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A real‐time FRET assay to measure the temporal order of events in the clamp loading reaction catalyzed by the Escherichia coli gamma complex
Author(s) -
Bloom Linda B.,
Anderson Stephen G.,
Paschall Christopher O.,
Thompson Jennifer A.
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.592.6
Subject(s) - clamp , förster resonance energy transfer , protein subunit , loader , biophysics , chemistry , patch clamp , dna clamp , voltage clamp , atp hydrolysis , dna , gamma subunit , biochemistry , fluorescence , biology , enzyme , atpase , gene , rna , membrane potential , receptor , mechanical engineering , reverse transcriptase , clamping , physics , quantum mechanics , engineering
A fluorescence resonance energy transfer (FRET) assay was developed to monitor protein‐protein interactions between the E. coli γ complex clamp loader and the β clamp directly in solution and in real time. A fluorescent donor is placed on the δ’ subunit, a unique subunit in 7‐subunit clamp loader. The clamp is a homodimer, and each monomer is symmetrically labeled with a nonfluorescent quencher. This FRET assay, in combination with real‐time DNA binding and ATP hydrolysis assays, was used to dissect the temporal order of events in the clamp loading reaction that occur when a pre‐formed clamp loader•clamp complex binds DNA. DNA binding initiates hydrolysis of three molecules of ATP, which in turn, triggers the clamp loader to release DNA and then the clamp. These results will be discussed in terms of a model in which interactions that the clamp loader makes with individual molecules of ATP (or ADP), the clamp, and DNA promote a series of conformational changes in the clamp loader. These changes modulate interactions with the next binding partner to favor a defined temporal order of events that leads to a productive clamp loading reaction.