Analysis of Heterogeneous Interactions

doi:10.1016/S0076-6879(04)84013-8

Methods in Enzymology

Volume 384, 2004, Pages 212-232

https://doi.org/10.1016/S0076-6879(04)84013-8 Get rights and content

Publisher Summary

This chapter presents the mathematical formalism to describe heterogeneous equilibria, with particular attention to the relationship between the macroscopic and microscopic equilibrium constants. Identification and characterization of critical macromolecular interactions within the cell are central research problems of the postgenomic era. Quantitative methods capable of accurately defining the stoichiometry, affinity, cooperativity, and thermodynamics are required for a fundamental mechanistic understanding and to effectively target molecular interactions for therapeutic intervention. Although many biologically significant interactions involve association of identical subunits, a much larger class of binding events involves interactions of dissimilar partners. Rigorous investigation of heterogeneous interactions presents particular challenges for experimental design, data analysis, and interpretation. Heterogeneous interactions involve at least two distinct macromolecular components. The features of several biophysical methods that are commonly used to characterize heterogeneous interactions are compared. Equilibrium and velocity analytical ultracentrifugation are particularly useful as baseline methods to define assembly models and extract equilibrium parameters. The focus in this chapter is on sedimentation equilibrium and this approach is illustrated with an analysis of a nonspecific protein–nucleic acid interaction.

Introduction

Identification and characterization of critical macromolecular interactions within the cell are central research problems of the postgenomic era. High throughput mapping of protein–protein interactions is providing a global picture of the cellular interaction networks.¹ However, quantitative methods capable of accurately defining the stoichiometry, affinity, cooperativity, and thermodynamics are required for a fundamental mechanistic understanding and to effectively target molecular interactions for therapeutic intervention. Although many biologically significant interactions involve association of identical subunits (homogeneous interactions), a much larger class of binding events involves interactions of dissimilar partners (heterogeneous interactions or mixed interactions). Rigorous investigation of heterogeneous interactions presents particular challenges for experimental design, data analysis, and interpretation. In this chapter, we present the mathematical formalism to describe heterogeneous equilibria, with particular attention to the relationship between the macroscopic and microscopic equilibrium constants. We then compare features of several biophysical methods that are commonly used to characterize heterogeneous interactions. Equilibrium and velocity analytical ultracentrifugation are particularly useful as baseline methods to define assembly models and extract equilibrium parameters. A chapter in this volume describes new methods for analysis of interacting systems by velocity sedimentation²; here, we focus on sedimentation equilibrium and illustrate this approach with an analysis of a nonspecific protein–nucleic acid interaction.

Section snippets

Formalism for the Analysis of Heterogeneous Interactions

Heterogeneous interactions involve at least two distinct macromolecular components. For simplicity, we consider systems composed of just two components, designated as A and B, that combine to form one or more species, designated A_iB_j. In some cases A or B self-associate as well, and either i or j can be zero. Note that each species A_iB_j may also exist in multiple conformational states with distinct hydrodynamic properties; here we consider only equilibria among species with differing

Experimental Methods

When beginning a project to characterize a molecular interaction one is faced with the problem of choosing an appropriate method. This can be a daunting task, since there are many experimental biophysical approaches available for quantifying heterogeneous macromolecular interactions. Here, we briefly consider the factors that govern the choice of methodology and consider some of the unique capabilities of each approach. At the outset, it should be acknowledged that no single approach is

Example: Binding of Protein Kinase R to RNA

Protein kinase R (PKR) contains an N-terminal double-stranded RNA-binding domain (dsRBD) and a C-terminal kinase domain.⁶² PKR binds to RNA in a nonspecific manner. Here, we characterize binding of the N-terminal dsRBD domain (amino acids 1–184) with a 20-base pair RNA by sedimentation equilibrium. Previous experiments established a binding stoichiometry of three dsRBD⧸RNA.¹⁶ Based on these data, samples for sedimentation equilibrium were prepared at multiple concentrations of A (RNA) and B

Conclusion

A variety of biophysical techniques with a range of capabilities are available to probe heterogeneous macromolecular interactions. The choice of technique is governed chiefly by the type of information that is required and by the specific properties of the material under investigation. Analytical ultracentrifugation measurements are particularly useful at the beginning of a study to define an association model; subsequently, higher-throughput methods may be more convenient to analyze large

Acknowledgements

I thank Jason Ucci for his contributions to the studies of PKR and Jeff Lary for careful reading of this manuscript. This work was supported by the University of Connecticut's Research Advisory Council Programs.

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Analysis of Heterogeneous Interactions

Publisher Summary

Introduction

Section snippets

Formalism for the Analysis of Heterogeneous Interactions

Experimental Methods

Example: Binding of Protein Kinase R to RNA

Conclusion

Acknowledgements

Methods Enzymol.

Methods Enzymol.

Methods Enzymol.

J. Theor. Biol.

J. Mol. Biol.

J. Mol. Biol.

J. Biol. Chem.

Biophys. Chem.

Biophys. J.

Structure

Methods Enzymol.

Methods

Methods Enzymol.

Anal. Biochem.

Methods Enzymol.

Curr. Opin. Struct. Biol.

Methods Enzymol.

Anal. Biochem.

Methods Enzymol.

Curr. Opin. Struct. Biol.

Anal. Biochem.

Methods Enzymol.

Anal. Biochem.

Comp. Phys. Commun.

Appl. Math. Comput.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Mol. Biol.

Biophys. Chem.

Biophys. J.