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Abstract

The bipartite structure of the proteasome raises the question of functional significance. A rational design for unraveling mechanistic details of the highly symmetrical degradation machinery from Thermoplasma acidophilum pursues orientated immobilization at metal‐chelating interfaces via affinity tags fused either around the pore apertures or at the sides. End‐on immobilization of the proteasome demonstrates that one pore is sufficient for substrate entry and product release. Remarkably, a ‘dead‐end’ proteasome can process only one substrate at a time. In contrast, the side‐on immobilized and free proteasome can bind two substrates, presumably one in each antechamber, with positive cooperativity as analyzed by surface plasmon resonance and single‐molecule cross‐correlation spectroscopy. Thus, the two‐stroke engine offers the advantage of speeding up degradation without enhancing complexity.