Solution-Phase Hydrogen/Deuterium Exchange Monitored by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Solution-Phase Hydrogen/Deuterium Exchange Monitored by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

4.11 - 1251 ratings - Source



ABSTRACT: Proteins are vital macromolecules of every living cell. They undertake a variety of tasks that are essential for life. Most proteins function in assemblies rather than individually. Protein function is strongly correlated to structure and elucidation of both aspects of the protein is indispensable to understand its mechanism of action. Among several structural characterization techniques, solution-phase hydrogen/deuterium exchange monitored by mass spectrometry has emerged in the last 25 years as a powerful tool to study protein-protein interaction and dynamics. In the first half of this dissertation, we describe several aspects of method development that allowed tackling of previously inconceivable biological problems. In the second half, we demonstrate how this tool is applied to study muscle biochemistry; particularly, the troponin complex and myosin. In Chapter 1, we introduce the method, its mechanism, each of its sequential steps improvements which overcame multiple limitations and permitted its application to wide variety of biological problems. In Chapter 2, we describe the optimization of the separation step to minimize back-exchange, a major drawback of the technique. Chapter 3. demonstrates how isotopic depletion of proteins could be advantageous for peptide fragment identification, reduction of the mass spectral complexity, and the increase of the signal-to-noise ratio. In a different aspect of method development, we present a global analysis algorithm in Chapter 4, to manage the enormous amount of data, increase throughput, and most importantly, take advantage of peptide fragment overlap to increase sequence resolution. On the other hand, the second half of the dissertation is dedicated to muscle biochemistry. Troponin is a heterotrimeric complex involved in muscle regulation. The structure of a truncated version of the cardiac isoform of the complex was only solved in the calcium-saturated state. In Chapter 5, we apply hydrogen/deuterium exchange on full length troponin in the calcium-saturated and the calcium-free states to understand how the troponin subunits fit together and what are the conformational changes induced by calcium. Moreover, Chapter 6 describes the allosteric changes induced by phosphorylation the inhibitory subunit N-terminal extension, specific to the cardiac isoform of the complex. Finally, in Chapter 7, we elucidate the conformational changes that accompany the transition of myosin V (a molecular motor) from the post-rigor (ATP-analog bound) to the rigor-like (nucleotide-free) states, thus characterizing new features of the powerstroke.ABSTRACT: Proteins are vital macromolecules of every living cell.


Title:Solution-Phase Hydrogen/Deuterium Exchange Monitored by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry
Author: George Michel Bou-Assaf
Publisher: - 2011
ISBN-13:

You must register with us as either a Registered User before you can Download this Book. You'll be greeted by a simple sign-up page.

Once you have finished the sign-up process, you will be redirected to your download Book page.

How it works:
  • 1. Register a free 1 month Trial Account.
  • 2. Download as many books as you like (Personal use)
  • 3. Cancel the membership at any time if not satisfied.


Click button below to register and download Ebook
Privacy Policy | Contact | DMCA