Two-substrate association with the 20S proteasome at single-molecule level.
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.
Hutschenreiter
Silke
S
Institute of Biochemistry, Biocenter, Johann Wolfgang Goethe-University Frankfurt, Frankfurt a. M., Germany.
Tinazli
Ali
A
Model
Kirstin
K
Tampé
Robert
R
D000345:Affinity Labels / Q000378:metabolism; D015153:Blotting, Western; D002364:Caseins / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D019793:Fluorescein / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D006868:Hydrolysis; D008051:Lipid Bilayers / Q000378:metabolism; D008433:Mathematics; D008670:Metals / Q000494:pharmacology; D008956:Models, Chemical; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance; D013822:Thermoplasma / Q000378:metabolism; D013997:Time Factors
D000345:Affinity Labels; D002364:Caseins; D002614:Chelating Agents; D008051:Lipid Bilayers; D008670:Metals; D006639:Histidine; D004492:Edetic Acid; D003546:Cysteine Endopeptidases; D019793:Fluorescein