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Peter Schuck - One of the best experts on this subject based on the ideXlab platform.
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Sedimentation in a time varying centrifugal field for rapid attainment of Sedimentation equilibrium
Biophysical Journal, 2015Co-Authors: Peter Schuck, Michael A Metrick, Huaying ZhaoAbstract:Sedimentation equilibrium (SE) analytical ultracentrifugation is a gold standard for the rigorous thermodynamic study of buoyant molecular weight and reversible interactions of macromolecules in solution. A significant drawback is the long experiment time, as it takes days to attain SE with standard solution columns. We have developed a new method for using a time-varying centrifugal field optimized such as to attain SE in significantly shorter time than usually required. Experimental data show that this permits long-column SE experiments to be carried out in times comparable to Sedimentation velocity experiments, approximately fivefold shorter than standard SE. In contrast to the classical initial overspeeding method, which uses a single initial speed, we employ a freely varying rotor speed profile during an initial phase, for example, parameterized as a step-wise modulated exponential decay to the desired SE rotor speed. The rotor speed schedule is computationally optimized on the basis of numerical Lamm equation solutions for given macromolecular Sedimentation parameter estimates, with the goal to provide a rapid attainment of equilibrium without the drawback of strong transient sample pre-concentration at the base of the solution column. The resulting rotor speed schedule frequently includes both over- and under-speeding sequences, and can be conveniently implemented on the Optima XLA/I analytical ultracentrifuge. We extended AUC data analysis models in SEDFIT to permit the analysis of concentration profiles in arbitrarily time-varying fields, to make it possible to exploit the migration in the initially high centrifugal field for estimates on macromolecular Sedimentation parameters, which may be used in real-time to refine the prediction of the rotor speed schedule, so that the SE experiment can be optimized in both information content and time efficiency.
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overview of current methods in Sedimentation velocity and Sedimentation equilibrium analytical ultracentrifugation
Current protocols in protein science, 2013Co-Authors: Huaying Zhao, Chad A Brautigam, Rodolfo Ghirlando, Peter SchuckAbstract:Modern computational strategies have allowed for the direct modeling of the Sedimentation process of heterogeneous mixtures, resulting in Sedimentation velocity (SV) size-distribution analyses with significantly improved detection limits and strongly enhanced resolution. These advances have transformed the practice of SV, rendering it the primary method of choice for most existing applications of analytical ultracentrifugation (AUC), such as the study of protein self- and hetero-association, the study of membrane proteins, and applications in biotechnology. New global multisignal modeling and mass conservation approaches in SV and Sedimentation equilibrium (SE), in conjunction with the effective-particle framework for interpreting the Sedimentation boundary structure of interacting systems, as well as tools for explicit modeling of the reaction/diffusion/Sedimentation equations to experimental data, have led to more robust and more powerful strategies for the study of reversible protein interactions and multiprotein complexes. Furthermore, modern mathematical modeling capabilities have allowed for a detailed description of many experimental aspects of the acquired data, thus enabling novel experimental opportunities, with important implications for both sample preparation and data acquisition. The goal of the current unit is to describe the current tools for the study of soluble proteins, detergent-solubilized membrane proteins and their interactions by SV and SE.
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diffusion of the reaction boundary of rapidly interacting macromolecules in Sedimentation velocity
Biophysical Journal, 2010Co-Authors: Peter SchuckAbstract:Sedimentation velocity analytical ultracentrifugation combines relatively high hydrodynamic resolution of macromolecular species with the ability to study macromolecular interactions, which has great potential for studying dynamically assembled multiprotein complexes. Complicated Sedimentation boundary shapes appear in multicomponent mixtures when the timescale of the chemical reaction is short relative to the timescale of Sedimentation. Although the Lamm partial differential equation rigorously predicts the evolution of concentration profiles for given reaction schemes and parameter sets, this approach is often not directly applicable to data analysis due to experimental and sample imperfections, and/or due to unknown reaction pathways. Recently, we have introduced the effective particle theory, which explains quantitatively and in a simple physical picture the Sedimentation boundary patterns arising in the Sedimentation of rapidly interacting systems. However, it does not address the diffusional spread of the reaction boundary from the coSedimentation of interacting macromolecules, which also has been of long-standing interest in the theory of Sedimentation velocity analytical ultracentrifugation. Here, effective particle theory is exploited to approximate the concentration gradients during the Sedimentation process, and to predict the overall, gradient-average diffusion coefficient of the reaction boundary. The analysis of the heterogeneity of the Sedimentation and diffusion coefficients across the reaction boundary shows that both are relatively uniform. These results support the application of diffusion-deconvoluting Sedimentation coefficient distributions c(s) to the analysis of rapidly interacting systems, and provide a framework for the quantitative interpretation of the diffusional broadening and the apparent molar mass values of the effective sedimenting particle in dynamically associating systems.
