been developed for the kinetic study of intermolecular interactions, 44 e.g., affinity capillary electrophoresis, 45 exponential decay method, 46 and kinetic capillary electrophoresis (KCE). [47][48][49][50][51] In these methods, ka and kd are determined by matching electropherograms or exponential decay curves numerically calculated by changing the rate constants to those experimentally measured. Macroscopic approach for studying kinetics at equilibrium (MASKE) was also developed for measuring equilibrium and kinetic parameters in the state of chemical equilibrium.…”

Section: Discussionmentioning

confidence: 99%

Moment Analysis Theory for Size Exclusion Capillary Electrochromatography with Chemical Reaction of Intermolecular Interaction

Miyabe

Suzuki

2017

ANAL. SCI.

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New moment equations were developed for size exclusion capillary electrochromatography (SECEC), in which intermolecular chemical reactions simultaneously took place. They explain how the first absolute and second central moments of elution peaks are correlated with some fundamental equilibrium and kinetic parameters of mass transfer and chemical reaction in SECEC column. In order to demonstrate the effectiveness of the moment equations, they were used to predict chromatographic behavior under hypothetical SECEC conditions. It was quantitatively studied how the association and dissociation rate constants of intermolecular interaction affected the position and spreading of elution peaks. It was indicated that both the intermolecular reaction kinetics and axial dispersion of solute molecules in a capillary column had a predominant contribution to the band broadening.

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“…[12][13][14] In KCE, ka and kd were determined by fitting elution curves numerically calculated to experimental data. The macroscopic approach for studying kinetics at equilibrium (MASKE) was also developed for measuring equilibrium and kinetic parameters in the state of chemical equilibrium.…”

Section: Introductionmentioning

confidence: 99%

Kinetic Study of Interaction between Solute Molecule and Surfactant Micelle

2015

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We developed moment analysis of affinity kinetics by chromatographic capillary electrophoresis (MKCCE) method for the kinetic study of intermolecular interactions. Association and dissociation rate constants of the interaction in a micellar electrokinetic chromatography (MEKC) system between thymol and sodium dodecylsulfate micelle were determined by the MKCCE method. It is a method based on the moment theory for the kinetic study of intermolecular interactions under the conditions that neither immobilization nor chemical modification of molecules is required. In CCE mode, experimental conditions are controlled so that the migration of solute-micelle complex is stopped and only solute molecules migrate in a capillary. Mass transfer behavior of solute molecules in the CCE system is analogous to that in a chromatographic system. However, because it was difficult in practice to really perform CE experiments under the CCE conditions, CE data were measured with changing experimental conditions, i.e., applied pressure, under the conditions that the migration velocity of solute-micelle complex was around zero. The rate constants could be analytically determined from the CE data. In the MKCCE method, it is not necessary to fit elution curves numerically calculated to those experimentally measured for the determination of the rate constants. Regarding the interaction between thymol and SDS micelles, association equilibrium constant and association and dissociation rate constants were determined as 6.35 × 10 3 dm 3 mol -1 , 5.6 × 10 4 dm 3 mol -1 s -1 , and 8.7 s -1 , respectively. It was demonstrated that the MKCCE method was effective for the kinetic study of intermolecular interactions.

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Kinetic Capillary Electrophoresis (KCE): A Conceptual Platform for Kinetic Homogeneous Affinity Methods

Cited by 138 publications

References 29 publications

AID Associates with Single-Stranded DNA with High Affinity and a Long Complex Half-Life in a Sequence-Independent Manner

AID Associates with Single-Stranded DNA with High Affinity and a Long Complex Half-Life in a Sequence-Independent Manner

Moment Analysis Theory for Size Exclusion Capillary Electrochromatography with Chemical Reaction of Intermolecular Interaction

Kinetic Study of Interaction between Solute Molecule and Surfactant Micelle

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