Influenza A virus M1 protein structure probed by in situ limited proteolysis with bromelain.
Influenza A virus matrix M1 protein is membrane associated and plays a crucial role in virus assembly and budding. The N-terminal two thirds of M1 protein was resolved by X-ray crystallography. The overall 3D structure as well as arrangement of the molecule in relation to the viral membrane remains obscure. Now a proteolytic digestion of virions with bromelain was used as an instrument for the in situ assessment of the M1 protein structure. The lipid bilayer around the subviral particles lacking glycoprotein spikes was partially disrupted as was shown by transmission electron microscopy. A phenomenon of M1 protein fragmentation inside the subviral particles was revealed by SDS-PAGE analysis followed by in-gel trypsin hydrolysis and MALDI-TOF mass spectrometry analysis of the additional bands. Putative bromelain-digestion sites appeared to be located at the surface of the M1 protein globule and could be used as landmarks for 3D molecular modeling.
Kordyukova
L V
LV
A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119991, Russia. kord@belozersky.msu.ru
Serebryakova
M V
MV
Polyakov
V Y
VY
Ovchinnikova
T V
TV
Smirnova
Yu A
YA
Fedorova
N V
NV
Baratova
L A
LA
D000595:Amino Acid Sequence; D001963:Bromelains / Q000378:metabolism; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006868:Hydrolysis; D053118:Influenza A Virus, H1N1 Subtype / Q000737:chemistry*; D053122:Influenza A Virus, H3N2 Subtype / Q000737:chemistry*; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data; D009439:Neuraminidase; D011487:Protein Conformation; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin; D014763:Viral Matrix Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014771:Virion / Q000737:chemistry* / Q000378:metabolism
D019267:Hemagglutinin Glycoproteins, Influenza Virus; C488936:M1 protein, Influenza A virus; D014763:Viral Matrix Proteins; D001963:Bromelains; D009439:Neuraminidase; D014357:Trypsin