The Experts below are selected from a list of 180 Experts worldwide ranked by ideXlab platform
Donald J. Winzor - One of the best experts on this subject based on the ideXlab platform.
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Allowance for effects of thermodynamic nonideality in Sedimentation Equilibrium distributions reflecting protein dimerization
Analytical biochemistry, 2011Co-Authors: Peter R. Wills, David J. Scott, Donald J. WinzorAbstract:This reexamination of a high-speed Sedimentation Equilibrium distribution for α-chymotrypsin under slightly acidic conditions (pH 4.1, I(M) 0.05) has provided experimental support for the adequacy of nearest-neighbor considerations in the allowance for effects of thermodynamic nonideality in the characterization of protein self-association over a moderate concentration range (up to 8 mg/mL). A widely held but previously untested notion about allowance for thermodynamic nonideality effects is thereby verified experimentally. However, it has also been shown that a greater obstacle to better characterization of protein self-association is likely to be the lack of a reliable estimate of monomer net charge, a parameter that has a far more profound effect on the magnitude of the measured Equilibrium constant than any deficiency in current procedures for incorporating the effects of thermodynamic nonideality into the analysis of Sedimentation Equilibrium distributions reflecting reversible protein self-association.
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Allowance for the effect of protein charge in the characterization of nonideal solute self-association by Sedimentation Equilibrium.
Biophysical chemistry, 2010Co-Authors: David J. Scott, Peter R. Wills, Donald J. WinzorAbstract:This theoretical investigation explores the use of statistical–mechanical approaches to characterize the reversible tetramerization of a protein monomer with the size and charge characteristics of serum albumin under conditions where consideration of nearest-neighbor interactions suffices to describe effects of thermodynamic non-ideality. Such analysis of simulated Sedimentation Equilibrium distributions points to the adequacy of both the scaled particle theory and potential-of-mean-force methods for determining the self-association constant. Although the latter method usually entails the assignment of a magnitude to monomer net charge, this requirement can be obviated to some extent by repeating the analysis for a range of monomer charges and identifying the most appropriate value as that associated with a minimum in the sum-of-squares-of-residuals (SSR) of the best-fit descriptions of the Sedimentation Equilibrium distribution. Reasonable estimates of the association constant are usually obtained from corresponding analyses of the same Sedimentation Equilibrium distributions with activity coefficients obtained by scaled particle theory, an approach which also involves the identification of parameters on the basis of a minimum in SSR. However, the value of monomer charge determined must be regarded as a curve-fitting parameter rather than a true measure of monomer charge. Similar qualifications are shown to prevail in the scaled particle theory approach, which also involves the identification of parameters (the effective monomer volume and the polymer/monomer volume ratio) on the basis of a minimum in SSR. We therefore recommend discontinuation of the practice whereby quite precise distinction between modes of self-association has been attempted on the grounds of the physical credibility of the magnitudes of these additional curve-fitting parameters.© 2010 Elsevier B.V.
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Comparison of Methods for Characterizing Nonideal Solute Self-Association by Sedimentation Equilibrium
Biophysical journal, 2009Co-Authors: David J. Scott, Donald J. WinzorAbstract:We have examined in detail analytical solutions of expressions for Sedimentation Equilibrium in the analytical ultracentrifuge to describe self-association under nonideal conditions. We find that those containing the radial dependence of total solute concentration that incorporate the Adams-Fujita assumption for composition-dependence of activity coefficients reveal potential shortcomings for characterizing such systems. Similar deficiencies are shown in the use of the NONLIN software incorporating the same assumption about the interrelationship between activity coefficients for monomer and polymer species. These difficulties can be overcome by iterative analyses incorporating expressions for the composition-dependence of activity coefficients predicted by excluded volume considerations. A recommendation is therefore made for the replacement of current software packages by programs that incorporate rigorous statistical-mechanical allowance for thermodynamic nonideality in Sedimentation Equilibrium distributions reflecting solute self-association.
