| Chapter title |
Mechanically Watching the ClpXP Proteolytic Machinery
|
|---|---|
| Chapter number | 12 |
| Book title |
Optical Tweezers
|
| Published in |
Methods in molecular biology, January 2017
|
| DOI | 10.1007/978-1-4939-6421-5_12 |
| Pubmed ID | |
| Book ISBNs |
978-1-4939-6419-2, 978-1-4939-6421-5
|
| Authors |
Juan Carlos Cordova, Adrian O. Olivares, Matthew J. Lang, Cordova, Juan Carlos, Olivares, Adrian O., Lang, Matthew J. |
| Abstract |
Energy-dependent protein degradation is studied through the dual bead ClpXP motility assay. Processing of folded proteins involves recognition, unfolding, translocation, and degradation stages. A dual optical trap, in a passive force-clamp geometry, exhibits bead-to-bead displacements that directly follow subprocesses underlying protein degradation. Discrete nanometer-scale displacements of the bead position reveal steps, dwells and pauses during the unfolding and translocation substeps. With a few structural modifications to the protease machinery and an engineered substrate, the assay represents a "chassis" for the measurement of a wide range of substrates and related machinery. The methods described faithfully record our assay as implemented, including substrate design, wet assay preparation, and the motility assay experiment protocol. The strategies herein permit adaptation of the ClpXP mechanical assay to a wide range of protein degradation systems. |
Mendeley readers
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| Unknown | 15 | 100% |
Demographic breakdown
| Readers by professional status | Count | As % |
|---|---|---|
| Student > Ph. D. Student | 3 | 20% |
| Professor | 2 | 13% |
| Student > Bachelor | 1 | 7% |
| Researcher | 1 | 7% |
| Professor > Associate Professor | 1 | 7% |
| Other | 0 | 0% |
| Unknown | 7 | 47% |
| Readers by discipline | Count | As % |
|---|---|---|
| Engineering | 3 | 20% |
| Chemical Engineering | 1 | 7% |
| Agricultural and Biological Sciences | 1 | 7% |
| Biochemistry, Genetics and Molecular Biology | 1 | 7% |
| Chemistry | 1 | 7% |
| Other | 1 | 7% |
| Unknown | 7 | 47% |