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Sergey N Krylov - One of the best experts on this subject based on the ideXlab platform.

  • determination of the Equilibrium Constant and rate Constant of protein oligonucleotide complex dissociation under the conditions of ideal filter capillary electrophoresis
    Analytical Chemistry, 2019
    Co-Authors: An T. H. Le, Svetlana M. Krylova, Sergey N Krylov
    Abstract:

    Ideal-filter capillary electrophoresis (IFCE) allows selection of protein binders from oligonucleotide libraries in a single step of partitioning in which protein-bound and unbound oligonucleotides move in the opposite directions. In IFCE, the unbound oligonucleotide does not reach the detector, imposing a problem for finding the Equilibrium Constant (Kd) and rate Constant (koff) of protein–oligonucleotide complex dissociation. We report a double-passage approach that allows finding Kd and koff under the IFCE conditions, i.e. near-physiological pH and ionic strength. First, a plug of the protein–oligonucleotide Equilibrium mixture passes to the detector in a pressure-driven flow, allowing for both the complex and free oligonucleotide to be detected as a single first peak. Second, the pressure is turned off and the voltage is applied to reverse the migration of only the complex which is detected as the second peak. The experiment is repeated with a lower voltage consequently resulting in longer travel time...

  • determination of the Equilibrium Constant and rate Constant of protein oligonucleotide complex dissociation under the conditions of ideal filter capillary electrophoresis
    Analytical Chemistry, 2019
    Co-Authors: Svetlana M. Krylova, Sergey N Krylov
    Abstract:

    Ideal-filter capillary electrophoresis (IFCE) allows selection of protein binders from oligonucleotide libraries in a single step of partitioning in which protein-bound and unbound oligonucleotides move in the opposite directions. In IFCE, the unbound oligonucleotide does not reach the detector, imposing a problem for finding the Equilibrium Constant ( Kd) and rate Constant ( koff) of protein-oligonucleotide complex dissociation. We report a double-passage approach that allows finding Kd and koff under the IFCE conditions, i.e. near-physiological pH and ionic strength. First, a plug of the protein-oligonucleotide Equilibrium mixture passes to the detector in a pressure-driven flow, allowing for both the complex and free oligonucleotide to be detected as a single first peak. Second, the pressure is turned off and the voltage is applied to reverse the migration of only the complex which is detected as the second peak. The experiment is repeated with a lower voltage consequently resulting in longer travel time of the complex to the detector, greater extent of complex dissociation, and the decreased area of the second peak. Finally, the peak areas are used to calculate the values of Kd and koff. Here we explain theoretical and practical aspects of the double-passage approach, prove its validity quantitatively, and, demonstrate its application to determine Kd and koff for an affinity complex between a protein and its DNA aptamer. The double-passage approach for finding Kd and koff of protein-oligonucleotide complexes under the IFCE conditions is a perfect complement for IFCE-based selection of protein binders from oligonucleotide libraries.

  • using nonEquilibrium capillary electrophoresis of Equilibrium mixtures neceem for simultaneous determination of concentration and Equilibrium Constant
    Analytical Chemistry, 2015
    Co-Authors: Mirzo Kanoatov, Svetlana M. Krylova, Victor A Galievsky, Leonid T Cherney, Hanna K Jankowski, Sergey N Krylov
    Abstract:

    NonEquilibrium capillary electrophoresis of Equilibrium mixtures (NECEEM) is a versatile tool for studying affinity binding. Here we describe a NECEEM-based approach for simultaneous determination of both the Equilibrium Constant, Kd, and the unknown concentration of a binder that we call a target, T. In essence, NECEEM is used to measure the unbound Equilibrium fraction, R, for the binder with a known concentration that we call a ligand, L. The first set of experiments is performed at varying concentrations of T, prepared by serial dilution of the stock solution, but at a Constant concentration of L, which is as low as its reliable quantitation allows. The value of R is plotted as a function of the dilution coefficient, and dilution corresponding to R = 0.5 is determined. This dilution of T is used in the second set of experiments in which the concentration of T is fixed but the concentration of L is varied. The experimental dependence of R on the concentration of L is fitted with a function describing t...

