Identification of subunit-subunit interaction sites in αA-WT crystallin and mutant αA-G98R crystallin using isotope-labeled cross-linker and mass spectrometry.

Cataract is characterized by progressive protein aggregation and loss of vision. α-Crystallins are the major proteins in the lens responsible for maintaining transparency. They exist in the lens as highly polydisperse oligomers with variable numbers of subunits, and mutations in α-crystallin are ass...

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Autores principales:	Rama Kannan, Puttur Santhoshkumar, Brian P Mooney, K Krishna Sharma
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/d2d668099d0140d8a7dae8283047cebd
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spelling	oai:doaj.org-article:d2d668099d0140d8a7dae8283047cebd2021-11-18T07:42:55ZIdentification of subunit-subunit interaction sites in αA-WT crystallin and mutant αA-G98R crystallin using isotope-labeled cross-linker and mass spectrometry.1932-620310.1371/journal.pone.0065610https://doaj.org/article/d2d668099d0140d8a7dae8283047cebd2013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23755258/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203Cataract is characterized by progressive protein aggregation and loss of vision. α-Crystallins are the major proteins in the lens responsible for maintaining transparency. They exist in the lens as highly polydisperse oligomers with variable numbers of subunits, and mutations in α-crystallin are associated with some forms of cataract in humans. Because the stability of proteins is dependent on optimal subunit interactions, the structural transformations and aggregation of mutant proteins that underlie cataract formation can be understood best by identifying the residue-specific inter- and intra-subunit interactions. Chemical crosslinking combined with mass spectrometry is increasingly used to provide structural insights into intra- and inter-protein interactions. We used isotope-labeled cross-linker in combination with LC-MS/MS to determine the subunit-subunit interaction sites in cataract-causing mutant αA-G98R crystallin. Peptides cross-linked by isotope-labeled (heavy and light forms) cross-linkers appear as doublets in mass spectra, thus facilitating the identification of cross-linker-containing peptides. In this study, we cross-linked wild-type (αA-WT) and mutant (αA-G98R) crystallins using the homobifunctional amine-reactive, isotope-labeled (d₀ and d₄) cross-linker-BS²G (bis[sulfosuccinimidyl]glutarate). Tryptic in-solution digest of cross-linked complexes generates a wide array of peptide mixtures. Cross-linked peptides were enriched using strong cation exchange (SCX) chromatography followed by both MS and MS/MS to identify the cross-linked sites. We identified a distinct intermolecular interaction site between K88-K99 in the β5 strand of the mutant αA-G98R crystallin that is not found in wild-type αA-crystallin. This interaction could explain the conformational instability and aggregation nature of the mutant protein that results from incorrect folding and assembly.Rama KannanPuttur SanthoshkumarBrian P MooneyK Krishna SharmaPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 6, p e65610 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Rama Kannan Puttur Santhoshkumar Brian P Mooney K Krishna Sharma Identification of subunit-subunit interaction sites in αA-WT crystallin and mutant αA-G98R crystallin using isotope-labeled cross-linker and mass spectrometry.
description	Cataract is characterized by progressive protein aggregation and loss of vision. α-Crystallins are the major proteins in the lens responsible for maintaining transparency. They exist in the lens as highly polydisperse oligomers with variable numbers of subunits, and mutations in α-crystallin are associated with some forms of cataract in humans. Because the stability of proteins is dependent on optimal subunit interactions, the structural transformations and aggregation of mutant proteins that underlie cataract formation can be understood best by identifying the residue-specific inter- and intra-subunit interactions. Chemical crosslinking combined with mass spectrometry is increasingly used to provide structural insights into intra- and inter-protein interactions. We used isotope-labeled cross-linker in combination with LC-MS/MS to determine the subunit-subunit interaction sites in cataract-causing mutant αA-G98R crystallin. Peptides cross-linked by isotope-labeled (heavy and light forms) cross-linkers appear as doublets in mass spectra, thus facilitating the identification of cross-linker-containing peptides. In this study, we cross-linked wild-type (αA-WT) and mutant (αA-G98R) crystallins using the homobifunctional amine-reactive, isotope-labeled (d₀ and d₄) cross-linker-BS²G (bis[sulfosuccinimidyl]glutarate). Tryptic in-solution digest of cross-linked complexes generates a wide array of peptide mixtures. Cross-linked peptides were enriched using strong cation exchange (SCX) chromatography followed by both MS and MS/MS to identify the cross-linked sites. We identified a distinct intermolecular interaction site between K88-K99 in the β5 strand of the mutant αA-G98R crystallin that is not found in wild-type αA-crystallin. This interaction could explain the conformational instability and aggregation nature of the mutant protein that results from incorrect folding and assembly.
format	article
author	Rama Kannan Puttur Santhoshkumar Brian P Mooney K Krishna Sharma
author_facet	Rama Kannan Puttur Santhoshkumar Brian P Mooney K Krishna Sharma
author_sort	Rama Kannan
title	Identification of subunit-subunit interaction sites in αA-WT crystallin and mutant αA-G98R crystallin using isotope-labeled cross-linker and mass spectrometry.
title_short	Identification of subunit-subunit interaction sites in αA-WT crystallin and mutant αA-G98R crystallin using isotope-labeled cross-linker and mass spectrometry.
title_full	Identification of subunit-subunit interaction sites in αA-WT crystallin and mutant αA-G98R crystallin using isotope-labeled cross-linker and mass spectrometry.
title_fullStr	Identification of subunit-subunit interaction sites in αA-WT crystallin and mutant αA-G98R crystallin using isotope-labeled cross-linker and mass spectrometry.
title_full_unstemmed	Identification of subunit-subunit interaction sites in αA-WT crystallin and mutant αA-G98R crystallin using isotope-labeled cross-linker and mass spectrometry.
title_sort	identification of subunit-subunit interaction sites in αa-wt crystallin and mutant αa-g98r crystallin using isotope-labeled cross-linker and mass spectrometry.
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/d2d668099d0140d8a7dae8283047cebd
work_keys_str_mv	AT ramakannan identificationofsubunitsubunitinteractionsitesinaawtcrystallinandmutantaag98rcrystallinusingisotopelabeledcrosslinkerandmassspectrometry AT puttursanthoshkumar identificationofsubunitsubunitinteractionsitesinaawtcrystallinandmutantaag98rcrystallinusingisotopelabeledcrosslinkerandmassspectrometry AT brianpmooney identificationofsubunitsubunitinteractionsitesinaawtcrystallinandmutantaag98rcrystallinusingisotopelabeledcrosslinkerandmassspectrometry AT kkrishnasharma identificationofsubunitsubunitinteractionsitesinaawtcrystallinandmutantaag98rcrystallinusingisotopelabeledcrosslinkerandmassspectrometry
_version_	1718423064729878528

Identification of subunit-subunit interaction sites in αA-WT crystallin and mutant αA-G98R crystallin using isotope-labeled cross-linker and mass spectrometry.

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