TY  - JOUR
AU  - Varghese, Sabu
AU  - Yang, Fei
AU  - Pacheco, Victor
AU  - Wrede, Kathrin
AU  - Medvedev, Alexander
AU  - Ogata, Hideaki
AU  - Knipp, Markus
AU  - Heise, Henrike
TI  - Expression, Purification, and Solid-State NMR Characterization of the Membrane Binding Heme Protein Nitrophorin 7 in Two Electronic Spin States
JO  - Biochemistry
VL  - 52
IS  - 40
SN  - 1520-4995
CY  - Columbus, Ohio
PB  - American Chemical Society
M1  - FZJ-2013-05971
SP  - 7031 - 7040
PY  - 2013
AB  - 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 13C,15N 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.
LB  - PUB:(DE-HGF)16
UR  - <Go to ISI:>//WOS:000326355500011
C6  - pmid:24033104
DO  - DOI:10.1021/bi401020t
UR  - https://juser.fz-juelich.de/record/140004
ER  -