scholarly journals Cataract-Associated New Mutants S175G/H181Q of βΒ2-Crystallin and P24S/S31G of γD-Crystallin Are Involved in Protein Aggregation by Structural Changes

2020 ◽  
Vol 21 (18) ◽  
pp. 6504
Author(s):  
In-Kang Song ◽  
Seungjin Na ◽  
Eunok Paek ◽  
Kong-Joo Lee
Keyword(s):  
Structural Changes ◽  
Hydrogen Deuterium Exchange ◽  
Structural Protein ◽  
Exchange Mass Spectrometry ◽  
Human Lenses ◽  
Hydrogen Deuterium ◽  
Proteomic Dataset ◽  
Deuterium Exchange Mass Spectrometry ◽  
Hdx Ms ◽  
New Mutations

β/γ-Crystallins, the main structural protein in human lenses, have highly stable structure for keeping the lens transparent. Their mutations have been linked to cataracts. In this study, we identified 10 new mutations of β/γ-crystallins in lens proteomic dataset of cataract patients using bioinformatics tools. Of these, two double mutants, S175G/H181Q of βΒ2-crystallin and P24S/S31G of γD-crystallin, were found mutations occurred in the largest loop linking the distant β-sheets in the Greek key motif. We selected these double mutants for identifying the properties of these mutations, employing biochemical assay, the identification of protein modifications with nanoUPLC-ESI-TOF tandem MS and examining their structural dynamics with hydrogen/deuterium exchange-mass spectrometry (HDX-MS). We found that both double mutations decrease protein stability and induce the aggregation of β/γ-crystallin, possibly causing cataracts. This finding suggests that both the double mutants can serve as biomarkers of cataracts.

scholarly journals Cataract-associated New Mutants S175G/H181Q of βB2-Crystallin and P24S/S31G of γD-Crystallin are Involved in Protein aggregation by Structural Changes

2020 ◽  
Author(s):  
In-Kang Song ◽  
Seungjin Na ◽  
Eunok Paek ◽  
Kong-Joo Lee
Keyword(s):  
Structural Changes ◽  
Structural Protein ◽  
Congenital Cataracts ◽  
Exchange Mass Spectrometry ◽  
Bioinformatic Tools ◽  
Human Lenses ◽  
Hydrogen Deuterium ◽  
Proteomic Dataset ◽  
Deuterium Exchange Mass Spectrometry ◽  
Hdx Ms

ABSTRACTβ/γ-Crystallins, the main structural protein in human lenses, have highly stable structure for keeping the lens transparent. Their mutations have been linked to congenital cataracts. In this study, we identified 10 new mutations of β/γ-crystallins in lens proteomic dataset of cataract patients using bioinformatic tools. Of these, two double mutants, S175G/H181Q of βΒ2-crystallin and P24S/S31G of γD-crystallin, were found mutations occurred in the largest loop linking the distant β-sheets in the Greek key motif. We selected these double mutants for identifying the properties of these mutations, employing biochemical assay, the identification of protein modifications with nanoUPLC-ESI-TOF tandem MS and examining their structural dynamics with hydrogen/deuterium exchange-mass spectrometry (HDX-MS). We found that both double mutations decrease protein stability and induce the aggregation of β/γ-crystallin, possibly causing cataracts. This finding suggests that both the double mutants can serve as biomarkers of congenital cataracts.

scholarly journals Protein Backbone and Average Particle Dynamics in Reconstituted Discoidal and Spherical HDL Probed by Hydrogen Deuterium Exchange and Elastic Incoherent Neutron Scattering

Biomolecules ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 121
Author(s):  
Valentin Gogonea ◽  
Judith Peters ◽  
Gary S. Gerstenecker ◽  
Celalettin Topbas ◽  
Liming Hou ◽  
...  
Keyword(s):  
Neutron Scattering ◽  
Deuterium Exchange ◽  
Average Particle ◽  
Hydrogen Deuterium Exchange ◽  
Exchange Mass Spectrometry ◽  
Incoherent Neutron Scattering ◽  
Hydrogen Deuterium ◽  
Deuterium Exchange Mass Spectrometry ◽  
Hdx Ms ◽  
Incoherent Neutron

Lipoproteins are supramolecular assemblies of proteins and lipids with dynamic characteristics critically linked to their biological functions as plasma lipid transporters and lipid exchangers. Among them, spherical high-density lipoproteins are the most abundant forms of high-density lipoprotein (HDL) in human plasma, active participants in reverse cholesterol transport, and associated with reduced development of atherosclerosis. Here, we employed elastic incoherent neutron scattering (EINS) and hydrogen-deuterium exchange mass spectrometry (HDX-MS) to determine the average particle dynamics and protein backbone local mobility of physiologically competent discoidal and spherical HDL particles reconstituted with human apolipoprotein A-I (apoA-I). Our EINS measurements indicated that discoidal HDL was more dynamic than spherical HDL at ambient temperatures, in agreement with their lipid-protein composition. Combining small-angle neutron scattering (SANS) with contrast variation and MS cross-linking, we showed earlier that the most likely organization of the three apolipoprotein A-I (apoA-I) chains in spherical HDL is a combination of a hairpin monomer and a helical antiparallel dimer. Here, we corroborated those findings with kinetic studies, employing hydrogen-deuterium exchange mass spectrometry (HDX-MS). Many overlapping apoA-I digested peptides exhibited bimodal HDX kinetics behavior, suggesting that apoA-I regions with the same amino acid composition located on different apoA-I chains had different conformations and/or interaction environments.

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 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.

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