title: Expression, purification, and solid-state NMR characterization of the membrane binding heme protein nitrophorin 7 in two electronic spin states.
abstract: The nitrophorins (NPs) comprise a group of NO transporting ferriheme b proteins found in the saliva of the blood sucking insect Rhodnius prolixus . In contrast to other nitrophorins (NP1-4), the recently identified membrane binding isoform NP7 tends to form oligomers and precipitates at higher concentrations in solution. Hence, solid-state NMR (ssNMR) was employed as an alternative method to gain structural insights on the precipitated protein. We report the expression and purification of (13)C,(15)N isotopically labeled protein together with the first ssNMR characterization of NP7. Because the size of NP7 (21 kDa) still provides a challenge for ssNMR, the samples were reverse labeled with Lys and Val to reduce the number of crosspeaks in two-dimensional spectra. The two electronic spin states with S = 1/2 and S = 0 at the ferriheme iron were generated by the complexation with imidazole and NO, respectively. ssNMR spectra of both forms are well resolved, which allows for sequential resonance assignments of 22 residues. Importantly, the ssNMR spectra demonstrate that aggregation does not affect the protein fold. Comparison of the spectra of the two electronic spin states allows the determination of paramagnetically shifted cross peaks due to pseudocontact shifts, which assists the assignment of residues close to the heme center.
authors.affiliation: ICS-6 Institute of Complex Systems-Structural Biochemistry, Forschungszentrum Jülich , D-2425 Jülich, Germany.
authors.forename: Sabu
authors.initials: S
authors.lastname: Varghese
authors.forename: Fei
authors.initials: F
authors.lastname: Yang
authors.forename: Victor
authors.initials: V
authors.lastname: Pacheco
authors.forename: Kathrin
authors.initials: K
authors.lastname: Wrede
authors.forename: Alexander
authors.initials: A
authors.lastname: Medvedev
authors.forename: Hideaki
authors.initials: H
authors.lastname: Ogata
authors.forename: Markus
authors.initials: M
authors.lastname: Knipp
authors.forename: Henrike
authors.initials: H
authors.lastname: Heise
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry; D018360:Crystallography, X-Ray; D000081062:Heme-Binding Proteins; D006420:Hemeproteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D019476:Insect Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D019906:Nuclear Magnetic Resonance, Biomolecular; D055503:Protein Multimerization; D012239:Rhodnius; D012471:Salivary Proteins and Peptides / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification
chemical_list: D002352:Carrier Proteins; D000081062:Heme-Binding Proteins; D006420:Hemeproteins; D019476:Insect Proteins; D012471:Salivary Proteins and Peptides; C090560:nitrophorin