  • Using NonEquilibrium Capillary Electrophoresis of Equilibrium Mixtures (NECEEM) for Simultaneous Determination of Concentration and Equilibrium Constant
    2015
    Co-Authors: Mirzo Kanoatov, Svetlana M. Krylova, Victor A Galievsky, Leonid T Cherney, Hanna K Jankowski, Sergey N Krylov
    Abstract:

    NonEquilibrium capillary electrophoresis of Equilibrium mixtures (NECEEM) is a versatile tool for studying affinity binding. Here we describe a NECEEM-based approach for simultaneous determination of both the Equilibrium Constant, Kd, and the unknown concentration of a binder that we call a target, T. In essence, NECEEM is used to measure the unbound Equilibrium fraction, R, for the binder with a known concentration that we call a ligand, L. The first set of experiments is performed at varying concentrations of T, prepared by serial dilution of the stock solution, but at a Constant concentration of L, which is as low as its reliable quantitation allows. The value of R is plotted as a function of the dilution coefficient, and dilution corresponding to R = 0.5 is determined. This dilution of T is used in the second set of experiments in which the concentration of T is fixed but the concentration of L is varied. The experimental dependence of R on the concentration of L is fitted with a function describing their theoretical dependence. Both Kd and the concentration of T are used as fitting parameters, and their sought values are determined as the ones that generate the best fit. We have fully validated this approach in silico by using computer-simulated NECEEM electropherograms and then applied it to experimental determination of the unknown concentration of MutS protein and Kd of its interactions with a DNA aptamer. The general approach described here is applicable not only to NECEEM but also to any other method that can determine a fraction of unbound molecules at Equilibrium

Svetlana M. Krylova - One of the best experts on this subject based on the ideXlab platform.

  • determination of the Equilibrium Constant and rate Constant of protein oligonucleotide complex dissociation under the conditions of ideal filter capillary electrophoresis
    Analytical Chemistry, 2019
    Co-Authors: An T. H. Le, Svetlana M. Krylova, Sergey N Krylov
    Abstract:

    Ideal-filter capillary electrophoresis (IFCE) allows selection of protein binders from oligonucleotide libraries in a single step of partitioning in which protein-bound and unbound oligonucleotides move in the opposite directions. In IFCE, the unbound oligonucleotide does not reach the detector, imposing a problem for finding the Equilibrium Constant (Kd) and rate Constant (koff) of protein–oligonucleotide complex dissociation. We report a double-passage approach that allows finding Kd and koff under the IFCE conditions, i.e. near-physiological pH and ionic strength. First, a plug of the protein–oligonucleotide Equilibrium mixture passes to the detector in a pressure-driven flow, allowing for both the complex and free oligonucleotide to be detected as a single first peak. Second, the pressure is turned off and the voltage is applied to reverse the migration of only the complex which is detected as the second peak. The experiment is repeated with a lower voltage consequently resulting in longer travel time...

  • determination of the Equilibrium Constant and rate Constant of protein oligonucleotide complex dissociation under the conditions of ideal filter capillary electrophoresis
    Analytical Chemistry, 2019
    Co-Authors: Svetlana M. Krylova, Sergey N Krylov
    Abstract:

