Аннотация:Nuclear export protein of influenza virus A (NEP) is involved in a number of
important phases in the virus life cycle. NEP is prone to aggregation, while
expressed in our earlier designed bacterial expression system. Preventing this
undesirable process requires an understanding the basic mechanisms of this protein
self-assembly.
The objective of this study was to investigate the peculiarities of NEP
aggregation and factors affecting this process. To this end, NEP variants with either
C- or N-terminal (His)6
-tags (NEP-C and NEP-N) were expressed in E.coli cells, thus
obtaining highly purified homogenous solutions after purification on Ni-NTA-agarose
column. The formation of oligomers in protein solutions using variable parameters
(temperature, pH, concentration, additives, potential disaggregating agents), was
analyzed with different techniques: dynamic light scattering (DLS), chemical crosslinking
with bifunctional reagent (glutaric aldehyde), gel-filtration chromatography,
atomic force microscopy (AFM). DLS measurements have shown the presence of polymeric
particles both in NEP-C and NEP-N solutions, with hydrodynamic radius being in the
range of 50-500 nm. AFM studies have revealed diverse morphology of the aggregates.
Spherical particles (diameter is about 12-14 nm) are mainly presented in NEP-N
solution, while fibrillar amyloid-like aggregates (height is 3.5-4.0 nm and length is
up to 500 nm) are predominated in case of NEP-C protein. Structures of the N- and Ctermini
were proposed to affect the propensity of NEP to aggregation. Analysis of
structural peculiarities of NEP, viz. amphipathicity of some secondary structure
elements accompanied with the concerted combination of outer exposed hydrophobic,
aromatic and charged basic amino acid residues, allows suggesting involvement these
specific domains in spontaneous aggregation process through multiple hydrophobic and
cation/dipole/π-π interacions.
The work was supported by RFBR, grant № 16-04-00563.