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characterizing protein protein interactions by Sedimentation velocity analytical ultracentrifugation
Current protocols in immunology, 2008Co-Authors: P Brown, Andrea Balbo, Peter SchuckAbstract:This unit introduces the basic principles and practice of Sedimentation velocity analytical ultracentrifugation for the study of reversible protein interactions, such as the characterization of self-association, heterogeneous association, multi-protein complexes, binding stoichiometry, and the determination of association constants. The analytical tools described include Sedimentation coefficient and molar mass distributions, multi-signal Sedimentation coefficient distributions, Gilbert-Jenkins theory, different forms of isotherms, and global Lamm equation modeling. Concepts for the experimental design are discussed, and a detailed step-by-step protocol guiding the reader through the experiment and the data analysis is available as an Internet resource. Curr. Protoc. Immunol. 81:18.15.1-18.15.39. © 2008 by John Wiley & Sons, Inc. Keywords: Sedimentation equilibrium; Sedimentation velocity; chemical equilibria; reversible interactions; multi-protein complex; analytical ultracentrifugation; size-distribution; Gilbert-Jenkins theory; Lamm equation; Bayesian analysis
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macromolecular size and shape distributions by Sedimentation velocity analytical ultracentrifugation
Biophysical Journal, 2006Co-Authors: P Brown, Peter SchuckAbstract:Abstract Sedimentation velocity analytical ultracentrifugation is an important tool in the characterization of macromolecules and nanoparticles in solution. The Sedimentation coefficient distribution c ( s ) of Lamm equation solutions is based on the approximation of a single, weight-average frictional coefficient of all particles, determined from the experimental data, which scales the diffusion coefficient to the Sedimentation coefficient consistent with the traditional s ∼ M 2/3 power law. It provides a high hydrodynamic resolution, where diffusional broadening of the Sedimentation boundaries is deconvoluted from the Sedimentation coefficient distribution. The approximation of a single weight-average frictional ratio is favored by several experimental factors, and usually gives good results for chemically not too dissimilar macromolecules, such as mixtures of folded proteins. In this communication, we examine an extension to a two-dimensional distribution of Sedimentation coefficient and frictional ratio, c ( s , f r ), which is representative of a more general set of size-and-shape distributions, including mass-Stokes radius distributions, c ( M , R S ), and Sedimentation coefficient-molar mass distributions c ( s , M ). We show that this can be used to determine average molar masses of macromolecules and characterize macromolecular distributions, without the approximation of any scaling relationship between hydrodynamic and thermodynamic parameters.
P Brown - One of the best experts on this subject based on the ideXlab platform.
