| Autor: |
Yongdae Shin, Davis, Joseph H., Brau, Ricardo R., Martin, Andreas, Kenniston, Jon A., Baker, Tania A., Sauer, Robert T., Lang, Matthew J. |
| Předmět: |
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| Zdroj: |
Proceedings of the National Academy of Sciences of the United States of America; 11/17/2009, Vol. 106 Issue 46, p19340-19345, 6p |
| Abstrakt: |
CIpXP is an ATP-fueled molecular machine that unfolds and degrades target proteins. CIpX, 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 CIpP for degradation. Here, we develop and apply single-molecule fluorescence assays to probe the kinetics of protein denaturation and degradation by CIpXP. 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), CIpXP 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 CIpX 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 CIpXP 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. [ABSTRACT FROM AUTHOR] |
| Databáze: |
Complementary Index |
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