title: Native mass spectrometry and ion mobility characterize the orange carotenoid protein functional domains.
abstract: Orange Carotenoid Protein (OCP) plays a unique role in protecting many cyanobacteria from light-induced damage. The active form of OCP is directly involved in energy dissipation by binding to the phycobilisome (PBS), the major light-harvesting complex in cyanobacteria. There are two structural modules in OCP, an N-terminal domain (NTD), and a C-terminal domain (CTD), which play different functional roles during the OCP-PBS quenching cycle. Because of the quasi-stable nature of active OCP, structural analysis of active OCP has been lacking compared to its inactive form. In this report, partial proteolysis was used to generate two structural domains, NTD and CTD, from active OCP. We used multiple native mass spectrometry (MS) based approaches to interrogate the structural features of the NTD and the CTD. Collisional activation and ion mobility analysis indicated that the NTD releases its bound carotenoid without forming any intermediates and the CTD is resistant to unfolding upon collisional energy ramping. The unfolding intermediates observed in inactive intact OCP suggest that it is the N-terminal extension and the NTD-CTD loop that lead to the observed unfolding intermediates. These combined approaches extend the knowledge of OCP photo-activation and structural features of OCP functional domains. Combining native MS, ion mobility, and collisional activation promises to be a sensitive new approach for studies of photosynthetic protein-pigment complexes.
authors.affiliation: Department of Chemistry, Washington University in St. Louis, St. Louis, MO 63130, USA; Photosynthetic Antenna Research Center (PARC), Washington University in St. Louis, St. Louis, MO 63130, USA.
authors.forename: Hao
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Photosynthetic Antenna Research Center (PARC), Washington University in St. Louis, St. Louis, MO 63130, USA; Department of Biology, Washington University in St. Louis, St. Louis, MO 63130, USA.
authors.forename: Haijun
authors.initials: H
authors.lastname: Liu
authors.affiliation: Department of Chemistry, Washington University in St. Louis, St. Louis, MO 63130, USA; Photosynthetic Antenna Research Center (PARC), Washington University in St. Louis, St. Louis, MO 63130, USA.
authors.forename: Yue
authors.initials: Y
authors.lastname: Lu
authors.affiliation: Department of Biology, Washington University in St. Louis, St. Louis, MO 63130, USA.
authors.forename: Nathan R
authors.initials: NR
authors.lastname: Wolf
authors.affiliation: Department of Chemistry, Washington University in St. Louis, St. Louis, MO 63130, USA; Photosynthetic Antenna Research Center (PARC), Washington University in St. Louis, St. Louis, MO 63130, USA.
authors.forename: Michael L
authors.initials: ML
authors.lastname: Gross
authors.affiliation: Department of Chemistry, Washington University in St. Louis, St. Louis, MO 63130, USA; Photosynthetic Antenna Research Center (PARC), Washington University in St. Louis, St. Louis, MO 63130, USA; Department of Biology, Washington University in St. Louis, St. Louis, MO 63130, USA. Electronic address: Blankenship@wustl.edu.
authors.forename: Robert E
authors.initials: RE
authors.lastname: Blankenship
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002338:Carotenoids / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D045524:Phycobilisomes / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D046939:Synechocystis / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D010446:Peptide Fragments; D045524:Phycobilisomes; C109473:orange carotenoid protein, Synechocystis; D002338:Carotenoids
keywords: Collision activation; Ion mobility; Native mass spectrometry; Orange carotenoid protein