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characterizing protein protein interactions by Sedimentation velocity analytical ultracentrifugation
Current protocols in immunology, 2008Co-Authors: P Brown, Andrea Balbo, Peter SchuckAbstract:This unit introduces the basic principles and practice of Sedimentation velocity analytical ultracentrifugation for the study of reversible protein interactions, such as the characterization of self-association, heterogeneous association, multi-protein complexes, binding stoichiometry, and the determination of association constants. The analytical tools described include Sedimentation coefficient and molar mass distributions, multi-signal Sedimentation coefficient distributions, Gilbert-Jenkins theory, different forms of isotherms, and global Lamm equation modeling. Concepts for the experimental design are discussed, and a detailed step-by-step protocol guiding the reader through the experiment and the data analysis is available as an Internet resource. Curr. Protoc. Immunol. 81:18.15.1-18.15.39. © 2008 by John Wiley & Sons, Inc. Keywords: Sedimentation equilibrium; Sedimentation velocity; chemical equilibria; reversible interactions; multi-protein complex; analytical ultracentrifugation; size-distribution; Gilbert-Jenkins theory; Lamm equation; Bayesian analysis
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macromolecular size and shape distributions by Sedimentation velocity analytical ultracentrifugation
Biophysical Journal, 2006Co-Authors: P Brown, Peter SchuckAbstract:Abstract Sedimentation velocity analytical ultracentrifugation is an important tool in the characterization of macromolecules and nanoparticles in solution. The Sedimentation coefficient distribution c ( s ) of Lamm equation solutions is based on the approximation of a single, weight-average frictional coefficient of all particles, determined from the experimental data, which scales the diffusion coefficient to the Sedimentation coefficient consistent with the traditional s ∼ M 2/3 power law. It provides a high hydrodynamic resolution, where diffusional broadening of the Sedimentation boundaries is deconvoluted from the Sedimentation coefficient distribution. The approximation of a single weight-average frictional ratio is favored by several experimental factors, and usually gives good results for chemically not too dissimilar macromolecules, such as mixtures of folded proteins. In this communication, we examine an extension to a two-dimensional distribution of Sedimentation coefficient and frictional ratio, c ( s , f r ), which is representative of a more general set of size-and-shape distributions, including mass-Stokes radius distributions, c ( M , R S ), and Sedimentation coefficient-molar mass distributions c ( s , M ). We show that this can be used to determine average molar masses of macromolecules and characterize macromolecular distributions, without the approximation of any scaling relationship between hydrodynamic and thermodynamic parameters.
German Rivas - One of the best experts on this subject based on the ideXlab platform.
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analytical ultracentrifugation for the study of protein association and assembly
Current Opinion in Chemical Biology, 2006Co-Authors: Geoffrey J Howlett, Allen P Minton, German RivasAbstract:Analytical ultracentrifugation remains pre-eminent among the methods used to study the interactions of macromolecules under physiological conditions. Recent developments in analytical procedures allow the high resolving power of Sedimentation velocity methods to be coupled to Sedimentation equilibrium approaches and applied to both static and dynamic associations. Improvements in global modeling based on numerical solutions of the Lamm equation have generated new Sedimentation velocity applications with an emphasis on data interpretation using Sedimentation coefficient or molar mass distributions. Procedures based on the use of multiple optical signals from absorption and interference optics for the analysis of the Sedimentation velocity and equilibrium behavior of more complex interactions have now been developed. New applications of tracer Sedimentation equilibrium experiments and the development of a fluorescence optical system for the analytical ultracentrifuge extend the accessible concentration range over several orders of magnitude and, coupled with the new analytical procedures, provide powerful new tools for studies of both weak and strong macromolecular interactions in solution.
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characterization of heterologous protein protein interactions using analytical ultracentrifugation
Methods, 1999Co-Authors: German Rivas, Walter F Stafford, Allen P MintonAbstract:Methods for quantitative characterization of heterologous protein–protein interactions by means of analytical ultracentrifugation (AUC) include Sedimentation equilibrium, tracer Sedimentation equilibrium, Sedimentation velocity, and analytical band Sedimentation. Fundamental principles governing the behavior of macromolecules in a centrifugal field are summarized, and the application of these principles to the interpretation of data obtained from each type of experiment is reviewed. Instrumentation and software for the acquisition and analysis of data obtained from different types of AUC experiments are described.
Allen P Minton - One of the best experts on this subject based on the ideXlab platform.
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analytical ultracentrifugation for the study of protein association and assembly
Current Opinion in Chemical Biology, 2006Co-Authors: Geoffrey J Howlett, Allen P Minton, German RivasAbstract:Analytical ultracentrifugation remains pre-eminent among the methods used to study the interactions of macromolecules under physiological conditions. Recent developments in analytical procedures allow the high resolving power of Sedimentation velocity methods to be coupled to Sedimentation equilibrium approaches and applied to both static and dynamic associations. Improvements in global modeling based on numerical solutions of the Lamm equation have generated new Sedimentation velocity applications with an emphasis on data interpretation using Sedimentation coefficient or molar mass distributions. Procedures based on the use of multiple optical signals from absorption and interference optics for the analysis of the Sedimentation velocity and equilibrium behavior of more complex interactions have now been developed. New applications of tracer Sedimentation equilibrium experiments and the development of a fluorescence optical system for the analytical ultracentrifuge extend the accessible concentration range over several orders of magnitude and, coupled with the new analytical procedures, provide powerful new tools for studies of both weak and strong macromolecular interactions in solution.
