Research - Structural
The surface layer (S-Layer) of archaea and prokaryotes plays a critical role in the organism's interaction with the external environment, nutrient uptake, cell excretion, signaling and surface interactions. Hence, this boundary layer is a major factor in determining microbial survival. To study the Methanosarcina acetivorans S-Layer (glyco)protein(s), we are employing in vivo biotinylation labeling. Streptavidin affinity purification together with SDS-PAGE and near-western blotting are used to select biotinylated proteins, followed by trypsin-digestion and LC-MS/MS analysis. The biotinylation experiments together with the LC-MS/MS and database searches revealed that many of the proteins found were constituents of transporter systems, ATPases, and chelatases. One of the proteins found is annotated in the genome as a hypothetical protein and it contained an S-Layer domain, two transmembrane domains and a signal peptide, consistent with a S-layer protein. Thus far, we have identified an S-layer protein candidate and we are characterizing its post-translational modifications.
Ogorzalek Loo RR and Loo JA.
Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry of Hydrophobic Proteins in Mixtures Using Formic Acid, Perfluorooctanoic Acid, and Sorbitol, Anal Chem 2007; 79: 1115-1125.
Alzheimer's disease (AD) affects more than 20 million individuals worldwide. Early pathogenic events in AD involves the increased production of β-Amyloid (Aβ), especially a 42 amino acid fragment of Aβ (Aβ42). Aβ42 aggregates to form fibrils which is believed to be neurotoxic and a direct cause to AD. Therefore, for the purpose of developing a treatment, it is critical to understand Aβ's process in folding and aggregation. However, Aβ is difficult to study using traditional structure determination techniques, namely X-ray crystallography and NMR, because it is metastable.
Vaults are among the largest known ribonucleoprotein structures found in nearly all eukaryotes. The precise function of these megadalton structures is unknown, although they have been considered as nucleocytoplasmic shuttles to transport cytoplasmic molecules to the nucleus. Elucidation of vault function is currently underway using a tandem affinity purification scheme of tagged major vault proteins for identification of vault-associated macromolecules by mass spectrometry. To understand the vault structure and dynamics we have chemically crosslinked the vaults and show that these structures remain intact even under denaturing conditions. Current efforts include identifying the residues involved in the crosslinking and determining the possible role of disulfide bonds in vault assembly.