title: Uncovering the stoichiometry of Pyrococcus furiosus RNase P, a multi-subunit catalytic ribonucleoprotein complex, by surface-induced dissociation and ion mobility mass spectrometry.
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 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.
authors.affiliation: Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University, Columbus, OH 43210 (USA).
authors.forename: Xin
authors.initials: X
authors.lastname: Ma
authors.forename: Lien B
authors.initials: LB
authors.lastname: Lai
authors.forename: Stella M
authors.initials: SM
authors.lastname: Lai
authors.forename: Akiko
authors.initials: A
authors.lastname: Tanimoto
authors.forename: Mark P
authors.initials: MP
authors.lastname: Foster
authors.forename: Vicki H
authors.initials: VH
authors.lastname: Wysocki
authors.forename: Venkat
authors.initials: V
authors.lastname: Gopalan
mesh_terms: D002384:Catalysis; D013058:Mass Spectrometry / Q000379:methods*; D020027:Pyrococcus furiosus / Q000201:enzymology*; D043262:Ribonuclease P / Q000737:chemistry / Q000378:metabolism*
chemical_list: D043262:Ribonuclease P
keywords: RNA-protein complexes; RNase P; ion mobility; native mass spectrometry; stoichiometry; surface-induced dissociation