    Ideal-filter capillary electrophoresis (IFCE) allows selection of protein binders from oligonucleotide libraries in a single step of partitioning in which protein-bound and unbound oligonucleotides move in the opposite directions. In IFCE, the unbound oligonucleotide does not reach the detector, imposing a problem for finding the Equilibrium Constant ( Kd) and rate Constant ( koff) of protein-oligonucleotide complex dissociation. We report a double-passage approach that allows finding Kd and koff under the IFCE conditions, i.e. near-physiological pH and ionic strength. First, a plug of the protein-oligonucleotide Equilibrium mixture passes to the detector in a pressure-driven flow, allowing for both the complex and free oligonucleotide to be detected as a single first peak. Second, the pressure is turned off and the voltage is applied to reverse the migration of only the complex which is detected as the second peak. The experiment is repeated with a lower voltage consequently resulting in longer travel time of the complex to the detector, greater extent of complex dissociation, and the decreased area of the second peak. Finally, the peak areas are used to calculate the values of Kd and koff. Here we explain theoretical and practical aspects of the double-passage approach, prove its validity quantitatively, and, demonstrate its application to determine Kd and koff for an affinity complex between a protein and its DNA aptamer. The double-passage approach for finding Kd and koff of protein-oligonucleotide complexes under the IFCE conditions is a perfect complement for IFCE-based selection of protein binders from oligonucleotide libraries.

  • using nonEquilibrium capillary electrophoresis of Equilibrium mixtures neceem for simultaneous determination of concentration and Equilibrium Constant
    Analytical Chemistry, 2015
    Co-Authors: Mirzo Kanoatov, Svetlana M. Krylova, Victor A Galievsky, Leonid T Cherney, Hanna K Jankowski, Sergey N Krylov
    Abstract:

    NonEquilibrium capillary electrophoresis of Equilibrium mixtures (NECEEM) is a versatile tool for studying affinity binding. Here we describe a NECEEM-based approach for simultaneous determination of both the Equilibrium Constant, Kd, and the unknown concentration of a binder that we call a target, T. In essence, NECEEM is used to measure the unbound Equilibrium fraction, R, for the binder with a known concentration that we call a ligand, L. The first set of experiments is performed at varying concentrations of T, prepared by serial dilution of the stock solution, but at a Constant concentration of L, which is as low as its reliable quantitation allows. The value of R is plotted as a function of the dilution coefficient, and dilution corresponding to R = 0.5 is determined. This dilution of T is used in the second set of experiments in which the concentration of T is fixed but the concentration of L is varied. The experimental dependence of R on the concentration of L is fitted with a function describing t...

  • Using NonEquilibrium Capillary Electrophoresis of Equilibrium Mixtures (NECEEM) for Simultaneous Determination of Concentration and Equilibrium Constant
    2015
    Co-Authors: Mirzo Kanoatov, Svetlana M. Krylova, Victor A Galievsky, Leonid T Cherney, Hanna K Jankowski, Sergey N Krylov
    Abstract:

    NonEquilibrium capillary electrophoresis of Equilibrium mixtures (NECEEM) is a versatile tool for studying affinity binding. Here we describe a NECEEM-based approach for simultaneous determination of both the Equilibrium Constant, Kd, and the unknown concentration of a binder that we call a target, T. In essence, NECEEM is used to measure the unbound Equilibrium fraction, R, for the binder with a known concentration that we call a ligand, L. The first set of experiments is performed at varying concentrations of T, prepared by serial dilution of the stock solution, but at a Constant concentration of L, which is as low as its reliable quantitation allows. The value of R is plotted as a function of the dilution coefficient, and dilution corresponding to R = 0.5 is determined. This dilution of T is used in the second set of experiments in which the concentration of T is fixed but the concentration of L is varied. The experimental dependence of R on the concentration of L is fitted with a function describing their theoretical dependence. Both Kd and the concentration of T are used as fitting parameters, and their sought values are determined as the ones that generate the best fit. We have fully validated this approach in silico by using computer-simulated NECEEM electropherograms and then applied it to experimental determination of the unknown concentration of MutS protein and Kd of its interactions with a DNA aptamer. The general approach described here is applicable not only to NECEEM but also to any other method that can determine a fraction of unbound molecules at Equilibrium

Claude Leforestier - One of the best experts on this subject based on the ideXlab platform.

