Proteomics

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Structural characterization of the oligomerization of full-length Hantaan virus polymerase into symmetric dimers and hexamers


ABSTRACT: Hantaan virus is a dangerous human pathogen whose segmented negative-stranded RNA genome is replicated and transcribed by a virally-encoded multi-functional polymerase. To describe the complete cryo-electron microscopy structure of Hantaan virus polymerase in several oligomeric forms, a full-length wild-type (WT) HTNV-L construct comprising an N-terminal his-tag was expressed in Hi5 insect cells and subsequently purified. Through a combination of size-exclusion chromatography and mass photometry analysis, the presence of distinct HTNV-L oligomers was observed. In buffers containing 250mM NaCl, several species were automatically detected including monomers (53%), dimers (17%), higher molecular weight oligomers up to hexamers (2%) and an unidentified 150kDa protein (18%). To characterize the proteins present in the 150kDa band, a bottom-up mass spectrometry (MS)-based proteomic analysis was performed. For this, proteins present in the 150 kDa band were in-gel digested with trypsin before analysis by nano liquid chromatography coupled to MS/MS. Obtained result revealed that the most abundant protein in this band was HTNV-L. The identified peptides were covering the entire HTNV-L sequence, suggesting that the 150kDa band may contain different cleaved forms of HTNV-L co-migrating in this particular SDS-PAGE gel band.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Hantaan Virus 76-118 Trichoplusia Ni

SUBMITTER: Yohann Couté  

LAB HEAD: Yohann Couté

PROVIDER: PXD049232 | Pride | 2024-02-07

REPOSITORIES: Pride

Dataset's files

Source:
Action DRS
Result.mzid.gz Mzid
Sample.mgf Mgf
Sample.raw Raw
checksum.txt Txt
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