Article
- The EMBO Journal (2004) 23, 2488 - 2497
- doi:10.1038/sj.emboj.7600262
Published online: 3 June 2004
Subject Categories:
Two-substrate association with the 20S proteasome at single-molecule level
Silke Hutschenreiter1, Ali Tinazli1, Kirstin Model2 and Robert Tampé1
- Institute of Biochemistry, Biocenter, Johann Wolfgang Goethe-University Frankfurt, Frankfurt a. M., Germany
- Department of Structural Biology, Max-Planck-Institute of Biophysics, Frankfurt a. M., Germany
Correspondence to:
Robert Tampé, Institute of Biochemistry, Biocenter, Johann Wolfgang Goethe-University Frankfurt, Marie-Curie Str. 9–11, 60439 Frankfurt a. M., Germany. Tel.: +49 69 798 29476; Fax: +49 69 798 29495; E-mail: tampe@em.uni-frankfurt.de
Received 24 March 2004; Accepted 10 May 2004
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.
Keywords:
- molecular machines,
- multicatalytic proteases,
- protein degradation,
- self-compartmentalization,
- single-molecule analysis
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