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@ARTICLE{Varghese:140004,
author = {Varghese, Sabu and Yang, Fei and Pacheco, Victor and Wrede,
Kathrin and Medvedev, Alexander and Ogata, Hideaki and
Knipp, Markus and Heise, Henrike},
title = {{E}xpression, {P}urification, and {S}olid-{S}tate {NMR}
{C}haracterization of the {M}embrane {B}inding {H}eme
{P}rotein {N}itrophorin 7 in {T}wo {E}lectronic {S}pin
{S}tates},
journal = {Biochemistry},
volume = {52},
number = {40},
issn = {1520-4995},
address = {Columbus, Ohio},
publisher = {American Chemical Society},
reportid = {FZJ-2013-05971},
pages = {7031 - 7040},
year = {2013},
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 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.},
cin = {ICS-6},
ddc = {570},
cid = {I:(DE-Juel1)ICS-6-20110106},
pnm = {452 - Structural Biology (POF2-452)},
pid = {G:(DE-HGF)POF2-452},
typ = {PUB:(DE-HGF)16},
UT = {WOS:000326355500011},
pubmed = {pmid:24033104},
doi = {10.1021/bi401020t},
url = {https://juser.fz-juelich.de/record/140004},
}