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Uncovering the Stoichiometry of Pyrococcus furiosus RNase P, a Multi‐Subunit Catalytic Ribonucleoprotein Complex, by Surface‐Induced Dissociation and Ion Mobility Mass Spectrometry
Author(s) -
Ma Xin,
Lai Lien B.,
Lai Stella M.,
Tanimoto Akiko,
Foster Mark P.,
Wysocki Vicki H.,
Gopalan Venkat
Publication year - 2014
Publication title -
angewandte chemie international edition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.831
H-Index - 550
eISSN - 1521-3773
pISSN - 1433-7851
DOI - 10.1002/anie.201405362
Subject(s) - pyrococcus furiosus , ribonucleoprotein , rnase p , protein subunit , chemistry , rna , dissociation (chemistry) , rnase ph , pyrococcus horikoshii , mass spectrometry , biochemistry , rnase h , stoichiometry , crystallography , biophysics , enzyme , biology , archaea , chromatography , gene
We demonstrate that surface‐induced dissociation (SID) coupled with ion mobility mass spectrometry (IM‐MS) is a powerful tool for determining the stoichiometry of a multi‐subunit ribonucleoprotein (RNP) complex assembled in a solution containing Mg 2+ . We investigated Pyrococcus furiosus (Pfu) RNase P, an archaeal RNP that catalyzes tRNA 5′ maturation. Previous step‐wise, Mg 2+ ‐dependent reconstitutions of Pfu RNase P with its catalytic RNA subunit and two interacting protein cofactor pairs (RPP21⋅RPP29 and POP5⋅RPP30) revealed functional RNP intermediates en route to the RNase P enzyme, but provided no information on subunit stoichiometry. Our native MS studies with the proteins showed RPP21⋅RPP29 and (POP5⋅RPP30) 2 complexes, but indicated a 1:1 composition for all subunits when either one or both protein complexes bind the cognate RNA. These results highlight the utility of SID and IM‐MS in resolving conformational heterogeneity and yielding insights on RNP assembly.