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nonequivalence of second virial coefficients from Sedimentation Equilibrium and static light scattering studies of protein solutions
Biophysical Chemistry, 2007Co-Authors: Donald J. Winzor, Stephen E Harding, Marcin Deszczynski, Peter R. WillsAbstract:Experimental data for ovalbumin and lysozyme are presented to highlight the nonequivalence of second virial coefficients obtained for proteins by Sedimentation Equilibrium and light scattering. Theoretical considerations confirm that the quantity deduced from Sedimentation Equilibrium distributions is B(22), the osmotic second virial coefficient describing thermodynamic nonideality arising solely from protein self-interaction. On the other hand, the virial coefficient determined by light scattering is shown to reflect the combined contributions of protein-protein and protein-buffer interactions to thermodynamic nonideality of the protein solution. Misidentification of the light scattering parameter as B(22) accounts for published reports of negative osmotic second virial coefficients as indicators of conditions conducive to protein crystal growth. Finally, textbook assertions about the equivalence of second virial coefficients obtained by Sedimentation Equilibrium and light scattering reflect the restriction of consideration to single-solute systems. Although Sedimentation Equilibrium distributions for buffered protein solutions are, indeed, amenable to interpretation in such terms, the same situation does not apply to light scattering measurements because buffer constituents cannot be regarded as part of the solvent: instead they must be treated as non-scattering cosolutes.
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Characterization of weak protein dimerization by direct analysis of Sedimentation Equilibrium distributions: The INVEQ approach
Analytical biochemistry, 2007Co-Authors: Donald J. Winzor, Peter R. WillsAbstract:Closer scrutiny has been accorded a recently reported procedure for characterizing weak protein dimerization by Sedimentation Equilibrium (INVEQ) in which the Equilibrium distribution is analyzed as a dependence of radial distance on solute concentration rather than of solute concentration on radial distance. By demonstrating theoretically that the fundamental parameter derived from the analysis is simply the difference between the dimerization constant and the osmotic second virial coefficient for monomer-monomer interaction, this investigation refutes the original claim that independent estimates of these two parameters can be obtained by nonlinear curve fitting of the Sedimentation Equilibrium distribution. This criticism also applies to conventional analyses of Sedimentation distributions by the commonly employed Beckman Origin and NONLIN software. Numerically simulated distributions are then analyzed to demonstrate limitations of the procedure and also to indicate a means of improving the reliability of the returned estimate of the dimerization constant. These features are illustrated by applying the original and revised analytical procedures to a Sedimentation Equilibrium distribution for α-chymotrypsin (pH 4.0, I 0.05 M).
Peter R. Wills - One of the best experts on this subject based on the ideXlab platform.
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Allowance for effects of thermodynamic nonideality in Sedimentation Equilibrium distributions reflecting protein dimerization
Analytical biochemistry, 2011Co-Authors: Peter R. Wills, David J. Scott, Donald J. WinzorAbstract:This reexamination of a high-speed Sedimentation Equilibrium distribution for α-chymotrypsin under slightly acidic conditions (pH 4.1, I(M) 0.05) has provided experimental support for the adequacy of nearest-neighbor considerations in the allowance for effects of thermodynamic nonideality in the characterization of protein self-association over a moderate concentration range (up to 8 mg/mL). A widely held but previously untested notion about allowance for thermodynamic nonideality effects is thereby verified experimentally. However, it has also been shown that a greater obstacle to better characterization of protein self-association is likely to be the lack of a reliable estimate of monomer net charge, a parameter that has a far more profound effect on the magnitude of the measured Equilibrium constant than any deficiency in current procedures for incorporating the effects of thermodynamic nonideality into the analysis of Sedimentation Equilibrium distributions reflecting reversible protein self-association.
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Allowance for the effect of protein charge in the characterization of nonideal solute self-association by Sedimentation Equilibrium.
