RESUMO
Carboranes 1 and 2 were designed and synthesized for hydrophobic tag (HyT)-induced degradation of HaloTag fusion proteins. The levels of the hemagglutinin (HA)-HaloTag2-green fluorescent protein (EGFP) stably expressed in Flp-In 293 cells were significantly reduced by HyT13, HyT55, and carboranes 1 and 2, with expression levels of 49, 79, 43, and 65%, respectively, indicating that carborane is an alternative novel hydrophobic tag (HyT) for protein degradation under an intracellular environment. To clarify the mechanism of HyT-induced proteolysis, bovine serum albumin (BSA) was chosen as an extracellular protein and modified with maleimide-conjugated m-carborane (MIC). The measurement of the ζ-potentials and the lysine residue modification with fluorescein isothiocyanate (FITC) of BSA-MIC conjugates suggested that the conjugation of carborane induced the exposure of lysine residues on BSA, resulting in the degradation via ubiquitin E3 ligase-related proteasome pathways in the cell.
Assuntos
ProteóliseRESUMO
Gas phase spectroscopy is a powerful tool for examining fundamental chemical structures and properties free from solvent molecules. We developed a gas-phase resonance Raman spectroscopy combined with IR-laser ablation of a droplet beam, which allowed us to elucidate local structures around chromophores in gas-phase proteins and DNAs. To demonstrate the potential of this approach, we applied this method to myoglobin, one of the heme proteins, and elucidated its structures in the gas phase and in aqueous solution. The experimental spectra are compared with calculated spectra of stable heme structures for the structural determination. These results show the oxidation/spin states of the Fe atom in myoglobin in the gas phase and were compared with the aqueous solution from the obtained resonant Raman spectra. The present method gives an important tool to investigate the gas-phase structure of large biomolecules.