  • water dimer Equilibrium Constant calculation a quantum formulation including metastable states
    Journal of Chemical Physics, 2014
    Co-Authors: Claude Leforestier
    Abstract:

    We present a full quantum evaluation of the water second virial coefficient B(T) based on the Takahashi-Imada second order approximation. As the associated trace Tr[e−βHAB−e−βHABo] is performed in the coordinate representation, it does also include contribution from the whole continuum, i.e., resonances and collision pairs of monomers. This approach is compared to a Path Integral Monte Carlo evaluation of this coefficient by Schenter [J. Chem. Phys. 117, 6573 (2002)] for the TIP4P potential and shown to give extremely close results in the low temperature range (250–450 K) reported. Using a recent ab initio flexible potential for the water dimer, this new formulation leads to very good agreement with experimental values over the whole range of temperatures available. The virial coefficient is then used in the well known relation Kp(T) = −(B(T) − bM)/RT where the excluded volume bM is assimilated to the second virial coefficient of pure water monomer vapor and approximated from the inner repulsive part of t...

  • water dimers in the atmosphere iii Equilibrium Constant from a flexible potential
    Journal of Physical Chemistry A, 2006
    Co-Authors: Yohann Scribano, Nir Goldman, Richard J Saykally, Claude Leforestier
    Abstract:

    We present new results for the water dimer Equilibrium Constant Kp(T) in the range 190−390 K, using a flexible potential energy surface fitted to spectroscopical data. The increased numerical complexity due to explicit consideration of the monomer vibrations is handled via an adiabatic (6 + 6)d decoupling between intra- and intermolecular modes. The convergence of the canonical partition function of the dimer is ensured by computing all energy levels up to dissociation for total angular momentum values J = 0−5 and using an extrapolation scheme to higher values. The newly calculated values for Kp(T) are in very good agreement with available experimental data at room temperature. At higher temperatures, an analysis of the convergence of the partition function reveals that quasi-bound states are likely to contribute to the Equilibrium Constant. Additional thermodynamical quantities (ΔG, ΔH, ΔS, and Cp) have also been determined and fit to quadratic expressions a + bT + cT2.

  • water dimers in the atmosphere Equilibrium Constant for water dimerization from the vrt asp w potential surface
    Journal of Physical Chemistry A, 2001
    Co-Authors: Nir Goldman, Claude Leforestier, R S Fellers, Richard J Saykally
    Abstract:

    The Equilibrium Constant for water dimerization (KP) was determined as a function of temperature via rigorous calculation of the canonical (H2O)2 partition function using the recently developed split Wigner pseudo-spectral method and the VRT(ASP-W) pair potential. Our KP(T) values are significantly larger than those from previous theoretical treatments but somewhat smaller than literature experimental values, which exist, however, only over a very limited temperature range. These results indicate that water dimers can exist in sufficient concentrations (e.g., 1016 cm-3 at 40 °C and 100% relative humidity) to affect physical and chemical processes in the atmosphere.

An T. H. Le - One of the best experts on this subject based on the ideXlab platform.

  • determination of the Equilibrium Constant and rate Constant of protein oligonucleotide complex dissociation under the conditions of ideal filter capillary electrophoresis
    Analytical Chemistry, 2019
    Co-Authors: An T. H. Le, Svetlana M. Krylova, Sergey N Krylov
    Abstract:

    Ideal-filter capillary electrophoresis (IFCE) allows selection of protein binders from oligonucleotide libraries in a single step of partitioning in which protein-bound and unbound oligonucleotides move in the opposite directions. In IFCE, the unbound oligonucleotide does not reach the detector, imposing a problem for finding the Equilibrium Constant (Kd) and rate Constant (koff) of protein–oligonucleotide complex dissociation. We report a double-passage approach that allows finding Kd and koff under the IFCE conditions, i.e. near-physiological pH and ionic strength. First, a plug of the protein–oligonucleotide Equilibrium mixture passes to the detector in a pressure-driven flow, allowing for both the complex and free oligonucleotide to be detected as a single first peak. Second, the pressure is turned off and the voltage is applied to reverse the migration of only the complex which is detected as the second peak. The experiment is repeated with a lower voltage consequently resulting in longer travel time...

Ioannis Anastopoulos - One of the best experts on this subject based on the ideXlab platform.