Biophysical chemistry, 2010Co-Authors: David J. Scott, Peter R. Wills, Donald J. WinzorAbstract:This theoretical investigation explores the use of statistical–mechanical approaches to characterize the reversible tetramerization of a protein monomer with the size and charge characteristics of serum albumin under conditions where consideration of nearest-neighbor interactions suffices to describe effects of thermodynamic non-ideality. Such analysis of simulated Sedimentation Equilibrium distributions points to the adequacy of both the scaled particle theory and potential-of-mean-force methods for determining the self-association constant. Although the latter method usually entails the assignment of a magnitude to monomer net charge, this requirement can be obviated to some extent by repeating the analysis for a range of monomer charges and identifying the most appropriate value as that associated with a minimum in the sum-of-squares-of-residuals (SSR) of the best-fit descriptions of the Sedimentation Equilibrium distribution. Reasonable estimates of the association constant are usually obtained from corresponding analyses of the same Sedimentation Equilibrium distributions with activity coefficients obtained by scaled particle theory, an approach which also involves the identification of parameters on the basis of a minimum in SSR. However, the value of monomer charge determined must be regarded as a curve-fitting parameter rather than a true measure of monomer charge. Similar qualifications are shown to prevail in the scaled particle theory approach, which also involves the identification of parameters (the effective monomer volume and the polymer/monomer volume ratio) on the basis of a minimum in SSR. We therefore recommend discontinuation of the practice whereby quite precise distinction between modes of self-association has been attempted on the grounds of the physical credibility of the magnitudes of these additional curve-fitting parameters.© 2010 Elsevier B.V.
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nonequivalence of second virial coefficients from Sedimentation Equilibrium and static light scattering studies of protein solutions
Biophysical Chemistry, 2007Co-Authors: Donald J. Winzor, Stephen E Harding, Marcin Deszczynski, Peter R. WillsAbstract:Experimental data for ovalbumin and lysozyme are presented to highlight the nonequivalence of second virial coefficients obtained for proteins by Sedimentation Equilibrium and light scattering. Theoretical considerations confirm that the quantity deduced from Sedimentation Equilibrium distributions is B(22), the osmotic second virial coefficient describing thermodynamic nonideality arising solely from protein self-interaction. On the other hand, the virial coefficient determined by light scattering is shown to reflect the combined contributions of protein-protein and protein-buffer interactions to thermodynamic nonideality of the protein solution. Misidentification of the light scattering parameter as B(22) accounts for published reports of negative osmotic second virial coefficients as indicators of conditions conducive to protein crystal growth. Finally, textbook assertions about the equivalence of second virial coefficients obtained by Sedimentation Equilibrium and light scattering reflect the restriction of consideration to single-solute systems. Although Sedimentation Equilibrium distributions for buffered protein solutions are, indeed, amenable to interpretation in such terms, the same situation does not apply to light scattering measurements because buffer constituents cannot be regarded as part of the solvent: instead they must be treated as non-scattering cosolutes.
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Characterization of weak protein dimerization by direct analysis of Sedimentation Equilibrium distributions: The INVEQ approach
Analytical biochemistry, 2007Co-Authors: Donald J. Winzor, Peter R. WillsAbstract:Closer scrutiny has been accorded a recently reported procedure for characterizing weak protein dimerization by Sedimentation Equilibrium (INVEQ) in which the Equilibrium distribution is analyzed as a dependence of radial distance on solute concentration rather than of solute concentration on radial distance. By demonstrating theoretically that the fundamental parameter derived from the analysis is simply the difference between the dimerization constant and the osmotic second virial coefficient for monomer-monomer interaction, this investigation refutes the original claim that independent estimates of these two parameters can be obtained by nonlinear curve fitting of the Sedimentation Equilibrium distribution. This criticism also applies to conventional analyses of Sedimentation distributions by the commonly employed Beckman Origin and NONLIN software. Numerically simulated distributions are then analyzed to demonstrate limitations of the procedure and also to indicate a means of improving the reliability of the returned estimate of the dimerization constant. These features are illustrated by applying the original and revised analytical procedures to a Sedimentation Equilibrium distribution for α-chymotrypsin (pH 4.0, I 0.05 M).