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characterization of heterologous protein protein interactions using analytical ultracentrifugation
Methods, 1999Co-Authors: German Rivas, Walter F Stafford, Allen P MintonAbstract:Methods for quantitative characterization of heterologous protein–protein interactions by means of analytical ultracentrifugation (AUC) include Sedimentation equilibrium, tracer Sedimentation equilibrium, Sedimentation velocity, and analytical band Sedimentation. Fundamental principles governing the behavior of macromolecules in a centrifugal field are summarized, and the application of these principles to the interpretation of data obtained from each type of experiment is reviewed. Instrumentation and software for the acquisition and analysis of data obtained from different types of AUC experiments are described.
Huaying Zhao - One of the best experts on this subject based on the ideXlab platform.
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Sedimentation in a time varying centrifugal field for rapid attainment of Sedimentation equilibrium
Biophysical Journal, 2015Co-Authors: Peter Schuck, Michael A Metrick, Huaying ZhaoAbstract:Sedimentation equilibrium (SE) analytical ultracentrifugation is a gold standard for the rigorous thermodynamic study of buoyant molecular weight and reversible interactions of macromolecules in solution. A significant drawback is the long experiment time, as it takes days to attain SE with standard solution columns. We have developed a new method for using a time-varying centrifugal field optimized such as to attain SE in significantly shorter time than usually required. Experimental data show that this permits long-column SE experiments to be carried out in times comparable to Sedimentation velocity experiments, approximately fivefold shorter than standard SE. In contrast to the classical initial overspeeding method, which uses a single initial speed, we employ a freely varying rotor speed profile during an initial phase, for example, parameterized as a step-wise modulated exponential decay to the desired SE rotor speed. The rotor speed schedule is computationally optimized on the basis of numerical Lamm equation solutions for given macromolecular Sedimentation parameter estimates, with the goal to provide a rapid attainment of equilibrium without the drawback of strong transient sample pre-concentration at the base of the solution column. The resulting rotor speed schedule frequently includes both over- and under-speeding sequences, and can be conveniently implemented on the Optima XLA/I analytical ultracentrifuge. We extended AUC data analysis models in SEDFIT to permit the analysis of concentration profiles in arbitrarily time-varying fields, to make it possible to exploit the migration in the initially high centrifugal field for estimates on macromolecular Sedimentation parameters, which may be used in real-time to refine the prediction of the rotor speed schedule, so that the SE experiment can be optimized in both information content and time efficiency.
-
overview of current methods in Sedimentation velocity and Sedimentation equilibrium analytical ultracentrifugation
Current protocols in protein science, 2013Co-Authors: Huaying Zhao, Chad A Brautigam, Rodolfo Ghirlando, Peter SchuckAbstract:Modern computational strategies have allowed for the direct modeling of the Sedimentation process of heterogeneous mixtures, resulting in Sedimentation velocity (SV) size-distribution analyses with significantly improved detection limits and strongly enhanced resolution. These advances have transformed the practice of SV, rendering it the primary method of choice for most existing applications of analytical ultracentrifugation (AUC), such as the study of protein self- and hetero-association, the study of membrane proteins, and applications in biotechnology. New global multisignal modeling and mass conservation approaches in SV and Sedimentation equilibrium (SE), in conjunction with the effective-particle framework for interpreting the Sedimentation boundary structure of interacting systems, as well as tools for explicit modeling of the reaction/diffusion/Sedimentation equations to experimental data, have led to more robust and more powerful strategies for the study of reversible protein interactions and multiprotein complexes. Furthermore, modern mathematical modeling capabilities have allowed for a detailed description of many experimental aspects of the acquired data, thus enabling novel experimental opportunities, with important implications for both sample preparation and data acquisition. The goal of the current unit is to describe the current tools for the study of soluble proteins, detergent-solubilized membrane proteins and their interactions by SV and SE.