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Footprinting Methods to Examine the Structure and Dynamics of Proteins

Structural Determination Techniques (DNA, RNA and Protein)

  1. Jing-Qu Guan1,3,
  2. Mark R. Chance1,2,3

Published Online: 15 SEP 2006

DOI: 10.1002/3527600906.mcb.200300025

Reviews in Cell Biology and Molecular Medicine

Reviews in Cell Biology and Molecular Medicine

How to Cite

Guan, J.-Q. and Chance, M. R. 2006. Footprinting Methods to Examine the Structure and Dynamics of Proteins. Reviews in Cell Biology and Molecular Medicine. .

Author Information

  1. 1

    Albert Einstein College of Medicine, Departments of Physiology & Biophysics, Bronx, New York, USA

  2. 2

    Albert Einstein College of Medicine, Department of Biochemistry, Bronx, New York, USA

  3. 3

    Albert Einstein College of Medicine, Center for Synchrotron Biosciences, Bronx, New York, USA

Publication History

  1. Published Online: 15 SEP 2006

Abstract

“Footprinting” refers to assays that examine ligand binding and conformational changes by determining the solvent accessibility of the backbone, bases, or side-chain structures of macromolecules through their sensitivity to chemical or enzymatic cleavage or modification reactions. Protein footprinting methods have been developed to examine protein structure and conformational changes by monitoring solvent accessibility using either modification or cleavage reactions. Examples of such methods include acetylation, nonspecific limited proteolysis, or probing accessibility of a range of sites that can be specifically or nonspecifically cleaved or modified. The basis of these diverse chemical approaches is to monitor the change in accessibility of susceptible residues as a function of relevant conformational fluctuations. In addition, the amounts of material required for exploring the structure and function of proteins using footprinting is in the femtomole to picomole level for detailed analysis. Protein footprinting methods based on protease cleavage have been used to map protein structure, nucleic acid–protein interactions and protein folding intermediates. However, cleavage techniques using proteases suffer from limited structural resolution due to the large size of the probe. Hydroxyl radical methods of cleavage and modification have been pursued to overcome this drawback. In this article, we document the history of protein footprinting and the development of new technologies based on mass spectrometry to accurately locate and quantify protein structure and conformational changes upon ligand binding.

Keywords:

  • One-hit Kinetics;
  • Footprinting;
  • Hydroxyl Radical;
  • Fenton Chemistry;
  • Radiolysis;
  • RNA Polymerase;
  • Mass Spectrometry;
  • Western Blot