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Analysis of Sedimentation Equilibrium Distributions Reflecting Nonideal Macromolecular Associations
Biophysical Journal, 2000Co-Authors: Peter R. Wills, Michael P. Jacobsen, Donald J. WinzorAbstract:A rigorous statistical-mechanical approach is adopted to derive general quantitative expressions that allow for the effects of thermodynamic nonideality in Equilibrium measurements reflecting interaction between dissimilar macromolecular reactants. An analytical procedure based on these expressions is then formulated for obtaining global estimates of Equilibrium constants and the corresponding reference thermodynamic activities of the free reactants in each of several Sedimentation Equilibrium experiments. The method is demonstrated by application to results from an ultracentrifugal study of an electrostatic interaction between ovalbumin and cytochrome c (Winzor, D. J., M. P, Jacobsen, and P. R. Wills. 1998. Biochemistry. 37:2226-2233). It is demonstrated that reliable estimates of relevant thermodynamic parameters are extracted from the data through statistical analysis by means of a simple nonlinear fitting procedure.
Stephen E Harding - One of the best experts on this subject based on the ideXlab platform.
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nonequivalence of second virial coefficients from Sedimentation Equilibrium and static light scattering studies of protein solutions
Biophysical Chemistry, 2007Co-Authors: Donald J. Winzor, Stephen E Harding, Marcin Deszczynski, Peter R. WillsAbstract:Experimental data for ovalbumin and lysozyme are presented to highlight the nonequivalence of second virial coefficients obtained for proteins by Sedimentation Equilibrium and light scattering. Theoretical considerations confirm that the quantity deduced from Sedimentation Equilibrium distributions is B(22), the osmotic second virial coefficient describing thermodynamic nonideality arising solely from protein self-interaction. On the other hand, the virial coefficient determined by light scattering is shown to reflect the combined contributions of protein-protein and protein-buffer interactions to thermodynamic nonideality of the protein solution. Misidentification of the light scattering parameter as B(22) accounts for published reports of negative osmotic second virial coefficients as indicators of conditions conducive to protein crystal growth. Finally, textbook assertions about the equivalence of second virial coefficients obtained by Sedimentation Equilibrium and light scattering reflect the restriction of consideration to single-solute systems. Although Sedimentation Equilibrium distributions for buffered protein solutions are, indeed, amenable to interpretation in such terms, the same situation does not apply to light scattering measurements because buffer constituents cannot be regarded as part of the solvent: instead they must be treated as non-scattering cosolutes.
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A comparison of molecular mass determination of hyaluronic acid using SEC/MALLS and Sedimentation Equilibrium
European Biophysics Journal, 2003Co-Authors: Sanya Hokputsa, Catherine Alexander, Kornelia Jumel, Stephen E HardingAbstract:Hyaluronic acid (HA) is a natural polysaccharide with importance in the pharmaceutical, medical and cosmetic industries. Determining factors in its final applications are its physicochemical properties, particularly molecular mass. A high molecular mass HA was degraded using five different hydroxyl free-radical starting concentrations chemically produced from ascorbic acid and hydrogen peroxide. The aims of the study were to investigate the effect of different hydroxyl free-radical concentrations on the chain length of HA and compare the molecular masses obtained from analytical ultracentrifugation using Sedimentation Equilibrium experiments and size exclusion chromatography/multi-angle laser light scattering (SEC/MALLS). The results indicated that their molecular masses varied, depending on the degree of hydroxyl free-radical starting concentration. Molecular mass values obtained from Sedimentation Equilibrium experiments for each sample showed the same trend as those obtained from the SEC/MALLS in the range of molecular masses studied. The molecular masses obtained from Sedimentation Equilibrium for high molecular mass samples from reciprocal plots of apparent weight average molecular mass against concentration gave values similar to those obtained by SEC/MALLS. In contrast, the molecular mass from conventional plots for high molecular mass samples were much lower than those from SEC/MALLS, even when high ionic strength buffers were used.
