The effect of C- and N-terminal polyhistidin tag on aggregation of influenza A virus nuclear export protein

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Дәйексөз келтіру

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Аннотация

The nuclear export protein (NEP) of the influenza A virus, being one of the key components of the virus life cycle, is a promising model for studying the characteristics of formation of amyloids by viral proteins. Using atomic force microscopy, comparative studies of the aggregation properties of recombinant NEP variants, including the protein of natural structure, as well as modified variants with N- and C-terminal affinity His6-tags, were carried out. All protein variants under physiological conditions are capable of forming aggregates of various morphologies: micelle-like nanoparticles, flexible protofibrils, rigid amyloid fibrils, etc. The His6-tag attached to the C-terminus has the greatest effect on the aggregation kinetics and morphology of nanoparticles, which indicates the important role of the C-terminal domain in the process of protein self-assembly. Molecular dynamics simulation hasn’t revealed the substantial influence of His6-containing fragments on the protein structure but demonstrated some variations in the mobility of these fragments that may explain the observed differences in the aggregation kinetics of different NEP variants. Hypothetical mechanisms for the formation and interconversion of various aggregates are considered.

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Авторлар туралы

O. Koroleva

Lomonosov Moscow State University

Email: dubrovin@polly.phys.msu.ru

Faculty of Chemistry

Ресей, 119991 Moscow

N. Kuzmina

Frumkin Institute of Physical Chemistry and Electrochemistry, Russian Academy of Sciences

Email: dubrovin@polly.phys.msu.ru
Ресей, 119071 Moscow

A. Tolstova

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences

Email: dubrovin@polly.phys.msu.ru
Ресей, 119991 Moscow

E. Dubrovin

Lomonosov Moscow State University; National University of Science and Technology MISIS

Хат алмасуға жауапты Автор.
Email: dubrovin@polly.phys.msu.ru

Faculty of Physics, Lomonosov Moscow State University

Ресей, 119991 Moscow; 119049, Moscow

V. Drutsa

Lomonosov Moscow State University

Email: dubrovin@polly.phys.msu.ru

A. N. Belozersky Institute of Physico-Chemical Biology

Ресей, 119991 Moscow

Әдебиет тізімі

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2. Fig. 1. Recombinant variants of the NEP protein studied in the work. a – Schematic representation. In the protein structure, rectangles mark α-helices C1 and C2 (according to the X-ray structural analysis of the C-terminal fragment of NEP [24]), dotted line – unstructured region (UR), inscriptions “His” – polyhistidine-containing affinity tags MHHHHHHSGGT (for NEP-N) and DPGSHHHHHHL (for NEP-C). b – Electrophoretic analysis in 15% SDS-PAGE of freshly isolated (I) and after incubation for 3 months (II) at 4 °C preparations of NEP (lanes 1 and 4), NEP-N (lanes 2 and 5) and NEP-C (lanes 3 and 6); M – protein molecular weight markers

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3. Fig. 2. AFM images of the natural NEP protein and its aggregates. a and b – The shelf life of the preparation after isolation is several hours (i.e., they were studied on the day of isolation). c–e – The shelf life of the preparation after isolation is 5 months. d – The doubling effect is present. The surface profiles along the lines in the corresponding AFM images are shown at the bottom. The insets in panels (a) and (c) are enlarged surface areas highlighted by a square (the size of the insets is 300 × 300 and 150 × 150 nm2 for (a) and (c), respectively). g – Histogram of the distribution of object heights in panel (a)

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4. Fig. 3. AFM images of the NEP-N protein and its aggregates. The shelf life of the preparation after isolation was 1 day (a and b) and 10 days (c). The surface profiles along the lines in the corresponding AFM images are shown at the bottom. The inset in panel (b) is an enlarged surface area highlighted by a square (insert size 250 × 250 nm2). d – Histogram of the distribution of object heights in panel (a)

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5. Fig. 4. AFM images of NEP-C protein and its aggregates. The shelf life of the preparation after isolation was 1 day (a), 8 days (b and c) and 14 days (d). The surface profiles along the lines in the corresponding AFM images are shown at the bottom. The inset in panel (b) is an enlarged surface area highlighted by a square (insert size 65 × 100 nm2). d – Histogram of the distribution of object heights in panel (a)

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6. Fig. 5. Schematic illustrating the formation of different aggregates of three NEP protein variants over time.

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7. Fig. 6. Representative three-dimensional structures of NEP (a), NEP-N (b) and NEP-C (c) molecules obtained as a result of MD simulations (200 ns). The positions of the N- and C-terminal regions are indicated.

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