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 2
0 S proteasome from Thermoplasma acidophilum have been investigated by atom
ic force microscopy (AFM) and surface plasmon resonance (SPR). Due to engin
eered histidine tags introduced at defined positions, the proteasome comple
x was pre-oriented at ultra-flat chelator lipid membranes allowing for high
-resolution imaging by AFM. Within these two-dimensional protein arrays, th
e overall structure of the proteasome and the organization of individual su
bunits was resolved under native conditions without fixation or crosslinkin
g. 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 substr
ate-proteasome complex were analyzed during proteolysis of the polypeptide
chain. By blocking the active sites with a specific inhibitor, the substrat
e binding step could be dissected from the degradation step thus resolving
mechanistic details of substrate recognition and cleavage by the 20 S prote
asome. (C) 1999 Academic Press.