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a comparison of molecular mass determination of hyaluronic acid using sec malls and Sedimentation Equilibrium
European Biophysics Journal, 2003Co-Authors: Sanya Hokputsa, Catherine Alexander, Kornelia Jumel, Stephen E HardingAbstract:Hyaluronic acid (HA) is a natural polysaccharide with importance in the pharmaceutical, medical and cosmetic industries. Determining factors in its final applications are its physicochemical properties, particularly molecular mass. A high molecular mass HA was degraded using five different hydroxyl free-radical starting concentrations chemically produced from ascorbic acid and hydrogen peroxide. The aims of the study were to investigate the effect of different hydroxyl free-radical concentrations on the chain length of HA and compare the molecular masses obtained from analytical ultracentrifugation using Sedimentation Equilibrium experiments and size exclusion chromatography/multi-angle laser light scattering (SEC/MALLS). The results indicated that their molecular masses varied, depending on the degree of hydroxyl free-radical starting concentration. Molecular mass values obtained from Sedimentation Equilibrium experiments for each sample showed the same trend as those obtained from the SEC/MALLS in the range of molecular masses studied. The molecular masses obtained from Sedimentation Equilibrium for high molecular mass samples from reciprocal plots of apparent weight average molecular mass against concentration gave values similar to those obtained by SEC/MALLS. In contrast, the molecular mass from conventional plots for high molecular mass samples were much lower than those from SEC/MALLS, even when high ionic strength buffers were used.
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The correct analysis of low-speed Sedimentation Equilibrium distributions recorded by the Rayleigh interference optical system in a Beckman XL-I ultracentrifuge
Progress in colloid and polymer science, 1999Co-Authors: Damien Hall, Stephen E Harding, Donald J. WinzorAbstract:The molecular mass of ovalbumin, a well-characterized protein, has been determined from low-speed Sedimentation Equilibrium distributions recorded by the absorption and Rayleigh optical systems of a Beckman XL-I ultracentrifuge in order to assess the reliability of various procedures for analyzing the Rayleigh interfero-metric records. Despite assertions to the contrary, the present results demonstrate the importance of establishing a concentration distribution in terms of absolute fringe displacement, J(r) versus r, before quantitative analysis of the distribution is atttempted in terms of the basic Sedimentation Equilibrium expression for a single solute: this consideration is particularly important in experiments where the con-centration at the meniscus is sizeable in relation to the concentration difference across the Equilibrium distribution. In that regard the incorporation of a synthetic boundary experiment into the protocol seems to provide the most reliable means of ascertaining the absolute concentration distribution from Rayleigh interferometric records of low-speed Sedimentation Equilibrium experiments, and is an essential prerequisite for the quantitative characterization of interacting systems.
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MSTARA and MSTARI: interactive PC algorithms for simple, model independent evaluation of Sedimentation Equilibrium data
European Biophysics Journal, 1997Co-Authors: Helmut Cölfen, Stephen E HardingAbstract:This paper describes a program available for PC's for the evaluation of molecular weights from Sedimentation Equilibrium. This program, in its two forms – MSTARA for absorption optical records and MSTARI for interference optical records – requires no prior assumption of the nature of the system (ideal, non-ideal, monodisperse, polydisperse, self-associating etc.) and takes into consideration the whole solute distribution (i.e. from solution meniscus to cell base) in the ultracentrifuge cell rather than just a selected data-set. MSTARA or MSTARI are therefore recommended as a first analysis programme of Sedimentation Equilibrium data coming off an absorption or interference based analytical ultracentrifuge. These programmes are therefore particularly well suited if heterogeneity (polydispersity or interaction phenomena) or non-ideality is suspected. Their use is demonstrated for a series of data-set types (ideal, non-ideal, polydisperse and self-associating). Although MSTARA and MSTARI are model independent, they provide the basis for more detailed analysis of interactions, polydisperse distributions or non-ideality via easy export of ASCII datafiles to model dependent routines.
