scholarly journals Pore dynamics and asymmetric cargo loading in an encapsulin nanocompartment revealed by Cryo-EM and hydrogen/deuterium exchange mass spectrometry

2021 ◽  
Author(s):  
Jennifer Ross ◽  
Zak McIver ◽  
Thomas Lambert ◽  
Cecilia Piergentili ◽  
Kelly J. Gallagher ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Fixed Number ◽  
Deuterium Exchange ◽  
Hydrogen Deuterium Exchange ◽  
Iron Storage ◽  
Single Particle Reconstruction ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry ◽  
Hdx Ms

AbstractEncapsulins (Enc) are protein nanocompartments which house various cargo enzymes, including a family of decameric ferritin-like proteins. Previously, we elucidated the structure and activity of these ferritin-like proteins (EncFtn) and demonstrated that they must be encapsulated in a nanocompartment for iron storage. Here, we study a recombinant Haliangium ochraceum Enc:EncFtn complex using electron cryo-microscopy (Cryo-EM) and hydrogen/deuterium exchange mass spectrometry (HDX-MS) to gain insight into the structural relationship between Enc and EncFtn. An asymmetric single particle reconstruction reveals four EncFtn decamers in a tetrahedral arrangement within the encapsulin nanocompartment. This leads to a symmetry mismatch between the EncFtn cargo and the icosahedral encapsulin shell. The EncFtn decamers are offset from the interior face of the encapsulin shell and are resolved at a much lower overall resolution in the final reconstruction. This flexibility, and the fixed number of EncFtn decamers sequestered within, implies that the loading of the encapsulin nanocompartment is limited by the steric effect of both engaged and free encapsulin localization sequences. Using a combination of focused refinements and HDX-MS, we observed dynamic behavior of the major five-fold pore, and show the pore opening via the movement of the encapsulin A-domain. These data can accelerate efforts to engineer the sequestration of heterologous cargo proteins and to alter the permeability of the encapsulin shell via pore modifications.

scholarly journals Hydrogen-Deuterium Exchange Mass Spectrometry: A Novel Structural Biology Approach to Structure, Dynamics and Interactions of Proteins and Their Complexes

Life ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 286
Author(s):  
Oliver Ozohanics ◽  
Attila Ambrus
Keyword(s):  
Mass Spectrometry ◽  
Membrane Proteins ◽  
Structural Biology ◽  
Deuterium Exchange ◽  
Hydrogen Deuterium Exchange ◽  
Ligand Interaction ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry ◽  
Hdx Ms

Hydrogen/Deuterium eXchange Mass Spectrometry (HDX-MS) is a rapidly evolving technique for analyzing structural features and dynamic properties of proteins. It may stand alone or serve as a complementary method to cryo-electron-microscopy (EM) or other structural biology approaches. HDX-MS is capable of providing information on individual proteins as well as large protein complexes. Owing to recent methodological advancements and improving availability of instrumentation, HDX-MS is becoming a routine technique for some applications. When dealing with samples of low to medium complexity and sizes of less than 150 kDa, conformation and ligand interaction analyses by HDX-MS are already almost routine applications. This is also well supported by the rapid evolution of the computational (software) background that facilitates the analysis of the obtained experimental data. HDX-MS can cope at times with analytes that are difficult to tackle by any other approach. Large complexes like viral capsids as well as disordered proteins can also be analyzed by this method. HDX-MS has recently become an established tool in the drug discovery process and biopharmaceutical development, as it is now also capable of dissecting post-translational modifications and membrane proteins. This mini review provides the reader with an introduction to the technique and a brief overview of the most common applications. Furthermore, the most challenging likely applications, the analyses of glycosylated and membrane proteins, are also highlighted.

scholarly journals Mass Spectrometry Reveals the Assembly Pathway of Encapsulated Ferritins and Highlights a Dynamic Ferroxidase Interface

2019 ◽  
Author(s):  
Jennifer Ross ◽  
Thomas Lambert ◽  
Cecilia Piergentili ◽  
Didi He ◽  
Kevin J. Waldron ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Storage System ◽  
Native Mass Spectrometry ◽  
Deuterium Exchange ◽  
Hydrogen Deuterium Exchange ◽  
Iron Storage ◽  
Exchange Mass Spectrometry ◽  
Assembly Pathway ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry

Encapsulated ferritins (EncFtn) are a recently characterised member of the ferritin superfamily. EncFtn proteins are sequestered within encapsulin nanocompartments and form a unique biological iron storage system. Here, we use native mass spectrometry and hydrogen-deuterium exchange mass spectrometry to elucidate the metal-mediated assembly pathway of EncFtn.

scholarly journals Construction of a Dual Protease Column, Subzero (-30 oC) Chromatography System and Multi-channel Precision Temperature Controller for Hydrogen-Deuterium Exchange Mass Spectrometry

2020 ◽  
Vol 125 ◽  
Author(s):  
Jeffrey W. Hudgens
Keyword(s):  
Mass Spectrometry ◽  
Three Dimensional ◽  
Deuterium Exchange ◽  
Hydrogen Deuterium Exchange ◽  
Exchange Mass Spectrometry ◽  
Precision Temperature ◽  
Hydrogen Deuterium ◽  
3D Printed ◽  
Deuterium Exchange Mass Spectrometry ◽  
Hdx Ms

This tutorial provides mechanical drawings, electrical schematics, parts lists, stereolithography (STL) files for producing three-dimensional (3D)-printed parts, initial graphics exchange specification (IGS) files for automated machining, and instructions necessary for construction of a dual protease column, subzero, liquid chromatography system for hydrogen-deuterium exchange mass spectrometry (HDX-MS). Electro-mechanical schematics for construction of two multi-zone temperature controllers that regulate to ±0.05 oC are also included in this tutorial.

scholarly journals Analysis of phosphoinositide 3-kinase inhibitors by bottom-up electron-transfer dissociation hydrogen/deuterium exchange mass spectrometry

2017 ◽  
Vol 474 (11) ◽  
pp. 1867-1877 ◽  
Author(s):  
Glenn R. Masson ◽  
Sarah L. Maslen ◽  
Roger L. Williams
Keyword(s):  
Mass Spectrometry ◽  
Electron Transfer ◽  
Electron Transfer Dissociation ◽  
Deuterium Exchange ◽  
Hydrogen Deuterium Exchange ◽  
Exchange Mass Spectrometry ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry ◽  
Phosphoinositide 3 Kinase ◽  
Hdx Ms

Until recently, one of the major limitations of hydrogen/deuterium exchange mass spectrometry (HDX-MS) was the peptide-level resolution afforded by proteolytic digestion. This limitation can be selectively overcome through the use of electron-transfer dissociation to fragment peptides in a manner that allows the retention of the deuterium signal to produce hydrogen/deuterium exchange tandem mass spectrometry (HDX-MS/MS). Here, we describe the application of HDX-MS/MS to structurally screen inhibitors of the oncogene phosphoinositide 3-kinase catalytic p110α subunit. HDX-MS/MS analysis is able to discern a conserved mechanism of inhibition common to a range of inhibitors. Owing to the relatively minor amounts of protein required, this technique may be utilised in pharmaceutical development for screening potential therapeutics.

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