Conformational Dynamics of Free and Catalytically Active Thermolysin Are Indistinguishable by Hydrogen/Deuterium Exchange Mass Spectrometry†

Biochemistry ◽  
2008 ◽  
Vol 47 (24) ◽  
pp. 6342-6351 ◽  
Author(s):  
Yu-Hong Liu ◽  
Lars Konermann
Keyword(s):  
Mass Spectrometry ◽  
Conformational Dynamics ◽  
Deuterium Exchange ◽  
Hydrogen Deuterium Exchange ◽  
Exchange Mass Spectrometry ◽  
Catalytically Active ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry

scholarly journals Load-dependent destabilization of the γ-rotor shaft in FOF1 ATP synthase revealed by hydrogen/deuterium-exchange mass spectrometry

2016 ◽  
Vol 113 (9) ◽  
pp. 2412-2417 ◽  
Author(s):  
Siavash Vahidi ◽  
Yumin Bi ◽  
Stanley D. Dunn ◽  
Lars Konermann
Keyword(s):  
Mass Spectrometry ◽  
Molecular Motor ◽  
Conformational Dynamics ◽  
Bound State ◽  
Deuterium Exchange ◽  
Hydrogen Deuterium Exchange ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Membrane Bound ◽  
Deuterium Exchange Mass Spectrometry

FoF1 is a membrane-bound molecular motor that uses proton-motive force (PMF) to drive the synthesis of ATP from ADP and Pi. Reverse operation generates PMF via ATP hydrolysis. Catalysis in either direction involves rotation of the γε shaft that connects the α3β3 head and the membrane-anchored cn ring. X-ray crystallography and other techniques have provided insights into the structure and function of FoF1 subcomplexes. However, interrogating the conformational dynamics of intact membrane-bound FoF1 during rotational catalysis has proven to be difficult. Here, we use hydrogen/deuterium exchange mass spectrometry to probe the inner workings of FoF1 in its natural membrane-bound state. A pronounced destabilization of the γ C-terminal helix during hydrolysis-driven rotation was observed. This behavior is attributed to torsional stress in γ, arising from γ⋅⋅⋅α3β3 interactions that cause resistance during γ rotation within the apical bearing. Intriguingly, we find that destabilization of γ occurs only when FoF1 operates against a PMF-induced torque; the effect disappears when PMF is eliminated by an uncoupler. This behavior resembles the properties of automotive engines, where bearings inflict greater forces on the crankshaft when operated under load than during idling.

scholarly journals Ligand-induced conformational dynamics of the Escherichia coli Na+/H+ antiporter NhaA revealed by hydrogen/deuterium exchange mass spectrometry

2017 ◽  
Vol 114 (44) ◽  
pp. 11691-11696 ◽  
Author(s):  
Martin Lorenz Eisinger ◽  
Aline Ricarda Dörrbaum ◽  
Hartmut Michel ◽  
Etana Padan ◽  
Julian David Langer
Keyword(s):  
Mass Spectrometry ◽  
Escherichia Coli ◽  
Conformational Dynamics ◽  
Ion Homeostasis ◽  
Deuterium Exchange ◽  
Hydrogen Deuterium Exchange ◽  
Exchange Mass Spectrometry ◽  
Ion Translocation ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry

Na+/H+ antiporters comprise a family of membrane proteins evolutionarily conserved in all kingdoms of life and play an essential role in cellular ion homeostasis. The NhaA crystal structure of Escherichia coli has become the paradigm for this class of secondary active transporters. However, structural data are only available at low pH, where NhaA is inactive. Here, we adapted hydrogen/deuterium-exchange mass spectrometry (HDX-MS) to analyze conformational changes in NhaA upon Li+ binding at physiological pH. Our analysis revealed a global conformational change in NhaA with two sets of movements around an immobile binding site. Based on these results, we propose a model for the ion translocation mechanism that explains previously controversial data for this antiporter. Furthermore, these findings contribute to our understanding of related human transporters that have been linked to various diseases.

scholarly journals PyHDX: Derivation and visualization of protection factors from Hydrogen-Deuterium Exchange Mass Spectrometry at near residue resolution

2020 ◽  
Author(s):  
Jochem H. Smit ◽  
Srinath Krishnamurthy ◽  
Bindu Y. Srinivasu ◽  
Spyridoula Karamanou ◽  
Anastassios Economou
Keyword(s):  
Mass Spectrometry ◽  
Local Equilibrium ◽  
Conformational Dynamics ◽  
Deuterium Exchange ◽  
Hydrogen Deuterium Exchange ◽  
Free Energies ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry ◽  
Hdx Ms

AbstractHydrogen Deuterium Exchange Mass Spectrometry (HDX-MS) is a powerful technique to monitor the intrinsic and conformational dynamics of proteins. Most HDX-MS experiments compare protein states (e.g. apoprotein vs liganded) and provide detailed information on differential dynamics between them obtained from multiple overlapping peptides. However, differential dynamics are difficult to compare across protein derivatives, oligomeric assemblies, homologues and samples treated under different buffer and protease conditions. A main reason is that peptide-based D-uptake differences do not inform on absolute intrinsic dynamics at the level of single aminoacyl residues. Such information is offered by protection factors, i.e. the position of the local equilibrium between the D-exchange-competent ‘open’ state and the non-exchanging ‘closed’ state. We present PyHDX, a software tool to calculate protection factors and Gibbs free energies typically within minutes from HDX-MS-derived peptide lists. PyHDX provides intrinsic information on the thermodynamics of protein dynamics at single-residue level. An interactive web interface further streamlines the process of transforming peptide lists to either coloured linear sequence maps or 3D structures of Gibbs free energies/protection factors.AvailabilityPyHDX source code is released under the MIT license and can be accessed at the project’s GitHub page.

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