Allen P Minton - One of the best experts on this subject based on the ideXlab platform.
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Detection and Quantitative Characterization of Macromolecular Heteroassociations via Composition Gradient Sedimentation Equilibrium
Analytical Ultracentrifugation, 2016Co-Authors: Allen P MintonAbstract:A novel approach to the detection and characterization of macromolecular heteroassociations is described, based upon a combination of short-column Sedimentation Equilibrium measurements and the utilization of a composition gradient spanning the entire range of mole fractions of interacting components. Simulations are presented to demonstrate that the method described provides a high degree of discrimination between alternative models for heteroassociation equilibria. Comparison with experimental protocols utilized previously shows that the new approach provides equivalent information with less sample and a considerably shorter duration of centrifugation.
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beyond the second virial coefficient Sedimentation Equilibrium in highly non ideal solutions
Methods, 2011Co-Authors: German Rivas, Allen P MintonAbstract:The general theory of Sedimentation Equilibrium (SE), applicable to mixtures of interacting sedimentable solutes at arbitrary concentration, is summarized. Practical techniques for the acquisition of SE data suitable for analysis are described. Experimental measurements and analyses of SE in concentrated protein solutions are reviewed. The method of non-ideal tracer Sedimentation Equilibrium (NITSE) is described. Experimental studies using NITSE to detect and quantitatively characterize intermolecular interactions in mixtures of dilute tracer species and concentrated proteins or polymers are reviewed.
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Quantitative characterization of polymer-polymer, protein-protein, and polymer-protein interaction via tracer Sedimentation Equilibrium.
Journal of Physical Chemistry B, 2010Co-Authors: Adedayo A. Fodeke, Allen P MintonAbstract:Quantitative analysis of the composition dependence of the concentration gradient of each species of macromolecule within a solution mixture at Sedimentation Equilibrium permits the quantitative characterization of self- and heterointeractions between sedimenting solutes. Sedimentation Equilibrium experiments were conducted on solutions containing a trace concentration of FITC-labeled BSA in varying concentrations of Ficoll 70 and on solutions containing a trace concentration of FITC-labeled Ficoll 70 in varying concentrations of BSA. The Equilibrium gradient of each solute component in each mixture was measured independently. Analysis of the resulting gradients resulted in evaluation of the dependence of the activity coefficient of Ficoll upon the concentrations of Ficoll and BSA at concentrations of up to 100 g/L and the dependence of the activity coefficient of BSA upon the concentrations of Ficoll and BSA at concentrations of up to 100 g/L. The activity coefficients of both species increase significan...
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Quantitative characterization of reversible macromolecular associations via Sedimentation Equilibrium: an introduction
Experimental & Molecular Medicine, 2000Co-Authors: Allen P MintonAbstract:The measurement and analysis of Sedimentation Equilibrium provides one of the most powerful and widely applicable methods for the characterization of reversible associations of macromolecules in solution. Recent developments in instrumentation, experimental design, and data analysis have substantially broadened the range of systems to which this technique may be applied, simplified its application, and reduced the cost of acquiring analytical capability.
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New developments in the study of biomolecular associations via Sedimentation Equilibrium
Trends in biochemical sciences, 1993Co-Authors: German Rivas, Allen P MintonAbstract:Abstract The measurement and analysis of Sedimentation Equilibrium provide on of the most powerful techniques for quantitative characterization of reversible and irreversible macromolecular associations in solution. The use of this technique by nonspecialists has been greatly helped in recent years by the development of new instrumentation, new types of experiments and new PC-based software for computer-aided analysis of experimental results.
James L Cole - One of the best experts on this subject based on the ideXlab platform.
