Single-molecule denaturation and degradation of proteins by the AAA+ ClpXP protease
This article contains supporting information online at www.pnas.org/cgi/content/full/ 0910484106/DCSupplemental.
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National Academy of Sciences
2010
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| Online Access: | http://hdl.handle.net/1721.1/58579 https://orcid.org/0000-0002-4614-251X https://orcid.org/0000-0002-1719-5399 |
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| author | Shin, Yongdae Brau, Ricardo R. Martin, Andreas Kenniston, Jon A. Lang, Matthew J. Baker, Tania Davis, Joseph Harry Sauer, Robert T |
| author2 | Massachusetts Institute of Technology. Department of Biological Engineering |
| author_facet | Massachusetts Institute of Technology. Department of Biological Engineering Shin, Yongdae Brau, Ricardo R. Martin, Andreas Kenniston, Jon A. Lang, Matthew J. Baker, Tania Davis, Joseph Harry Sauer, Robert T |
| author_sort | Shin, Yongdae |
| collection | MIT |
| description | This article contains supporting information online at www.pnas.org/cgi/content/full/
0910484106/DCSupplemental. |
| first_indexed | 2024-09-23T11:12:04Z |
| format | Article |
| id | mit-1721.1/58579 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T11:12:04Z |
| publishDate | 2010 |
| publisher | National Academy of Sciences |
| record_format | dspace |
| spelling | mit-1721.1/585792022-10-01T01:58:56Z Single-molecule denaturation and degradation of proteins by the AAA+ ClpXP protease Shin, Yongdae Brau, Ricardo R. Martin, Andreas Kenniston, Jon A. Lang, Matthew J. Baker, Tania Davis, Joseph Harry Sauer, Robert T Massachusetts Institute of Technology. Department of Biological Engineering Massachusetts Institute of Technology. Department of Biology Massachusetts Institute of Technology. Department of Mechanical Engineering Lang, Matthew J. Shin, Yongdae Brau, Ricardo R. Martin, Andreas Kenniston, Jon A. Sauer, Robert T. Lang, Matthew J. Baker, Tania Davis, Joseph Harry molecular machine protein degradation single-molecule fluorescence protein unfolding protein translocation This article contains supporting information online at www.pnas.org/cgi/content/full/ 0910484106/DCSupplemental. ClpXP is an ATP-fueled molecular machine that unfolds and degrades target proteins. ClpX, an AAA+ enzyme, recognizes specific proteins, and then uses cycles of ATP hydrolysis to denature any native structure and to translocate the unfolded polypeptide into ClpP for degradation. Here, we develop and apply single-molecule fluorescence assays to probe the kinetics of protein denaturation and degradation by ClpXP. These assays employ a single-chain variant of the ClpX hexamer, linked via a single biotin to a streptavidin-coated surface, and fusion substrates with an Nterminal fluorophore and a C-terminal GFP-titin-ssrA module. In the presence of adenosine 5'-[γ-thio]triphosphate (ATPγS), ClpXP degrades the titin-ssrA portion of these substrates but stalls when it encounters GFP. Exchange into ATP then allows synchronous resumption of denaturation and degradation of GFP and any downstream domains. GFP unfolding can be monitored directly, because intrinsic fluorescence is quenched by denaturation. The time required for complete degradation coincides with loss of the substrate fluorophore from the protease complex. Fitting singlemolecule data for a set of related substrates provides time constants for ClpX unfolding, translocation, and a terminal step that may involve product release. Comparison of these single-molecule results with kinetics measured in bulk solution indicates similar levels of microscopic and macroscopic ClpXP activity. These results support a stochastic engagement/unfolding mechanism that ultimately results in highly processive degradation and set the stage for more detailed single-molecule studies of machine function. Howard Hughes Medical Institute National Institutes of Health (U.S) (AI-15706) National Science Foundation (U.S.) (Career Award 0643745) Samsŏng Munhwa Chaedan (Korea) (Samsung Scholarship) 2010-09-17T13:36:36Z 2010-09-17T13:36:36Z 2009-11 2009-09 Article http://purl.org/eprint/type/JournalArticle 0027-8424 http://hdl.handle.net/1721.1/58579 Yongdae Shin, Joseph H. Davis, Ricardo R. Brau, Andreas Martin, Jon A. Kenniston, Tania A. Baker, Robert T. Sauer, and Matthew J. Lang (2009). Single-molecule denaturation and degradation of proteins by the AAA+ ClpXP protease. PNAS 106: 19340-19345. ©2009 by the National Academy of Sciences 19892734 https://orcid.org/0000-0002-4614-251X https://orcid.org/0000-0002-1719-5399 en_US http://dx.doi.org/10.1073/pnas.0910484106 Proceedings of the National Academy of Sciences of the United States of America Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. application/pdf National Academy of Sciences PNAS |
| spellingShingle | molecular machine protein degradation single-molecule fluorescence protein unfolding protein translocation Shin, Yongdae Brau, Ricardo R. Martin, Andreas Kenniston, Jon A. Lang, Matthew J. Baker, Tania Davis, Joseph Harry Sauer, Robert T Single-molecule denaturation and degradation of proteins by the AAA+ ClpXP protease |
| title | Single-molecule denaturation and degradation of proteins by the AAA+ ClpXP protease |
| title_full | Single-molecule denaturation and degradation of proteins by the AAA+ ClpXP protease |
| title_fullStr | Single-molecule denaturation and degradation of proteins by the AAA+ ClpXP protease |
| title_full_unstemmed | Single-molecule denaturation and degradation of proteins by the AAA+ ClpXP protease |
| title_short | Single-molecule denaturation and degradation of proteins by the AAA+ ClpXP protease |
| title_sort | single molecule denaturation and degradation of proteins by the aaa clpxp protease |
| topic | molecular machine protein degradation single-molecule fluorescence protein unfolding protein translocation |
| url | http://hdl.handle.net/1721.1/58579 https://orcid.org/0000-0002-4614-251X https://orcid.org/0000-0002-1719-5399 |
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