ABSTRACT
This review considers principles of the use of mass spectrometry for the study of biological macromolecules. Some examples of protein identification, virion proteomics, testing vaccine preparations, and strain surveillance are represented. Possibilities of structural characterization of viral proteins and their posttranslational modifications are shown. The authors' studies by MALDI-MS on S-acylation of glycoproteins from various families of enveloped viruses and on oligomerization of the influenza virus hemagglutinin transmembrane domains are summarized.
Subject(s)
Glycoproteins/chemistry , Preventive Medicine , Viral Envelope Proteins/chemistry , Viruses/chemistry , Protein Conformation , Proteomics , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
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.
Subject(s)
Influenza A Virus, H1N1 Subtype/chemistry , Influenza A Virus, H3N2 Subtype/chemistry , Viral Matrix Proteins/chemistry , Virion/chemistry , Amino Acid Sequence , Bromelains/metabolism , Crystallography, X-Ray , Electrophoresis, Polyacrylamide Gel , Hemagglutinin Glycoproteins, Influenza Virus , Hydrolysis , Microscopy, Electron, Transmission , Molecular Sequence Data , Neuraminidase , Protein Conformation , Sequence Analysis, Protein , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Trypsin , Viral Matrix Proteins/genetics , Viral Matrix Proteins/metabolism , Virion/metabolismABSTRACT
The topography of bacteriorhodopsin (bR) in situ was earlier studied by using the tritium bombardment approach [Eur. J. Biochem. 178 (1988) 123]. Now, having the X-ray crystallography data of bR at atom resolution [Proc. Natl. Acad. Sci. 95 (1998) 11673], we estimated the influence of membrane environment (lipid and protein) on tritium incorporation into amino acid residues forming transmembrane helices. We have determined the tritium flux attenuation coefficients for residues 10-29 of helix A. They turned out to be low (0.04+/-0.02 A(-1)) for residues adjacent to the lipid matrix, and almost fourfold higher (0.15+/-0.05 A(-1)) for those oriented to the neighboring transmembrane helices. We believe that tritium incorporation data could help modeling transmembrane segment arrangement in the membrane.
Subject(s)
Bacteriorhodopsins/chemistry , Membrane Proteins/chemistry , Crystallography, X-Ray , TritiumABSTRACT
Bilayer liposomes from a mixture of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylethanolamine (DPPC: DPPE = 8:2, molar ratio) or DPPC labeled with 14C-DPPC (DPPC: 14C-DPPC) were bombarded with thermally activated tritium atoms. The tritiated liposomes were hydrolyzed by phospholipase C, and the tritium incorporation into different parts of the bilayer along its thickness was determined. The tritium flux attenuation coefficients were calculated for the headgroup (k1 = 0.176+/-0.032 A(-1)) and acylglycerol residue (k2 = 0.046+/-0.004 A(-1)) layers indicating a preferential attenuation of the tritium flux in the headgroup region and relative transparence of the membrane hydrophobic part. The finding is potentially important to apply tritium bombardment for investigation of spatial organization of transmembrane proteins in their native lipid environment.