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analytical ultracentrifugation Sedimentation velocity and Sedimentation Equilibrium
Methods in Cell Biology, 2008Co-Authors: James L Cole, Jeffrey W Lary, Thomas P Moody, Thomas M. LaueAbstract:Analytical ultracentrifugation (AUC) is a versatile and powerful method for the quantitative analysis of macromolecules in solution. AUC has broad applications for the study of biomacromolecules in a wide range of solvents and over a wide range of solute concentrations. Three optical systems are available for the analytical ultracentrifuge (absorbance, interference, and fluorescence) that permit precise and selective observation of Sedimentation in real time. In particular, the fluorescence system provides a new way to extend the scope of AUC to probe the behavior of biological molecules in complex mixtures and at high solute concentrations. In Sedimentation velocity (SV), the movement of solutes in high centrifugal fields is interpreted using hydrodynamic theory to define the size, shape, and interactions of macromolecules. Sedimentation Equilibrium (SE) is a thermodynamic method where Equilibrium concentration gradients at lower centrifugal fields are analyzed to define molecule mass, assembly stoichiometry, association constants, and solution nonideality. Using specialized sample cells and modern analysis software, researchers can use SV to determine the homogeneity of a sample and define whether it undergoes concentration-dependent association reactions. Subsequently, more thorough model-dependent analysis of velocity and Equilibrium experiments can provide a detailed picture of the nature of the species present in solution and their interactions.
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Global analysis of non-specific protein-nucleic interactions by Sedimentation Equilibrium.
Biophysical chemistry, 2004Co-Authors: Jason W Ucci, James L ColeAbstract:Protein-nucleic acid interactions govern a variety of processes, including replication, transcription, recombination and repair. These interactions take place in both sequence-specific and non-specific modes, and the latter occur in many biologically significant contexts. Analytical ultracentrifugation is a useful method for the detailed characterization of the stoichiometry and affinity of macromolecular interactions in free solution. There has been a resurgence of interest in the application of Sedimentation Equilibrium methods to protein-nucleic acid interactions. However, these studies have been generally focused on sequence-specific interactions. Here we describe an approach to analyze non-specific interactions using Sedimentation Equilibrium. We have adapted an existing model for non-specific interaction of proteins with finite, one-dimensional nucleic acid lattices for global fitting of multiwavelength Sedimentation Equilibrium data. The model is extended to accommodate protein binding to multiple faces of the nucleic acid, resulting in overlap of consecutive ligands along the sequence of the RNA or DNA. The approach is illustrated in a Sedimentation Equilibrium analysis of the interaction of the double-stranded RNA binding motif of protein kinase R with a 20-basepair RNA construct.
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Global analysis of non-specific protein–nucleic interactions by Sedimentation Equilibrium
Biophysical Chemistry, 2004Co-Authors: Jason W Ucci, James L ColeAbstract:Protein-nucleic acid interactions govern a variety of processes, including replication, transcription, recombination and repair. These interactions take place in both sequence-specific and non-specific modes, and the latter occur in many biologically significant contexts. Analytical ultracentrifugation is a useful method for the detailed characterization of the stoichiometry and affinity of macromolecular interactions in free solution. There has been a resurgence of interest in the application of Sedimentation Equilibrium methods to protein-nucleic acid interactions. However, these studies have been generally focused on sequence-specific interactions. Here we describe an approach to analyze non-specific interactions using Sedimentation Equilibrium. We have adapted an existing model for non-specific interaction of proteins with finite, one-dimensional nucleic acid lattices for global fitting of multiwavelength Sedimentation Equilibrium data. The model is extended to accommodate protein binding to multiple faces of the nucleic acid, resulting in overlap of consecutive ligands along the sequence of the RNA or DNA. The approach is illustrated in a Sedimentation Equilibrium analysis of the interaction of the double-stranded RNA binding motif of protein kinase R with a 20-basepair RNA construct.