High-resolution AFM-imaging and mechanistic analysis of the 20 S proteasome.
As macromolecular protease complex, the 20 S proteasome is responsible for the degradation of cellular proteins and the generation of peptide epitopes for antigen presentation. Here, structural and functional aspects of the 20 S proteasome from Thermoplasma acidophilum have been investigated by atomic force microscopy (AFM) and surface plasmon resonance (SPR). Due to engineered histidine tags introduced at defined positions, the proteasome complex was pre-oriented at ultra-flat chelator lipid membranes allowing for high-resolution imaging by AFM. Within these two-dimensional protein arrays, the overall structure of the proteasome and the organization of individual subunits was resolved under native conditions without fixation or crosslinking. In addition, the substrate-proteasome interaction was monitored in real-time by SPR using a novel approach. Instead of following enzyme activity by product formation, the association and dissociation kinetics of the substrate-proteasome complex were analyzed during proteolysis of the polypeptide chain. By blocking the active sites with a specific inhibitor, the substrate binding step could be dissected from the degradation step thus resolving mechanistic details of substrate recognition and cleavage by the 20 S proteasome.
D000327:Adsorption; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000502:physiology* / Q000648:ultrastructure*; D007328:Insulin / Q000378:metabolism; D007700:Kinetics; D008566:Membranes / Q000737:chemistry; D018625:Microscopy, Atomic Force; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000502:physiology* / Q000648:ultrastructure*; D046988:Proteasome Endopeptidase Complex; D011506:Proteins / Q000378:metabolism; D013997:Time Factors