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Single Molecule Studies of Conformational Switching and Nucleotide Binding in the AAA+ Protease ClpXP

dc.contributor.advisorLang, Matthew J
dc.creatorManning, Harris Walpole 2020
dc.description.abstractAAA+ (ATPases Associated with various cellular Activities) proteins are an ancient family of molecular motors found in all branches of life. AAA+ motors are essential components in numerous cellular processes such as DNA replication and membrane fusion. The AAA+ motor ClpX is a ring-shaped homohexamer that uses the energy of ATP binding and hydrolysis to unfold and translocate target proteins into the lumen of the ClpP peptidase for proteolysis. Structural studies have established that ClpX subunits can adopt different distinct conformations, but it is not known whether subunits interconvert between these conformations. Additionally, biochemical studies have determined the binding affinities of different subunit classes in ATPase defective motors, but it is unclear how individual subunits interact with nucleotides and coordinate their transactions with other subunits. Here, we used single-molecule techniques to characterize conformational switching and nucleotide binding in ClpX subunits. We developed a quantitative conformational motion assay by engineering ClpX motors labeled with a fluorophore and a dark quencher in a single subunit. We calibrated our inter-probe distances with a novel single-molecule DNA annealing technique and verified ClpX-ClpP binding using fluorescence colocalization. Our results show that three subunit classes exist whose proportions and transition rates depend on nucleotide type and the presence of ClpP and substrate. Interestingly, we observed unique intermediate conformational states not present in either X-ray crystal or cryo-EM structures of ClpX and ClpXP, respectively. We used a hydrolyzable ATP analog functionalized with a dark quencher moiety, ATPQ, to monitor nucleotide binding/unbinding to a fluorescently-labeled ClpX subunit. We discovered that a working ClpX motor can bind up to 5 nucleotides at a time at saturation. Moreover, experiments with doubly-labeled ClpX hexamers revealed that subunits oriented in ortho and meta configurations bind nucleotides cooperatively. Finally, combined optical trapping and single-molecule fluorescence results suggest that large conformational changes are not necessary for substrate unfolding but may play an important role when ClpX exits a translocation pause.
dc.titleSingle Molecule Studies of Conformational Switching and Nucleotide Binding in the AAA+ Protease ClpXP
dc.type.materialtext Engineering University Graduate School

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