Secondary structure, dynamics, and architecture of the p7 membrane protein from hepatitis C virus by NMR spectroscopy

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Abstract

P7 is a small membrane protein that is essential for the infectivity of hepatitis C virus. Solution-state NMR experiments on p7 in DHPC micelles, including hydrogen/deuterium exchange, paramagnetic relaxation enhancement and bicelle ‘q-titration,’ demonstrate that the protein has a range of dynamic properties and distinct structural segments. These data along with residual dipolar couplings yield a secondary structure model of p7. We were able to confirm previous proposals that the protein has two transmembrane segments with a short interhelical loop containing the two basic residues K33 and R35. The 63-amino acid protein has a remarkably complex structure made up of seven identifiable sections, four of which are helical segments with different tilt angles and dynamics. A solid-state NMR two-dimensional separated local field spectrum of p7 aligned in phospholipid bilayers provided the tilt angles of two of these segments. A preliminary structural model of p7 derived from these NMR data is presented.

Abbreviations

HCV
hepatitis C virus
NMR
nuclear magnetic resonance
DHPC
(1,2-dihexyl-1-sn-glycero-3-phosphocholine)
NOE
nuclear Overhauser effect
PRE
paramagnetic relaxation enhancement
RDC
residual dipolar couplings
SDS
sodium dodecyl sulfate
D2O
deuterium oxide
EDTA
ethylenediaminetetraacetic acid
HEPES
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
DMPC
1,2-dimyristoyl-sn-glycero-3-phosphocholine
6-O-PC
1,2-di-O-hexyl-sn-glycero-3-phosphocholine
IPAP
in-phase/anti-phase
CSI
chemical shift index
PISA
polar index slant angle
ER
endoplasmic reticulum

Keywords

Hepatitis C virus
p7
Membrane protein
Secondary structure
Nuclear magnetic resonance

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