Chapter 5. Mass Spectrometry-Based Approaches to Study Biomolecular Dynamics: Equilibrium Intermediates

  1. Igor A. Kaltashov and
  2. Stephen J. Eyles

Published Online: 27 JAN 2005

DOI: 10.1002/0471705179.ch5

Mass Spectrometry in Biophysics: Conformation and Dynamics of Biomolecules

Mass Spectrometry in Biophysics: Conformation and Dynamics of Biomolecules

How to Cite

Kaltashov, I. A. and Eyles, S. J. (2005) Mass Spectrometry-Based Approaches to Study Biomolecular Dynamics: Equilibrium Intermediates, in Mass Spectrometry in Biophysics: Conformation and Dynamics of Biomolecules, John Wiley & Sons, Inc., Hoboken, NJ, USA. doi: 10.1002/0471705179.ch5

Author Information

  1. University of Massachusetts at Amherst, USA

Publication History

  1. Published Online: 27 JAN 2005
  2. Published Print: 6 APR 2005

Book Series:

  1. Wiley-Interscience Series in Mass Spectrometry

Book Series Editors:

  1. Dominic M. Desiderio2 and
  2. Nico M. M. Nibbering3

Series Editor Information

  1. 2

    Departments of Neurology and Biochemistry, University of Tennessee Health Science Center, USA

  2. 3

    Vrije Universiteit Amsterdam, The Netherlands

ISBN Information

Print ISBN: 9780471456025

Online ISBN: 9780471705178

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Keywords:

  • intermediate state trapping;
  • disulfide mapping;
  • charge state distributions;
  • hydrogen-deuterium exchange;
  • slow exchange;
  • back-exchange;
  • hydrogen scrambling

Summary

In the preceding chapter, we surveyed various mass spectrometry-based approaches to study higher order structure of proteins under native conditions. For many decades, such well-defined and highly organized structures were thought of as the most important (if not the only) determinants of the protein function. Protein folding was often considered a linear process leading from fully unstructured (and, therefore, dysfunctional) states to the highly organized native (function-competent) state. The advent of NMR has changed our perception of what “functional” protein states are, with the realization that native proteins are very dynamic species. Perhaps the most illustrious examples of the intimate link between protein dynamics and function were found in enzyme catalysis, where the chemical conversion of substrate to product is often driven by relatively small-scale dynamic events within (and often beyond) the active site. It became clear in recent years that large-scale macromolecular dynamics may also be an important determinant of protein function. A growing number of proteins are found to be either partially or fully unstructured under native conditions, and such flexibility (intrinsic disorder) appears to be vital for their function. Proteins that do have native folds under physiological conditions can also exhibit dynamic behavior via local structural fluctuations or by sampling alternative (higher-energy or “activated”) conformations transiently. In many cases such activated (non-native) states are functionally important despite their low Boltzmann weight. Realization of the importance of transient non-native protein structures for their function not only greatly advanced our understanding of processes as diverse as recognition, signaling and transport, but has also had profound practical implications, particularly for the design of drugs targeting specific proteins. Because of their transient nature, these non-native states present a great challenge vis-à-vis detection and characterization. This chapter will present a concise introduction to an array of techniques that are used to study structure and behavior of the so-called “equilibrium intermediate states.” We will begin our discussion by considering protein ion charge state distributions in ESI mass spectra as indicators of protein unfolding. We will then proceed to various “trapping” techniques that exploit protein reactivity to reveal structural details of various non-native states. We will conclude the Chapter with a detailed discussion of hydrogen exchange, arguably one of the most widely used methods to probe the structure and dynamics of non-native (partially unstructured) protein states.