Single molecule fluorescence spectroscopy of ClpXP-mediated substrate degradation

Energy-dependent proteases, such as ClpXP, are responsible for the regulated destruction of proteins in prokaryotes and organelles of eukaryotes. AAA+ ATPases in these proteases recognize protein substrates and power their mechanical denaturation and subsequent translocation into a sequestered degradation chamber where polypeptide cleavage occurs. Here, we present the single molecule fluorescence assay for probing the interaction between the ClpXP enzyme and its substrates. A covalently crosslinked ClpX hexamer maintain functionally stable form at the low concentration of single molecule level. Surface passivation through polyethylene glycol (PEG) remove unwanted nonspecific binding of substrates, providing specific immobilization of ClpXP protease on the glass surface illuminated by total internal reflection fluorescence (TIRF). Cy3-labeled engineered substrates containing nondegradable GFP in the prescence of ATP[gamma]S form stable pre-engaged substrate-ClpXP complexes where the whole substrate degradation pathway, from unfolding to egress of degraded products, can be monitored without competing with dissociation or additional background characteristic of free labeled substrate in solution. We directly observe some terminal processes that are encountered by ClpXP at the end of substrate degradation process. It is also shown that GFP tail domain stably bind to the ClpX in the presence of ATP[gamma]S and even in the absence of ATP hydrolysis. With the developement of single molecule assay for AAA+ protease, we can expand our knowledge on the mechanism of this crucial motor protein family.

Department dc:contributor.department

Massachusetts Institute of Technology. Dept. of Mechanical Engineering.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2009

Author dc:creator

• Shin, Yongdae

Advisor dc:contributor.advisor

• Matthew J. Lang.

• Mechanical Engineering.

Statement dc:rights

• M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/50562

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/50562