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Fluorescence Anisotropy-Based Salt-Titration Approach to Characterize Protein–Nucleic Acid Interactions

  • Tiffiny Rye-McCurdy
  • Ioulia Rouzina
  • Karin Musier-ForsythEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1259)

Abstract

Many proteins bind nucleic acids (NA) via cationic residues that interact electrostatically with the anionic phosphate backbone of RNA or DNA. These electrostatic interactions are often insensitive to NA sequence and structure, but confer strong salt dependence to the binding interactions. In contrast, salt-independent non-electrostatic contacts reflect more specific binding interactions. Proteins with multiple cationic NA-binding domains connected by flexible linkers, such as the HIV-1 Gag polyprotein, may bind different NA molecules in distinct ways. For example, Gag binding to the Psi-packaging signal of the HIV-1 RNA genome optimizes the specific non-electrostatic binding component of this protein–RNA interaction. In contrast, Gag binding to a non-psi RNA optimizes the electrostatic interactions at the expense of specific contacts. Here, we describe a fluorescence anisotropy-based salt-titration approach that allows complete characterization of both electrostatic and non-electrostatic binding components for any protein–NA complex in a quantitative manner within a single assay.

Key words

Fluorescence anisotropy Proteins Nucleic acids Specific binding Nonspecific binding Electrostatic interactions Non-electrostatic interactions HIV-1 Gag Nucleocapsid 

Notes

Acknowledgments

We thank Mr. Joseph A. Webb and Drs. Christopher Jones and Leslie Parent for their role in FA-based salt-titration method development and application, Ms. Roopa Comandur for determining the effect of glycerol on the FA signal, and Mr. Erik Olson and Ms. Weixin Wu for critical reading of this chapter prior to submission. This work was supported by NIH GM065056 (K.M.-F.) and T.R.-M. was supported by NIH T32-GM008512.

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Tiffiny Rye-McCurdy
    • 1
  • Ioulia Rouzina
    • 2
  • Karin Musier-Forsyth
    • 1
    Email author
  1. 1.Department of Chemistry and Biochemistry, Ohio State Biochemistry Program, Centers for Retroviral Research and RNA BiologyThe Ohio State UniversityColumbusUSA
  2. 2.Department of Biochemistry, Molecular Biology and BiophysicsUniversity of MinnesotaMinneapolisUSA

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