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- Title
Uncovering the Stoichiometry of Pyrococcus furiosus RNaseP, a Multi-Subunit Catalytic Ribonucleoprotein Complex, by Surface-Induced Dissociation and Ion Mobility Mass Spectrometry.
- Authors
Ma, Xin; Lai, Lien B.; Lai, Stella M.; Tanimoto, Akiko; Foster, Mark P.; Wysocki, Vicki H.; Gopalan, Venkat
- Abstract
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 multisubunit ribonucleoprotein (RNP) complex assembled in a solution containing Mg2+. We investigated Pyrococcus furiosus (Pfu) RNaseP, an archaeal RNP that catalyzes tRNA 5' maturation. Previous step-wise, Mg2+-dependent reconstitutions of Pfu RNaseP with its catalytic RNA subunit and two interacting protein cofactor pairs (RPP21·RPP29 and POP5·RPP30) revealed functional RNP intermediates en route to the RNaseP 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.
- Subjects
PYROCOCCUS furiosus; STOICHIOMETRY; RIBONUCLEASE P; NUCLEOPROTEINS; DISSOCIATION (Chemistry); ION mobility spectroscopy
- Publication
Angewandte Chemie, 2014, Vol 126, Issue 43, p11667
- ISSN
0044-8249
- Publication type
Article
- DOI
10.1002/ange.201405362